Serine Proteases: Role in Human Health and Disease 9783111325040, 9783111324982

Table of contents :
About the editors
Preface
Acknowledgments
Contents
List of contributing authors
Chapter 1 Serine proteases: classification, catalytic mechanism, types, and industrial applications
Chapter 2 Serine proteases and respiratory disorders
Chapter 3 Role of serine proteases in cancer progression and metastasis
Chapter 4 Role of serine proteases in lung diseases: a view from acute and chronic lung infection complications
Chapter 5 Inhibition of the serine exopeptidase DPP-IV as a means of mitigation of diabetic nephropathy
Chapter 6 Serine proteases in arthritis
Chapter 7 Serine protease HtrA: a promising therapeutic target to develop antimicrobial therapy
Chapter 8 Potential targeted therapy for SARS-CoV-2: host serine proteases
Chapter 9 Role of serine proteases in fertility
Chapter 10 Serine protease inhibitors and its therapeutics
Index

Citation preview

Serine Proteases

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Serine Proteases

Role in Human Health and Disease Edited by Anil K. Sharma and Poonam Bansal

Editors Prof. Dr. Anil K. Sharma Department of Bio-Sciences and Technology Maharishi Markandeshwar University (Deemed to be University) Mullana, Ambala 133207 Haryana India [email protected] Dr. Poonam Bansal Department of Bio-Sciences and Technology Maharishi Markandeshwar University (Deemed to be University) Mullana, Ambala 133207 Haryana India [email protected]

ISBN 978-3-11-132498-2 e-ISBN (PDF) 978-3-11-132504-0 e-ISBN (EPUB) 978-3-11-132528-6 Library of Congress Control Number: 2023941342 Bibliographic information published by the Deutsche Nationalbibliothek The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available on the Internet at http://dnb.dnb.de. © 2023 Walter de Gruyter GmbH, Berlin/Boston Cover image: Christoph Burgstedt/iStock/Getty Images Plus Typesetting: Integra Software Services Pvt. Ltd. Printing and binding: CPI books GmbH, Leck www.degruyter.com

Dedicated to My dear husband CA Pavit Singla Dr. Poonam Bansal Editor

About the editors Prof. Anil K. Sharma is having 25 years of research experience including industrial R&D, teaching and administration. Currently, he is working as a Professor and Head in the Department of Bioscience and Technology at Maharishi Markandeshwar (M.M.) (Deemed to be University) Mullana, Ambala, Haryana, India for the past 13 years (2010–till date) and before this assignment worked as a Senior Research Specialist in Health Sciences (2008–2010) and a Post-Doctoral Research Fellow (Molecular biology) (2003–2010) in the Microbiology and Immunology Department at UIC College of Medicine Chicago, IL, USA. Dr. Sharma has worked in diverse scientific fields ranging from molecular biology, cancer biology, antimicrobial drug resistance, nanomedicines to the development of microbial strains for remediation of heavy metals, and pesticides. His contributions to Medical Biotechnology and Industrial cum Pharmaceutical Biotechnology have been greatly acknowledged. Dr. Sharma has published more than 205 articles in peer-reviewed, high impact journals of international repute with some of them in Journal of Biological Chemistry (ASBMB, USA) (impact factor ~4.57), Plant Biotechnology Journal (impact factor 13.4), Seminars in Cancer Biology (Elsevier, impact factor ~17.1), Anti-Cancer Agents in Medicinal Chemistry (impact factor 2.7), Immunology Cell Biology (Nature Publishing Group) (impact factor 4.2), Current Medicinal Chemistry (impact factor 3.8), etc. having more than 6,685 citations (H-index ~34;i10 index:72; cumulative impact factor ~475). He has filed 17 patent inventions and published 12 books from prestigious publishers including Springer, Pan-Stanford, Nova Publishers as well. Dr. Sharma has been felicitated with many awards for scientific excellence during his career such as Outstanding Scientist Award-2021, Abdul Kalam Azad Award-2018, BRICPL Eminent Scientist Award (2017 and 2018), Achiever Award-2017, Appreciation Award-2008 from UIC Illinois at Chicago, USA, MGIMS Young Scientist Award-2000, VCS Memorial Young Scientist-Award 1999, etc.

Anil K. Sharma Professor & Head, Biotechnology, Maharishi Markandeshwar (Deemed to be University) Mullana, Ambala 133207, Haryana, India; Phone:+91-8059777758 Email: [email protected] or [email protected] Google Scholar: Dr. Anil K Sharma,PhD, Sr. Res. Scientist Health Science (2008–2010) and PostDoc UIC USA (2003–2008) – Google Scholar SCOPUS ID: Sharma, Anil K. – Author details – Scopus Preview Web OF Science ID: Sharma, Anil K. – Web of Science Core Collection https://orcid.org/0000-0002-9768-1644 ORCID ID: Top 2% highly cited researchers in the world (2019, 2020, and 2021) by Stanford University California, USA Lead Guest Editor for special issues for Seminars in Cancer Biology (IF ~17.1) (2018 and 2021)

Dr. Poonam Bansal is currently working as an Assistant Professor in the Department of Bioscience and Technology at M.M. (Deemed to be University) Mullana, Ambala, Haryana, India. She is having 5 years of research experience including biosciences research, Teaching and Administration. Dr. Bansal have got her master’s (Biochemistry) and doctorate degree (Biochemistry) from Kurukshetra University, Kurukshetra. During her doctoral study, she worked on Phenotypic, Biochemical, Molecular and In silico Characterization of Pediococcus acidilactici NCDC 252 and screening it for its safety and efficacy assessment as per ICMR-DBT (INDIA) guidelines in DST-INDIA funded project as Research Fellow (2015–2019). This work helped in establishing Pediococcus acidilactici NCDC 252 strain as a useful probiotic strain. Dr. Bansal has worked in diversified scientific areas ranging from Protein Biochemistry, Molecular Biology to https://doi.org/10.1515/9783111325040-202

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About the editors

Genomics, Proteomics, Next Generation Sequencing, and Drug designing. She also published Genome Sequence of Pediococcus acidilactici NCDC 252 in National Center for Biotechnology Information (NCBI), 2019 and identified 19 novel genes in Pediococcus acidilactici NCDC 252 genome. Dr. Bansal has published 23 research articles in peer-reviewed, internationally repute journals with some of them in Process Biochemistry (4.88), World Journal of Microbiology and Biotechnology (4.25), Molecular Biology Report (2.74), and Catalysis Letter (2.93) having 103 citations (H-Index ~5;i10 index:4; cumulative impact factor: 26.201).

Dr. Poonam Bansal Assistant Professor, Maharishi Markandeshwar (Deemed to be University), Mullana, Ambala 133207, Haryana, India; Phone: +91-99965995552 Email: [email protected] Google Scholar: https://scholar.google.com/citations?user=r_NS6ksAAAAJ&hl=en Scopus: https://www.scopus.com/authid/detail.uri?authorId=57210319201 Web of Science: https://www.webofscience.com/wos/author/record/GPK-0746-2022 ORCID ID: (https://orcid.org/0000-0001-5756-1034)

Preface Proteases are the large and diverse cluster of hydrolytic enzymes that are classified based on their site of action, structure of active site, and particular reaction mechanisms. Proteases, also termed as proteinases or peptidases, are enzymes that carry out proteolysis. Proteases are widely distributed in nature and perform several important biological functions in living system such as growth, development, regulation, adaptation, germination, protein turnover, disease, and death. These enzymes evolved to perform various reactions by different mechanisms. These enzymes are classified on the basis of their mechanisms of catalysis, and totally seven known distinct classes of proteases are identified, that is, serine, metallo, cysteine, aspartic, glutaic, asparagine, and threonine proteases. Serine proteases (EC 3.4.21) are a family of proteases, utilize active serine (Ser) in its substrate-binding site, and hydrolyze peptide bonds. Serine proteases are divided into two main classes on the basis of their localization inside the extracellular matrix (ECM): cell-surface-anchored or membrane-anchored serine proteases and secreted serine proteases. Serine proteases are widespread and significantly played various roles in a physiology and human disease. Abnormal expression and activities of serine proteases have been significantly linked with pathogenesis of many diseases. The endeavor of the book entitled Serine Proteases is to present the association of serine proteases and human diseases. This book has 10 chapters that will help the readers to understand classification, catalytic mechanism, and types of serine proteases and their role in human disease pathogenesis and the underlying mechanism. The book covers the general introduction to serine, industrial importance, and its role in respiratory disorders. The chapters briefly explain the role of serine proteases in lungs infection, diabetic nephropathy, arthritis, fertility, and cancer. The book also covers the therapeutic importance of serine proteases as drug targets along with the mechanistic insights of serine proteases inhibitors. Serine protease is known to play crucial role in biological processes but disturbance in their equilibrium can result in serious health conditions. To maintain homeostasis, serine protease inhibitors come in action and inhibit proteases. Several serine protease inhibitors have been identified and many more are being designed as novel compounds for inhibitions of proteases that provide management of comorbidities. Therefore, this book will serve as a useful reference for students and researchers to understand physiological role of serine proteases and their association with initiation and progression of human diseases. It will also help to devise strategies to develop serine proteases inhibitors as drug target of serine proteases at cellular and molecular level. Editors

https://doi.org/10.1515/9783111325040-203

Acknowledgments The successful completion of this book would have been impossible without mentioning the names of those persons who helped me to make it possible. I thank Almighty for giving me support to complete this book. First and foremost, my heartfelt thanks to Dr. Anil K. Sharma, Head (Professor), Department of Bioscience and Technology, MM(DU), Ambala who worked as coeditor and helped me to edit this book. He always provided scientific advice and moral support throughout in editing this book. I wish to express my deep sense of gratitude to Dr. Jasbir Singh and Dr. Suman Dhanda, Professor, Department of Biochemistry, K.U.K., for their invaluable suggestions given time to time. I also extend my gratitude to all the authors who contributed in the completion of book. I am grateful to Maharishi Markandeshwar (Deemed to be University), Mullana, Ambala for providing the requisite facilities to complete this book. I am deeply thankful to my parents Mr. Sushil Kumar and Mrs. Satya Devi for their unconditional moral support. A special thanks to my dear husband CA Pavit Singla for being with me in every moment of ups and downs. Thanks to my beloved little angel Hinaya Singla for making me smile all the time. I extend my warm thanks to Dr. Raman Kumar, Department of Biochemistry, Kurukshetra University Kurukshetra for his support and timely help. Last but not the least, I would like to express my gratitude and respect to everyone who has helped in one way or the other in completing this book. Dr. Poonam Bansal Editor

https://doi.org/10.1515/9783111325040-204

Contents About the editors Preface

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Acknowledgments

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List of contributing authors

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Poonam Bansal, Anil K. Sharma, Raman Kumar, Preeti Chanalia, and Bhupesh Gupta Chapter 1 Serine proteases: classification, catalytic mechanism, types, and industrial applications 1 Priti, Poonam Bansal, Sonali Sangwan, and Shweta Dhanda Chapter 2 Serine proteases and respiratory disorders 17 Raman Kumar, Poonam Bansal, and Praveen Kumar Chapter 3 Role of serine proteases in cancer progression and metastasis

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Raman Kumar, Poonam Bansal, and Praveen Kumar Chapter 4 Role of serine proteases in lung diseases: a view from acute and chronic lung infection complications 51 Semim Akhtar Ahmed, Anuj Kumar Borah, and Jagat C. Borah Chapter 5 Inhibition of the serine exopeptidase DPP-IV as a means of mitigation of diabetic nephropathy 65 Preeti Chanalia, Poonam Bansal, and Dimpi Gandhi Chapter 6 Serine proteases in arthritis 83

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Kapil Singh Narayan, Reenu Kashyap, Raman Kumar, Anil K. Sharma, Anil Panwar, and Varruchi Sharma Chapter 7 Serine protease HtrA: a promising therapeutic target to develop antimicrobial therapy 101 Shweta Dhanda, Kiran Bala, Priti, Anil K. Sharma, Anil Panwar, and Varruchi Sharma Chapter 8 Potential targeted therapy for SARS-CoV-2: host serine proteases 109 Shweta Dhanda, Kiran Bala, Anil K. Sharma, Anil Panwar, and Varruchi Sharma Chapter 9 Role of serine proteases in fertility 129 Sonali Sangwan, Shikha Yashveer, Mahiti Gupta, Priti, Himani Punia, Jayanti Tokas, and Bhupesh Gupta Chapter 10 Serine protease inhibitors and its therapeutics 141 Index

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List of contributing authors Poonam Bansal Department of Biosciences and Technology Maharishi Markandeshwar (Deemed to be University) Mullana, Ambala 133207 Haryana India [email protected] Chs 1, 2, 3, 4, 6 Anil K. Sharma Department of Biosciences and Technology Maharishi Markandeshwar (Deemed to be University) Mullana, Ambala 133207 Haryana India Ch 1, 7, 8, 9 Raman Kumar Department of Biochemistry Kurukshetra University Kurukshetra 136119 Haryana India [email protected] Ch 1, 3, 4, 7 Preeti Chanalia Research and Innovation Department Shri Krishna AYUSH University Kurukshetra, Haryana India Ch 1, 6 Bhupesh Gupta Computer Science and Engineering Department MMEC Maharishi Markandeshwar (Deemed to be University) Mullana, Ambala 133207 Haryana India Ch 1, 10

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Priti Department of Biotechnology K. L. Mehta Dayanand College for Women Faridabad Haryana India Chs 2, 8, 10 Sonali Sangwan Department of Biosciences and Technology Maharishi Markandeshwar (Deemed to be University) Mullana, Ambala Haryana India Ch 2, 10 Shweta Dhanda National Centre for Veterinary Type Cultures ICAR-National Research Centre on Equines Hisar India [email protected] Ch 2, 8, 9 Praveen Kumar Department of Biochemistry Kurukshetra University Kurukshetra 136119 Haryana India Chs 3, 4 Semim Akhtar Ahmed Chemical Biology Lab Institute of Advanced Study in Science and Technology (IASST) Paschim Boragaon Guwahati 35 Assam India And Academy of Scientific and Innovative Research (AcSIR) India Ch 5

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Anuj Kumar Borah Department of Biochemistry Royal School of Bio-Sciences The Assam Royal Global University Betkuchi, NH-37 Guwahati Assam India Ch 5 Jagat C. Borah Chemical Biology Lab Institute of Advanced Study in Science and Technology (IASST) Guwahati-35 Assam India and Academy of Scientific and Innovative Research (AcSIR) Ghaziabad India Division of Life Sciences Vigyan Path Guwahati Assam-781035 [email protected] Ch 5 Preeti Chanalia Research Assistant Research and Innovation Department Shri Krishna AYUSH University Kurukshetra, Haryana India [email protected] Ch 6 Dimpi Gandhi Research associate I National Brain Research Centre Gurgaon Haryana India Ch 6

Kapil Singh Narayan Department of Clinical Studies School of Veterinary Medicine New Bolton Center University of Pennsylvania Kennett Square PA 19348 USA [email protected] Ch 7 Reenu Kashyap 382 West Street Road CAHP Building New Bolton Center School of Veterinary Medicine University of Pennsylvania PA 19348 Anil Panwar Department of Bioinformatics and Computational Biology College of Biotechnology CCS Haryana Agricultural University Hisar Haryana 125004 Varruchi Sharma Department of Biotechnology and Bioinformatics Sri Guru Gobind Singh College Sector-26 Chandigarh (UT) India Chs 7, 8, 9 Kiran Bala Department of Biochemistry Om Sterling Global University Hisar Haryana India Chs 8, 9

List of contributing authors

Shikha Yashveer Department of Molecular Biology and Biotechnology Department of Molecular Biology Biotechnology, and Bioinformatics College of Biotechnology Chaudhary Charan Singh Haryana Agricultural University Hisar 125004 Haryana India Ch 10 Mahiti Gupta Department of Biosciences and Technology Maharishi Markandeshwar (Deemed to be University) Mullana, Ambala 133207 Haryana India Ch 10

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Himani Punia Department of Sciences Chandigarh School of business Chandigarh Group of Colleges Jhanjeri 140307 Mohali India Ch 10 Jayanti Tokas Department of Biochemistry COBS&H Chaudhary Charan Singh Haryana Agricultural University Hisar 125004 Haryana India Ch 10

Poonam Bansal✶, Anil K. Sharma, Raman Kumar, Preeti Chanalia, and Bhupesh Gupta

Chapter 1 Serine proteases: classification, catalytic mechanism, types, and industrial applications Abstract: Proteases, also named as peptidases, proteinases, or proteolytic enzymes, are hydrolytic enzymes that hydrolyze peptide bonds within protein molecules. Serine proteases are considered as the largest proteolytic class belonging to S1 family of the PA clan superfamily which comprises the largest number of serine proteases. Serine proteases utilize activated serine in its substrate-binding site to catalytically hydrolyze peptide bonds. Serine proteases are involved in many important developmental and normal physiological processes of living systems including humans. Abnormal expression of these proteases leads to several destructive diseases, inflammatory disorders as well as cancer. These serine proteases are not only important for physiological functioning of living organisms, but they also have commercial significance. Proteases from microbes are preferred over other sources because of their ease of availability, economic advantages, and rapid growth. Serine proteases have wide range of applications in leather, detergent, pharmaceutical, and food processing industry. Modern techniques including protein engineering and metagenomics open up avenues for discovery of new enzymes with modified catalytic properties. Keywords: serine proteases, classification, catalyst, mechanism, commercial importance

Introduction Proteases are the largest and diverse cluster of hydrolytic enzymes that are classified based on their specific site of action, active site structure, and specific reaction mechanisms (Ward, 2011). The term “proteases” defines enzymes that digest proteins. It was in-

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Corresponding author: Poonam Bansal, Department of Biosciences and Technology, Maharishi Markandeshwar (Deemed to be University), Mullana, Ambala 133207, Haryana, India, e-mail: [email protected] Anil K. Sharma, Department of Biosciences and Technology, Maharishi Markandeshwar (Deemed to be University), 133207, Mullana, Ambala, Haryana, India Raman Kumar, Department of Biochemistry, Kurukshetra University, Kurukshetra, India Preeti Chanalia, Research & Innovation Department, Shri Krishna AYUSH University, Kurukshetra, India Bhupesh Gupta, Computer Science and Engineering Department, MMEC, Maharishi Markandeshwar (Deemed to be University), Mullana, Ambala, 133207, Haryana, India https://doi.org/10.1515/9783111325040-001

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troduced in 1903 from University of Oxford by S. H. Vines (Professor of Botany). Moreover, the term “peptidase” was introduced later by Petersen and Short (PhilippsWiemann, 2018). Proteases are ubiquitous in all living systems that catalyze hydrolysis of proteins to peptides and their constituent amino acids (Bansal et al., 2021). Proteases are present nearly in all animals, plants, and microbes. In higher organisms, approximately 2% of the gene codes are formed by proteases. Proteases are ubiquitous and perform a major role in the physiological functioning of living organisms and also in commercial fields. These enzymes possessed highly specialized proteolytic functions. Being ubiquitous in nature and found in all living organisms, these enzymes are essential for cell growth and differentiation. Proteases have great importance in the industrial sector and thus provide a lot of economic advantages (Rani et al., 2012). Generally, proteases are classified as endopeptidases and exopeptidases (Figure 1). Endopeptidases are proteases that mainly operate on the interior of its substrate, whereas exopeptidases operate on ends of the substrate (Gonçalves et al., 2016). Proteases are further classified based on their catalytic mechanism of action and type of functional group at their active site. There are total seven known distinct classes of proteases identified, that is, serine, cysteine, metallo, aspartic, glutamic, asparagine, and threonine proteases (Dong et al., 2021). Threonine (EC 3.4.25) and serine proteases (EC 3.4.21) have hydroxyl groups, while cysteine proteases (EC 3.4.22) have the sulfhydryl group as a nucleophile, whereas for glutamic (EC 3.4.19), aspartic (EC 3.4.23), and metallo-proteases (EC 3.4.24), activated water is a nucleophile in their catalytic system (Gonçalves et al., 2016). Proteases constitute a large and diversified enzymatic group which differs in enzymatic properties such as pH and temperature optima, stability, active site, substrate specificity, and catalytic mechanism. Enzymatic specificity of proteolytic enzymes is based on the type of amino acid and other functional groups (aliphatic/aromatic/sulfur-containing) close to the bond being hydrolyzed. Along with their role in different proteolytic reactions, these enzymes also regulate various enzyProteases

Exoproteases

Endoproteases Serine endopeptidases

Aminopeptidases Aspartic endopeptidases Metalloendopeptidases Carboxypeptidases

Gulatmic acid endopeptidases Cyteine/thiol endopeptidases Threonine endopeptidases

Figure 1: Classification of proteases.

Chapter 1 Serine proteases

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matic cascades, thus promoting all metabolic reactions involving in the living systems (i.e., breakdown of fats and carbohydrates) (Sumantha et al., 2006). Proteases are important mediators of initiation of different biological processes including in the growth and development. Enzymes participate in regulation of gene expression and thus control several physiological functioning of the host viz. cell division and differentiation, reproductive system, meiosis, senescence, epidermal cell fate, chloroplast biogenesis, and stomata development. Enzymes are also involved in the breaking down of unfolded proteins, protein processing and their targeting, and activation of zymogen and protein hormones via digestion of signal peptide digestion. Proteases also participate in apoptosis and local and systemic defense responses (Gonçalves et al., 2016).

Serine proteases Among all the classes of proteases, serine proteases are considered as the largest proteolytic class, containing 13 clans and 40 families. Over one-third of the identified proteases comprise serine proteases (Di Cera, 2009). Serine proteases are involved in many important developmental and physiological processes of the living system (Böttcher-Friebertshäuser, 2018).

General properties of serine proteases Serine proteases (EC 3.4.21) are a family of proteases which are named on the basis of nucleophilic serine (Ser) present in their substrate-binding site. The presence of Ser in its active site breaks down the peptide bond at its carboxyl terminus and produces an acyl-enzyme intermediate (Ekici et al., 2008). The enzyme activity of serine proteases is regulated at transcriptional, post-transcriptional, and post-translational levels (Craik et al., 2011). Generally, these enzymes act as zymogens to regulate enzymatic activities (Poddar et al., 2017). Serine proteases are present in all living forms including several viral genomes. But major differences are present in their existence in every clan across species. Proteases of the PA clan are highly characterized in eukaryotic species, but uncommon in prokaryotic and plant genomes (Page and Di Cera, 2008). Mostly, serine proteases are of endopeptidase type (Di Cera, 2009).

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Catalytic mechanism of serine proteases Serine proteases utilize activated Ser in its substrate-binding site to catalytically hydrolyze peptide bonds. Nucleophilic Ser is present in the active site and targets the carbonyl moiety of the peptide bond of the substrate to form a tetrahedral intermediate (acyl-enzyme intermediate). Enzymes contain three residues at their active site: Ser195, histidine57 (His), and an aspartate102 (Asp). The nucleophilicity of this Ser is mainly dependent on the catalytic triad of aspartate (Asp), histidine (His), and Ser. This is called as a charge relay system. This catalytic triad was first seen 30 years ago in chymotrypsin (Page and Di Cera, 2008). Amino acids present in this catalytic triad are very far from each other in the primary structure of the enzyme. However, upon protein folding, these residues come into close proximity to carry out specific enzyme catalysis (Poddar et al., 2017). The presence of this catalytic triad (Asp-His-Ser) in four different protein folds, that is, in subtilisin, trypsin, prolyl oligopeptidase, and ClpP peptidase, displayed the evolvement of this triad as a catalytic machinery in four different evolutionary pathways (Laskar et al., 2012). Other enzyme families such as esterases, asparaginases, lactamases, and acylases also utilize an identical catalytic triad for nucleophilic catalytic reaction mechanism (Page & Di Cera, 2008). Additionally, many serine proteases have also been reported to utilize dyad of Lys or His paired with Ser to catalyze the reaction mechanism. However, some initiated catalysis via a unique catalytic triad in which His pairs with the nucleophilic Ser (Di Cera, 2009). Through ping-pong catalysis, serine proteases form an enzyme-peptide intermediate (unstable), stabilize the reaction intermediate, and finally the release of peptide fragment. In the reaction mechanism, serine protease participates in acylation followed deacylation, where a nucleophilic attack of water molecule occurred on the intermediate and thus promote proteolysis. This whole process was mediated by the catalytic triad of serine protease (Poddar et al., 2017).

Conversion of zymogen to active serine proteases Among all serine protease families, the S1 family of the PA clan superfamily has the largest number of serine proteases (Tripathi and Sowdhamini, 2008). S1 family comprises the major group of similar proteases in human genome involved in various biological processes (Di Cera, 2009). Proteases of PA clan are well characterized in eukaryotes, participating in different extracellular functions. Mostly, these enzymes possessed trypsin-like substrate specificity that breakdown polypeptide on its carboxyl-terminus at lysine (Lys) or arginine (Arg) (Laskar et al., 2012). Several biological processes depend on clan PA proteases including blood coagulation and immunomodulation, which involve cascades of zymogen activation. S1 family of PA clan is divided into two parts, that is, S1A and S1B. Both these subfamilies evolutionarily differ

Chapter 1 Serine proteases

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from each other but have similar two β-barrel structures. S1B proteases are present in all living forms and are involved in intracellular protein turnover. However, S1A proteases are trypsin-like and thus mediate several extracellular processes (Di Cera, 2009). There is a need of proteolysis of inactive precursor to produce an active enzyme (Chakraborty et al., 2018). In all family members, the cleavage of zymogen occurred at the same position (Di Cera, 2009). N-terminal of nascent protein initiated conformational changes in enzyme via intramolecular electrostatic interaction with Asp194 to stabilize substrate-binding region and oxyanion hole. Enzyme stabilization in its active form is usually accompanied by disulfide bonds. Zymogen activation is a major regulatory mechanism to control peptidase activities, prevent premature enzyme inhibition, and release active enzymes to initiate proteolytic events. This proteolytic network of serine proteases is responsible for major biological processes of living system (Chakraborty et al., 2018; Page and Di Cera, 2008).

Types of serine proteases Based on the cleavage specificity of the peptide bonds, serine proteases are of three types: trypsin (cleaves Lys and Arg at the cleavage site), chymotrypsin (cleaves tyrosine, phenylalanine, or tryptophan (aromatic amino acids) at the cleavage site), and elastase (cleaves c-terminus of amino acids having side chain viz. valine, alanine, etc.) (Poddar et al., 2017). Trypsin-like, chymotrypsin-like, and elastase-like serine proteases are part of S1 family of serine proteases (Laskar et al., 2012). Serine proteases are classified into two broad classes on the basis of their localization within the extracellular matrix (ECM): membrane-anchored (cell-surface anchored) serine proteases and secreted serine proteases. Membrane-anchored serine proteases are unique subgroup of S1 serine proteases that are found to be directly anchored to the plasma membrane through its carboxy- or amino-terminus domains (Poddar et al., 2017). Membrane-anchored serine proteases are further subdivided into different subgroups on the basis of their structural characteristics. Enzymes are attached to cell membrane via three modes: (i) carboxy-terminus transmembrane domain (Type I transmembrane serine proteases (TTSPs)), (ii) amino-terminus transmembrane domain having cytoplasmic extension (TTSPs or Type II TTSPs), and (iii) carboxylterminus transmembrane domain with glycosyl phosphatidylinositol (GPI) linkage that is added post-translationally (Antalis et al., 2010; Martin and List, 2019). Membrane-anchored serine proteases have structurally conserved catalytic domain, belonging to the S1 peptidase family. Generally, such enzymes are present in their inactive form as zymogens. After autoactivation of these enzymes, the catalytic domain remains membrane-bound (Szabo and Bugge, 2011; Böttcher-Friebertshäuser, 2018). Mostly identified serine proteases in humans belong to type II TTSPs (Tanabe and

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List, 2017). This protease family is further divided into four subfamilies: (i) matriptase, (ii) hepsin/transmembrane protease/serine (TMPRSS), (iii) human airway trypsin-like (HAT)/differentially expressed in squamous cell carcinoma (DESC), and (iv) corin. TTSPs are synthesized as inactive precursors and need to be activated by proteolytic cleavage. Hepsin, matriptase, matriptase-2, TMPRSS2, TMPRSS3, TMPRSS4, and TMPRSS13 are of TTSPs types, capable of their own autoactivation which further suggest their basal activity as zymogens (Tanabe and List, 2017; Martin and List, 2019). Similarly, secreted serine proteases are also fully characterized family members of S1 serine proteases. The enzymes are generally released into the extracellular environment by secretory vesicles (Poddar et al., 2017). Secreted serine proteases such as fibroblast activation protein-α, urokinase plasminogen activator (uPA), kallikrein-related peptidase, HtrA, and granzymes are found to be associated with several biological events in living organisms (Rao et al., 1998). uPA and kallikreins participate in pericellular proteolysis either by binding with the coreceptors or by stimulating zymogens of other substrates (De Lorenzi et al., 2016; Poddar et al., 2017).

Serine proteases in human physiology and pathology Generally, proteases are connected to various physiological functions of human body such as cell proliferation in development and differentiation, tissue morphogenesis and remodeling, angiogenesis, unfolded protein responses, heat shock, autophagy, apoptosis, senescence, necrosis, inflammation, and immunity (López-Otín and Bond, 2008; Herszényi et al., 2014). These enzymes play a significant role in the breaking down and reconstitution of ECM in different physiological processes (Lu et al., 2011). Serine proteases are considered as the largest family of human proteases, which participate in several major physiological and developmental processes of humans including cellular growth, differentiation and development, digestion of proteins, blood coagulation, and regulation of complement system (Patel, 2017). Cell surface-localized proteases-mediated pericellular proteolysis has been considered as an important pathway where interaction of cells with their cellular microenvironment occurred. Several proteins of pericellular microenvironment viz. enzymes, growth factors, receptors, cell adhesion molecules, and cytokines are present in their inactive forms which further need to be activated by endo-proteolytic cleavage of peptide bonds. Activation of these inactive precursors is mediated by cell surface-localized proteases via protease receptors or directly by cell-surface anchored protease through membrane-anchoring domains (Antalis et al., 2010). Cell-surface-anchored serine proteases are known to contribute in several different types of physiological processes viz. fertilization, development of embryo, tissue morphogenesis, cell signaling, and maintenance of epithelial barrier (Szabo and Bugge, 2011). Secreted serine proteases are

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known to participate in many biological processes such as nutrients uptake, tissue repair, and immunity (Poddar et al., 2017). Studies revealed that serine proteases participate in the regulation of cell behavior, survival, and death. But abnormal expression of serine proteases and alterations in proteolytic systems of these proteases cause further pathological severities (LópezOtín and Bond, 2008; Herszényi et al., 2014). Dysregulation of pericellular and extracellular proteolysis is associated with the secreted and cell-surface-anchored serine proteases which is considered as a major hallmark in many diseases. Abnormal expression of these proteases leads to severe diseases including tumor development and metastasis (Herszényi et al., 2014).

Commercial serine proteases Being necessary for physiological functioning of host, proteases are found across all biological sources. These hydrolytic enzymes are present in all living organisms from eukaryotes like animals, plants, fungi, protists to prokaryotic domains of archaea, and bacteria. Viruses are also reported to encode proteases in their life cycle (Solanki et al., 2021; Rani et al., 2012). Proteases account for approximately 60% of the total enzyme market in the world. These enzymes play a very significant role in industrial sector. Industrial advantages of proteases covered several industries viz. leather, textile and detergent, food processing, and pharmaceuticals (David Troncoso et al., 2022). Among all proteases, serine proteases have received more attention because these enzymes have wide-spectrum applications in industrial processes from food to pharmaceuticals (Bansal et al., 2021). Serine proteases are known to possess unique properties like higher stability, comparatively low specificity for substrate, higher microbial secretion, and easy downstream processing. Owing to these properties, serine proteases have attracted world-wide attention in an attempt to increase their production from different sources for biotechnological applications (Rao et al., 1998).

Plant-based serine proteases Serine proteases are considered as the largest class of proteases in plants. Studies showed that plant serine proteases are involved in various processes including regulation of plant development and defense responses. However, some plant serine carboxypeptidases belong to S10 family, which function as acyltransferases instead of hydrolases. This suggested the diversification of function of proteolytic enzymes to regulate secondary metabolism of plants (Tripathi and Sowdhamini, 2006). Plant serine proteases are widely distributed among taxonomic groups, that is, from trees and

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crops to legumes and herbs. Serine proteases are found in nearly all parts of plants, but appear to be higher in fruits. Plant origin serine proteases are known to be most active and stable enzymes because of their stability at higher temperature, pH, in the presence of surfactants and oxidizing agents, and thus considered as most economical for industrial purpose (Thakur and Kumar, 2018). Serine proteases have been successfully extracted from barley, jackfruit, maize, and melon etc. (Antão and Malcata, 2005). Seeds have also been reported to be a good source of serine proteases. Enzymes have been isolated from soy, mung, and jack bean. These enzymes are trypsin-like and showed milk-clotting activity. Solanaceae family members have subtilase-type serine proteases. Plant pollens have also been known to harbor subtilisin-like serine proteases (Patel, 2017). Many serine proteases have been productively produced by in vivo methodology from a wide range of tissues. Examples are seeds (Arabidopsis thaliana, barley, and rice), flowers (cardoon), and leaves (potato and tomato) (Troncoso et al., 2022).

Microbial-based serine proteases Microbes are considered as outstanding source of enzymes because of their large biochemical diversity and their susceptibility to genetic manipulations (Adrio and Demain, 2014). Proteases from microbial source have become more popular and have distinct advantages associated with them. Microorganisms, as proteases source, offer many benefits viz. higher productive rate, low cost of investment with respect to time and land requirements, and not being influenced by climate. Nowadays, proteases of microbes represent two-third part of the total proteases used in industrial sector (Solanki et al., 2021). Microbial proteases are preferred more than that of animals and plants because of the presence of all desired properties for industrial uses including their easy availability, economic advantages, and faster growth rate (Bansal et al., 2021). Serine proteases from microorganisms have attracted wider interest since last decade owing to their uniqueness and broad industrial applications (Raj et al., 2017). Alkaline serine proteases are one of the most important groups of industrial enzymes (Bhunia et al., 2012). Alkaline serine proteases shared two-third part of the worldwide protease market because of higher stability, low specificity for substrate, and increased microbial secretion (Matkawala et al., 2021). Enzymes are produced by various types of fermentation techniques using microorganisms (Bhunia et al., 2012). The quantity of enzymes produced from microbes varies between species to species. It also depends on the type of culture medium used for the microbial growth (Soccol et al., 2017). Serine alkaline proteases are active in neutral to alkaline pH range (Matkawala et al., 2021). These enzymes are produced by several bacteria, fungi, yeasts, and molds. Most commonly used microorganisms for the production of alkaline serine proteases belong to Bacillus sp. having broad applications in leather, detergent,

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pharmaceutical, and food processing industry (Thakur and Kumar, 2018; Bhunia et al., 2012). Microbial species used for alkaline serine proteases production are listed in Table 1. Enzymes can also be produced by some other bacteria including Streptomyces, Arthrobacter, and Flavobacterium spp. as well (Rao et al., 1998). Table 1: List of some major microbial species used for alkaline serine proteases production. Microorganisms

Source

Property

References

Stenotrophomonas maltophilia strain S-

Soil

Heat stability

Miyaji et al., 

Bacillus alcalophilus TCCC

Alkaline soil

Stability toward detergents

Cheng et al., 

Bacillus licheniformis NMS-

Soil

Thermostable

Mathew and Gunathilaka, 

Bacillus caseinilyticus

Lake

Thermotolerant

Mothe and Sultanpuram, 

Anoxybacillus kamchatkensis MV

Hot water springs

Thermostable

Mechri et al., 

Neocosmospora ramosa N

Soil

Thermostable

Matkawala et al., a

Citricoccus sp.

Agricultural soil

Compatible with detergents

Verma and Pandey, 

Bacillus pumilus AR 

Soil

Thermotolerant, solvent stable

Jagadeesan et al., 

Bacillus halodurans RSCVSPF

Poultry farm soil

Alkali stable

Chauhan and Mishra, 

Gracilibacillus boraciitolerans LO

Lakes sediments

Salt and solvent tolerance

Ouelhadj et al., 

Neurospora crassa CGMCC

Okara

Organic solvent stable

Zheng et al., 

Arthrobacter sp. KFS-

Dump site

Keratinase activity

Nnolim et al., 

Bacillus licheniformis strain KB

Sediments of mangrove forest

Work in harsh conditions

Foophow et al., 

Serine proteases from lactic acid bacteria Lactic acid bacteria (LAB) have shown very fast developments especially in the biotechnology industry from the past few decades. LABs are very important and promising for economic and industrial successes (Kieliszek et al., 2021). LABs are generally

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regarded as safe and received wider attention as a substitute source of proteases. Many LABs are known (e.g., Lactobacillus genus) that possessed proteolytic properties (Bansal et al., 2021). Alkaline serine proteases from different LAB were well characterized with the highest activity in Lactobacillus plantarum subsp. plantarum PTCC 1896 (Mustafa et al., 2020) and in L. plantarum FNCC 0,270 (Trismilah et al., 2015; Margono et al., 2014). Another extracellular alkaline serine protease isolated from Pediococcus acidilactici NCDC 252 was further purified and characterized for its catalytic properties (Bansal et al., 2021).

Applications of commercial serine proteases Microbial proteases are considered as the most important hydrolytic enzymes while alkaline proteases hold a significant rank in the enzyme market (Razzaq et al., 2019). Microbial proteases have great importance for the industrial bioprocess development. Many different industrial products are produced from various microbial enzymes, and this demand of industrially important enzymes from microbes is continuously rising day by day for desired product development. Nowadays, several new molecular techniques such as protein-engineering and metagenomics have been applied in order to get a better-quality product and enzyme performance in many industrial applications (Thakur and Kumar, 2018). Studies revealed that serine proteases are used in food and detergent industries since long time. Both chymotrypsin and trypsin were established as active ingredients in detergent for breaking down proteinaceous stains. BioDestain-ALKP (L), Addclean PRO L/PRO S, BIOTOUCH® ROC 250 LC, and Opticlean are some of the commercially available alkaline serine proteases used in detergent industries (Matkawala et al., 2021). The enzymes possess greater ionic ability, higher temperature and pH stability, and prolonged shelf life that improved the effectiveness of detergents (Solanki et al., 2021). Alkaline serine proteases are also known to be involved in leather processing such as soaking, dehairing, bating, and tanning as alternative source than conventional methods (chemical treatment) to improve leather quality (Thakur and Kumar, 2018). SEBZyme AP 200 and BioTan-ALKP are commercial alkaline serine proteases used in leather industries (Matkawala et al., 2021). These enzymes also play an important role in the waste management from different sources such as agriculture, poultry, meat, and fish industries. These enzymes are known to degrade the proteinaceous waste as well. Hydrolysis of feathers into soluble proteins and amino acids served as productive organic fertilizers (Singh and Bajaj, 2017). Alkaline serine proteases have been widely utilized in medical and pharmaceutical sector. Food and Drug Administration (FDA) has approved several proteases to treat many medical conditions and several protease-associated remedies are under investigation. Immobilized subtilisin has been developed as ointments, soft gel-based formulas, bandage materials, and

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nonwoven tissues to treat burn injuries and wounds. Enzymes have also been used to treat muscle cramps (botulinum toxin A and B), hemophilia (factor VIIa), thrombolysis (fibrinolytic, urokinase), purulent wound drainage and abscesses (elastoterase), sepsis (activated protein C), and lymphocytic leukemia (aspaginase) (Craik et al., 2011; Matkawala et al., 2021). Proteases from microbes have been widely used as food enzymes in baking, food processing, and dairy industries since long time (Thakur and Kumar, 2018). Protein hydrolysates are used as food additives and alkaline serine proteases are mostly utilized in breaking down plant and animal proteins to obtain hydrolysates having high nutritional importance. This protein hydrolysate is further used in processed foods, dietary products, and newborn food formulations and is also utilized as bioactive component in nutraceuticals. Savinase, Alcalase, Esperase, Flavourzyme, SEBDigest F59 P, and COROLASE are some of the commercial enzymes used in food processing industries (Contesini et al., 2018; Matkawala et al., 2021).

Conclusions and future perspective About one-third of all known proteases are serine proteases and considered as the largest proteolytic class containing 40 families and 13 clans. Serine proteases are broadly distributed in nature and found in all kingdoms of cellular life and are classified into clans that shared structural homology. Nucleophilicity of the catalytic Ser is usually dependent on a catalytic triad of Asp, His, and Ser residues. Serine proteases are well-characterized, widespread and play a significant role in physiological and pathological processes in host. Enzymes are involved in the regulation of physiological processes like digestion, cell signaling, blood coagulation, cytokines signaling, and ECM remodeling. Abnormal expression of serine proteases and alterations in their proteolytic activities causes further pathological severities. Along with their immense roles in physiological processes, proteases also have commercial importance. These enzymes account for around 60% of the total enzyme market worldwide. Because of their broad biochemical diversity, fast growth, limited space requirement for cultivation, and susceptibility to genetic manipulation, microbes are considered as excellent source of proteases. Microbial proteases have been continuously utilized in food, dairy, and detergent industries since long time. Among all proteases, serine proteases have received more attention because these enzymes have importance in industrial processes including food, detergent, and pharmaceutics. Serine proteases have both degradative and synthetic properties. Scientists are continuously searching for novel microorganisms from different microbial sources that have the ability to produce alkaline serine proteases with desired properties. Advancements in microbial genetic engineering by site-directed mutagenesis of cloned genes open new avenues for introducing predesigned changes, which result in

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the production of modified version of enzymes with novel and desired properties. Recent ongoing technologies in protein engineering, bioinformatics, and computationalbased approaches might be able to produce alkaline serine proteases with desired features and substrate specificities.

References Adrio, J. L., & Demain, A. L. (2014). Microbial enzymes: Tools for biotechnological processes. Biomolecules, 4(1), 117–139. Antalis, T. M., Buzza, M. S., Hodge, K. M., Hooper, J. D., & Netzel-Arnett, S. (2010). The cutting edge: Membrane-anchored serine protease activities in the pericellular microenvironment. Biochemical Journal, 428(3), 325–346. Antão, C. M., & Malcata, F. X. (2005). Plant serine proteases: Biochemical, physiological and molecular features. Plant Physiology and Biochemistry, 43(7), 637–650. Bansal, P., Kumar, R., Singh, J., & Dhanda, S. (2021). Production of extracellular alkaline serine protease from Pediococcus acidilactici NCDC 252: Isolation, purification, physicochemical and catalytic characterization. Catalysis Letters, 151(2), 324–337. Bhunia, B., Basak, B., & Dey, A. (2012). A review on production of serine alkaline protease by Bacillus spp. Journal of Biochemical Technology, 3(4), 448–457. Böttcher-Friebertshäuser, E. (2018). Membrane-anchored serine proteases: Host cell factors in proteolytic activation of viral glycoproteins. Activation of Viruses by Host Proteases, 153–203. Chakraborty, P., Acquasaliente, L., Pelc, L. A., & Di Cera, E. (2018). Interplay between conformational selection and zymogen activation. Scientific Reports, 8(1), 1–10. Chauhan, R. S., & Mishra, R. M. (2020). Characterization of alkaline protease producing Bacillus halodurans RSCVS–PF21 isolated from poultry farm soil. Biosciences Biotechnology Research Asia, 17(2), 385–392. Cheng, K., Lu, F. P., Li, M., Liu, L. L., & Liang, X. M. (2010). Purification and biochemical characterization of a serine alkaline protease TC4 from a new isolated Bacillus alcalophilus TCCC11004 in detergent formulations. African Journal of Biotechnology, 9(31), 4942–4953. Contesini, F. J., Melo, R. R. D., & Sato, H. H. (2018). An overview of Bacillus proteases: From production to application. Critical Reviews in Biotechnology, 38(3), 321–334. Craik, C. S., Page, M. J., & Madison, E. L. (2011). Proteases as therapeutics. Biochemical Journal, 435(1), 1–16. David Troncoso, F., Alberto Sánchez, D., & Luján Ferreira, M. (2022). Production of plant proteases and new biotechnological applications: An updated review. ChemistryOpen, 11(3), e202200017. De Lorenzi, V., Sarra Ferraris, G. M., Madsen, J. B., Lupia, M., Andreasen, P. A., & Sidenius, N. (2016). Urokinase links plasminogen activation and cell adhesion by cleavage of the RGD motif in vitronectin. EMBO Reports, 17(7), 982–998. Di Cera, E. (2009). Serine proteases. IUBMB Life, 61(5), 510–515. Dong, Z., Yang, S., & Lee, B. H. (2021). Bioinformatic mapping of a more precise Aspergillus niger degradome. Scientific Reports, 11(1), 1–21. Ekici, Ö. D., Paetzel, M., & Dalbey, R. E. (2008). Unconventional serine proteases: Variations on the catalytic Ser/His/Asp triad configuration. Protein Science, 17(12), 2023–2037. Foophow, T., Sittipol, D., Rukying, N., Phoohinkong, W., & Jongruja, N. (2022). Purification and Characterization of a novel extracellular haloprotease VPR from Bacillus licheniformis strain KB111. Food Technology and Biotechnology, 60(2), 225–236.

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Gonçalves, R. N., Gozzini Barbosa, S. D., & Silva-López, R. E. D. (2016). Proteases from Canavalia ensiformis: Active and thermostable enzymes with potential of application in biotechnology. Biotechnology Research International, 2016. Herszényi, L., Barabás, L., Hritz, I., István, G., & Tulassay, Z. (2014). Impact of proteolytic enzymes in colorectal cancer development and progression. World Journal of Gastroenterology: WJG, 20(37), 13246. Jagadeesan, Y., Meenakshisundaram, S., Saravanan, V., & Balaiah, A., (2020). Sustainable production, biochemical and molecular characterization of thermo-and-solvent stable alkaline serine keratinase from novel Bacillus pumilus AR57 for promising poultry solid waste management. International Journal of Biological Macromolecules, 163, 135–146. Kieliszek, M., Pobiega, K., Piwowarek, K., & Kot, A. M. (2021). Characteristics of the proteolytic enzymes produced by lactic acid bacteria. Molecules, 26(7), 1858. Laskar, A., Rodger, E. J., Chatterjee, A., & Mandal, C. (2012). Modeling and structural analysis of PA clan serine proteases. BMC Research Notes, 5(1), 1–11. López–Otín, C., & Bond, J. S. (2008). Proteases: Multifunctional enzymes in life and disease. Journal of Biological Chemistry, 283(45), 30433–30437. Lu, P., Takai, K., Weaver, V. M., & Werb, Z. (2011). Extracellular matrix degradation and remodeling in development and disease. Cold Spring Harbor Perspectives in Biology, 3(12), a005058. Margono, T., Sumaryono, W., Malik, A., & Sadikin, M. (2014). Characterization of trypsin-like protease of Lactobacillus plantarum FNCC 0270. HAYATI Journal of Biosciences, 21(2), 87–94. Martin, C. E., & List, K. (2019). Cell surface-anchored serine proteases in cancer progression and metastasis. Cancer and Metastasis Reviews, 38(3), 357–387. Mathew, C. D., & Gunathilaka, R. M. S. (2015). Production, purification and characterization of a thermostable alkaline serine protease from Bacillus lichniformis NMS-1. International Journal of Biotechnology and Molecular Biology Research, 6(3), 19–27. Matkawala, F., Nighojkar, S., Kumar, A., & Nighojkar, A. (2019). A novel thiol-dependent serine protease from Neocosmospora sp. N1. Heliyon, 5(8), e02246. Matkawala, F., Nighojkar, S., Kumar, A., & Nighojkar, A. (2021). Microbial alkaline serine proteases: Production, properties and applications. World Journal of Microbiology and Biotechnology, 37(4), 1–12. Mechri, S., Bouacem, K., Zaraî Jaouadi, N., Rekik, H., Ben Elhoul, M., Omrane Benmrad, M., Hacene, H., Bejar, S., Bouanane-Darenfed, A., & Jaouadi, B. (2019). Identification of a novel protease from the thermophilic Anoxybacillus kamchatkensis M1V and its application as laundry detergent additive. Extremophiles, 23(6), 687–706. Miyaji, T., Otta, Y., Shibata, T., Mitsui, K., Nakagawa, T., Watanabe, T., Niimura, Y., & Tomizuka, N. (2005). Purification and characterization of extracellular alkaline serine protease from Stenotrophomonas maltophilia strain S‐1. Letters in Applied Microbiology, 41(3), 253–257. Mothe, T., & Sultanpuram, V. R. (2016). Production, purification and characterization of a thermotolerant alkaline serine protease from a novel species Bacillus caseinilyticus. 3 Biotech, 6(1), 1–10. Mustafa, M. H., Soleimanian-Zad, S., & Sheikh-Zeinoddin, M., (2020). Characterization of a trypsin-like protease 1 produced by a probiotic Lactobacillus plantarum subsp. plantarum PTCC 1896 from skimmed milk based medium. LWT, 119, 108818. Nnolim, N. E., Ntozonke, N., Okoh, A. I., & Nwodo, U. U., (2020). Exoproduction and characterization of a detergent-stable alkaline keratinase from Arthrobacter sp. KFS-1. Biochimie, 177, 53–62. Ouelhadj, A., Bouacem, K., Asmani, K. L., Allala, F., Mechri, S., Yahiaoui, M., & Jaouadi, B., (2020). Identification and homology modeling of a new biotechnologically compatible serine alkaline protease from moderately halotolerant Gracilibacillus boraciitolerans strain LO15. International Journal of Biological Macromolecules, 161, 1456–1469. Page, M. J., & Di Cera, E. (2008). Serine peptidases: Classification, structure and function. Cellular and Molecular Life Sciences, 65(7), 1220–1236.

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Patel, S. (2017). A critical review on serine protease: Key immune manipulator and pathology mediator. Allergologia et Immunopathologia, 45(6), 579–591. Philipps-Wiemann, P. (2018). Proteases – general aspects. In Enzymes in Human and Animal Nutrition (pp. 257–266). Eds Carlos Simões Nunes, Vikas Kumar. Academic Press. Paperback ISBN: 9780128054192, eBook ISBN: 9780128094266. Poddar, N. K., Maurya, S. K., & Saxena, V. (2017). Role of serine proteases and inhibitors in cancer. In Proteases in Physiology and Pathology, (pp. 257–287). Eds Sajal Chakraborti, Naranjan S. Dhalla. Springer, Singapore. Hardcover ISBN: 978-981-10-2512-9, eBook ISBN: 978-981-10-2513-6. Raj, T., Nikhil, S., & Savitri, B. T., (2017). Bacterial serine proteases: Computational and statistical approach to understand temperature adaptability. Journal Proteomics Bioinform, 10, 329–334. Rani, K., Rana, R., & Datt, S. (2012). Review on latest overview of proteases. International Journal of Life Sciences, 2(1), 12–18. Rao, M. B., Tanksale, A. M., Ghatge, M. S., & Deshpande, V. V. (1998). Molecular and biotechnological aspects of microbial proteases. Microbiology and Molecular Biology Reviews, 62(3), 597–635. Razzaq, A., Shamsi, S., Ali, A., Ali, Q., Sajjad, M., Malik, A., & Ashraf, M., (2019). Microbial proteases applications. Frontiers in Bioengineering and Biotechnology, 7, 110. Singh, S., & Bajaj, B. K. (2017). Potential application spectrum of microbial proteases for clean and green industrial production. Energy, Ecology and Environment, 2(6), 370–386. Soccol, C. R., da Costa, E. S. F., Letti, L. A. J., Karp, S. G., Woiciechowski, A. L., & de Souza Vandenberghe, L. P. (2017). Recent developments and innovations in solid state fermentation. Biotechnology Research and Innovation, 1(1), 52–71. Solanki, P., Putatunda, C., Kumar, A., Bhatia, R., & Walia, A. (2021). Microbial proteases: Ubiquitous enzymes with innumerable uses. 3 Biotech, 11(10), 1–25. Sumantha, A., Larroche, C., & Pandey, A. (2006). Microbiology and industrial biotechnology of food-grade proteases: A perspective. Food Technology and Biotechnology, 44(2), 211–220. Szabo, R., & Bugge, T. H., (2011). Membrane-anchored serine proteases in vertebrate cell and developmental biology. Annual Review of Cell and Developmental Biology, 27, 213. Tanabe, L. M., & List, K. (2017). The role of type II transmembrane serine protease‐mediated signaling in cancer. The FEBS Journal, 284(10), 1421–1436. Thakur, P., & Kumar, V. (2018). Serine proteinase industrial applications: A review. Journal of Information and Computational Science, 5(7), 316–328. Tripathi, L. P., & Sowdhamini, R. (2006). Cross genome comparisons of serine proteases in Arabidopsis and rice. BMC Genomics, 7(1), 1–31. Tripathi, L. P., & Sowdhamini, R. (2008). Genome-wide survey of prokaryotic serine proteases: Analysis of distribution and domain architectures of five serine protease families in prokaryotes. BMC Genomics, 9(1), 1–28. Trismilah, T., Nurhasanah, A., Sumaryono, W., Malik, A., & Sadikin, M. (2015). Optimization of trypsin-like protease production by Lactobacillus plantarum FNCC 0270 using response surface methodology. Makara Journal of Science, 19(2), 64–72. Verma, J., & Pandey, S., (2019). Characterization of partially purified alkaline protease secreted by halophilic bacterium Citricoccus sp. isolated from agricultural soil of northern India. Biocatalysis and Agricultural Biotechnology, 17, 605–612. Ward, O. P. (2011). Proteases. Comprehensive Biotechnology, 571. Zheng, L., Yu, X., Wei, C., Qiu, L., Yu, C., Xing, Q., Fan, Y., & Deng, Z., (2020). Production and characterization of a novel alkaline protease from a newly isolated Neurospora crassa through solidstate fermentation. LWT, 122, 108990.

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Biographical Sketch of the Corresponding author Name: Dr. Poonam Bansal Affiliation: Assistant Professor, Department of Biosciences and Technology, Maharishi Markandeshwar (Deemed to be University), Mullana Ambala Education: M.Sc. & Ph.D. Biochemistry

Research and Professional Experience – –

Assistant Professor in Department of Biotechnology, Maharishi Markandeshwar (Deemed to be University), Mullana Ambala 133207, Department of Biosciences and Technology, India. Worked on Phenotypic, Biochemical, Molecular and In silico Characterization of Pediococcus acidilactici NCDC 252.

Publications Poonam Bansal, Raman Kumar and Suman Dhanda (2022). Characterization of starter cultures and nutritional properties of Pediococcus acidilactici NCDC 252: A potential probiotic of dairy origin. Journal of Food Processing and Preservation, DOI: https://doi.org/10.1111/jfpp.16817. Poonam Bansal, Raman Kumar, Jasbir Singh and Suman Dhanda (2021). Production of Extracellular Alkaline Serine Protease from Pediococcus acidilactici NCDC 252: Isolation, Purification, Physicochemical and Catalytic Characterization. Catalysis Letters, 151: 324-337. Poonam Bansal, Raman Kumar, Jasbir Singh and Suman Dhanda✶ (2020). In silico analysis of Pediococcus acidilactici NCDC 252 genome revealed nineteen novel genes. Gene Reports, 21, 100849. Raman Kumar✶, Poonam Bansal✶, Jasbir Singh, Suman Dhanda and Jitender Kumar Bhardwaj (2020). Aggregation, adhesion and efficacy studies of probiotic candidate Pediococcus acidilactici NCDC 252: A strain of dairy origin. World Journal of Microbiology and Biotechnology, 36(1):10. Priti Duhan, Poonam Bansal and Sulekha Rani (2020). Isolation, identification and characterization of endophytic bacteria from medicinal plant Tinospora cordifolia. South African Journal of Botany, 1–7.

Priti✶, Poonam Bansal, Sonali Sangwan, and Shweta Dhanda

Chapter 2 Serine proteases and respiratory disorders Abstract: The enormous variety of enzyme proteases has attracted attention on a global scale due to their physiological and biotechnological applications. The one-third populations of proteolytic enzymes are known as serine proteases. Serine acts as the nucleophilic amino acid at the active site of serine proteases, which are often endoproteases that cleave peptide bonds in proteins. Serine proteases play a crucial role in human health and respiratory diseases. Proteases maintain their homeostatic function to regulate regeneration and repair of lungs. The extracellular or intracellular regulation of processes such as microbial eradication, formation of mucin, remodeling of tissue, and neutrophil chemotaxis is possible with lung proteases. Additionally, they regulate lung inflammation and infection. The dysregulation of balancing of proteases and antiproteases is crucial for the demonstration of diverse forms of lung disorders. In the present chapter, we provide a quick overview of the various proteases and antiproteases in regulating various lung disorders. Keywords: serine proteases, antiproteases, lung disorders, inflammation

Introduction Proteases are a special class of enzymes because they have enormous physiological and commercial significance. Proteases, commonly referred to as proteolytic enzymes or proteinases, are a class of enzymes that break down the peptide links in proteins. The selective amendments of proteins via limited cleavage by proteolytic enzymes include blood clotting and the destruction of fibrin clots and also in the processing and transit of secretory proteins beyond the membranes and activation of zymogens (Rani et al., 2012). An already significant group of enzymes that are also utilized in various biological activities like control of metabolism, enzyme adaptations, photogenicity, apoptotic processes, and in the complement system have made these properties even more interesting.

✶ Corresponding author: Priti, Department of Biotechnology, K. L. Mehta Dayanand College for Women, Faridabad, Haryana, India, e-mail: [email protected] Poonam Bansal, Sonali Sangwan, Department of Biosciences and Technology, Maharishi Markandeshwar (Deemed to be University), Mullana, Ambala, Haryana, India Shweta Dhanda, National Centre for Veterinary Type Cultures, ICAR-National Research Centre on Equines, Hisar, Haryana, India

https://doi.org/10.1515/9783111325040-002

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Serine proteases make up the one-third population of proteolytic enzymes. These enzymes are abundant in nature and also found in all cellular life kingdoms. Serine proteases are endoproteases which catalyze the hydrolysis of the bond in middle part of polypeptide chain. In 2005, Puente et al. reported the 2–4% of the genes in a normal genome code for the proteolytic enzymes. These proteases are more prevalent and functionally distinct, having evolved during the course of evolution (Page et al., 2008a; Page et al., 2008b). These enzymes are extensive in nature and perform a very important role in physiological and commercial fields including coagulation processes, fertilization, developmental processes, inflammation, malignancy, and neuromuscular patterning processes (Jackson and Nemerson, 1980). They are also helpful in the posttranslational processing and modifications of polypeptides. The enzyme serine proteases are defined by the presence of serine group in their active site. At their active site, each serine protease has three residues: a serine, a histidine, and an aspartate which makes a distinctive catalytic triad. They are abundant and common among bacteria, eukaryotes, and viruses, indicating that they are essential to the organisms. The exopeptidase, endopeptidase, oligopeptidase, and omega peptidase families all contain serine proteases. The activity of serine proteases was reported not only at neutral pH but also at alkaline pH. The optimal pH range is between 7 and 11 for this enzyme. Nucleophilic serine, which is present in the active site of enzymes, attacks the carbonyl moiety of peptide bond of substrate and an acyl enzyme intermediate is generated (Hedstrom, 2002). The nucleophilicity of the catalytic serine depends on the catalytic triad known as the charge-relay system (Blow et al., 1969). This family of proteins plays crucial functions in the preservation of homoeostasis and also in the digestive process. These proteases take role in the regulation of key amplification cascades via proteolytic activation of zymogen precursor. The protease substrate inside these cascades is in the inactive form (zymogen) of a downstream serine protease. Blood coagulation (Davie et al., 1991), kinin production (Proud and Kaplan, 1988), and the complement system (Reid and Porter, 1981) are a few instances of regulation mediated by serine-protease enzyme. Regulation of blood coagulation is also the key stone for maintaining homeostasis. The kinin-kallikrein system and the complement system are believed to be playing an important role during inflammation and various immune reactions (Proud and Kaplan, 1988; Reid and Porter, 1981). This enzyme plays a vital role in human health and respiratory diseases. Proteases maintain their homeostatic function to regulate lung’s regeneration and repair inside normal lungs. The extracellular or intracellular regulation of processes, formation of mucin, remodeling of tissue, neutrophil chemotaxis process, and eradication of microbes are very much possible with lung proteases. Additionally, they regulate lung inflammation and infection. The dysregulation of balancing of proteases and antiproteases is crucial for the demonstration of diverse forms of lung disorders. In an effort to take advantage of

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their physiological and biotechnological implications, these enzymes have attracted the attention of researchers on a global scale. In the present chapter, we provide a quick overview of various proteases and antiproteases in regulating various lung disorders.

Biological importance of proteases The importance of proteases in lung health and disease control cannot be overstated. Proteases continue to perform their homeostatic roles, which control procedures like regeneration and repair, in healthy lungs. The emergence of various forms of lung disorders depends on the balance of proteases and antiproteases that are dysregulated. A notable increase in protease activities is linked to chronic inflammatory lung diseases. Therefore, for successful microbial infection and inflammation in the lung, blockage of antiproteolytic control mechanisms is just as critical as protease activities. The main protease classes found in the human lung are metalloproteases, aspartic, cysteine, and serine. In 2016, Lecaille et al. reported that the neutrophil serine proteases, for example, proteinase 3, elastase, matrix metalloproteinases (MMPs), and cathepsin G are important pathogenic determinants of diverse chronic lung disorders. The lung proteases collaborate with protease enzymes of invading microorganisms for the inactivation of antiproteases along with antimicrobial compounds, and as a result, they play a critical role in diverse respiratory disorders, for example, asthma, influenza, lung fibrosis, cystic fibrosis (CF), and tuberculosis. The different types of serine proteases with their biological role are summarized in Table 1. Table 1: Serine proteases and their biological activities. Proteinases

Sources

Biological activities

References

Neutrophill elastase Cathepsin G Proteinase-

PMNs P Monocytes

Growth factor activation, bacterial and fungal killing, lymphocytes, and platelets activation

Lecaille et al., 

Plasminogen activators

PMNs MNPs Endothelial cells

Convert plasminogen to plasmin; cell adhesion and migration

Rijken and Sakharov, 

Coagulation proteins (thrombin)

Plasma

Homeostasis

Davie et al., 

Complement components

Plasma

Complement activation

Reid and Porter, 

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Table 1 (continued) Proteinases

Sources

Biological activities

References

Trypsin

Mast cells

Activates protease activated receptors

Klenk et al., ; Lazarowitz and Choppin,

PMN: polymorphonuclear neutrophill; MNPs: mononuclear phagocytes.

Serine proteases and various respiratory disorders Here, in this section, we will discuss about the potential roles being played by various proteases and antiproteases in the regulation of lung disorders and some applications of serine proteases are depicted in Figure 1.

Asthma Activate Zymogen

Influenza

Cystic fibrosis

Application of Serine Proteases

Physiological processes

Lung Fibrosis

Tuberculosis Complement Activation

Figure 1: Applications of serine proteases.

Influenza The virus responsible for influenza is highly contagious and cause intense respiratory illnesses in both humans and other animals (Palese, 2004; Yewdell and Garcia-Sastre, 2002; Horimoto and Kawaoka, 2005; Garten et al., 2009). There are three types of influenza viruses: A, B, and C. The three major influenza outbreaks of the twentieth century, as well as the recent influenza pandemic of swine origin, were both brought on by influenza virus A, which is present in humans, birds, and mammals (Blanquer et al., 1991).

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Various influenza A-related deaths are attributed to secondary bacterial pneumonia (Lauderdale et al., 2005; Garten and Klenk, 2008). The haemagglutinin (HA) protein is essential for the influenza viruses’ pathogenicity. The influenza virus’s HA binds with the sialic acid which contains cell surface receptors. After being split into HA1 and HA2 subunits, HA is fused to the host cell membrane, initiating the infection process (Garten and Klenk, 2008; Klenk and Garten, 1994; Steinhauer, 1999; Skehel and Wiley, 2000). Although a single lysine amino acid has occasionally been seen there, the cleavage site for HA of avian and mammalian influenza viruses is a single arginine. Trypsin (Klenk et al., 1975; Lazarowitz and Choppin, 1975) proteases like plasmin (Goto and Kawaoka, 1998; Lazarowitz et al.,1973; LeBouder et al., 2008), bronchiolar epithelial cells, and mast cells’ tryptase (Kido et al., 2007) as well as bacterial proteases (Tashiro et al., 1987; Scheiblauer et al., 1992; Bahgat et al., 2011) can all cause cleavage extracellularly. Other proteases that are expressed in the lungs can also help in the spreading of influenza virus. In 2006, Böttcher et al. (2006) reported that the transmembrane serine proteases (TMPRSS2 and TMPRSS11D) also known as the human airway trypsin-like protease (HAT) activate these influenza viruses H1N1, H2N9, and H3N2 upon cleavage of HA and contributes in the pathogenicity of influenza viruses inside lungs (Böttcher et al., 2006; Chaipan et al., 2009). Extracellular proteases, for example, trypsin and tryptase activate the family of receptors called protease activated receptors which play a vital role in the replication of virus and also in innate immunity at virus replicating sites I microenvironment of respiratory tract (Kido et al., 2007; Riteau et al., 2006). Different proteases have been found to activate the four pathogen-associated receptors. After the receptors have been degraded by proteases enzymes, the newly formed amino-terminal sequence binds and inwardly activates the receptors (Chignard and Pidard, 2006). In airways of mice infected by IAV, an enhancement in PAR 2 following IFNc-mediated regulation plays a crucial role in the influenza pathogenesis (Lan et al., 2004; Khoufache et al., 2009). Numerous serine protease activities are thought to have a role in the transmission of the influenza virus. Mice are significantly protected from infection when the influenza A virus is prevented from infecting cultivated epithelial cells of lung by inhibitor of serine proteases, for example, aprotinin (Ovcharenko and Zhirnov, 1994).

Lung fibrosis Interstitial lung disease (lung fibrosis) is another name for the chronic disorder, which is characterized by an increase in the degradation of matrix and also the intraalveolar fibrosis that causes dyspnea, poor oxygen transfer, and also alveolar collapse (Quan et al., 2006; Katzenstein and Myers, 1998). Lung fibrosis is characterized as an accumulation of the differentiated fibroblasts and extracellular matrix (ECM) compo-

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nents which occurs inside the alveolar space and interstitium. Dysregulation of proteolytic activity is linked to lung fibrotic disease. Several reports have suggested that cathepsins are involved in this situation. CatB may be involved in fibrogenesis because increased TGF-β production was accompanied by increased proteolytic processing of CatB in the lungs (Moles et al., 2009). Microarray research has shown that MMP-7 (also known as matrilysin) is a crucial COPD marker in addition to NE. The extracellular proteoglycan decorin is broken down by MMP-7, which then releases decorin-bound TGF-β (Imai et al., 1997) and helps in its activation which is an important COPD sign (Van den Brûle et al., 2005).

Cystic fibrosis Protease activity may significantly become upregulated in CF, which can lead to progressive bronchiectasis, a condition in which the lungs’ bronchial tubes chronically swell and become permanently damaged due to infection in bronchi (Rowe et al., 2005; Gibson et al., 2013). The loss of expression or functional mutation in CFTR gene which is responsible for the autosomal recessive genetic disorder is known as cystic fibrosis (Tilly et al., 1992; Tilly et al., 1992). The pathology associated with CF is caused by its impact on respiratory system despite the fact that CF affects many other organs. The control of sodium and chloride ions across the epithelial membranes is prevented in CF patients due to nonfunctional CFTR channels, which increases the amount of dehydrated mucus discharges in the lungs (Rowe et al., 2005; Gibson et al., 2013; Tilly et al., 1992; Tilly et al., 1992; Kreda et al., 2012). Lung disease caused by CF is a genetic ailment that affects around 1 in 2,500 newborns. CF is a chronic lung infection that causes airway inflammation. Neutrophil counts are higher in CF patients’ sputum, which cause neutrophil serine proteases to be higher as well. In healthy human beings, proteases help in phagocytosis via digestion of microbial peptide and thus the protease enzymes are present in homeostatic equilibrium with the help of cognate antiprotease. The high concentration of neutrophil-derived proteases overwhelms cognate antiproteases as a result of the significant neutrophil load associated with CF. These antiprotease enzymes have been considered as potential therapy to reestablish the protease antiprotease balance. As a result, numerous endogenous and synthetic antiproteases have been tested as treatments for CF lung disease, with varying degrees of efficacy. Neutrophil serine protease is the main protease which is implicated in damage seen inside patient’s lungs. These include cathepsin G (Cat G), proteinase 3 (PR3), and neutrophil elastase (NE) in CF patients (Korkmaz et al., 2008). Neutrophils express all three chymotrypsin family members (Korkmaz et al., 2008). These proteases are referred as zymogens, which are precursor peptides that are inactive proteases. To create their active mature versions, all three serine proteases go through a two-stage

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posttranslational modification process, and the starting stage is the breakage of Nterminal signal peptide with the help of signal peptidase enzyme. The cysteine protease (cathepsin G) cleaves the Prodi peptide from N-terminal. This is necessary for the enzyme activity and also for the cleavage of propeptide from C-terminus. It is further urgent for the packaging of mature protein; this constitutes the second step (Pham and Ley, 1999; Wolters et al., 2001; Adkison et al., 2002; Sk“old et al., 2002; Gullberg et al., 1995). The cytoplasm of neutrophils is store house of azurophilic granules that store mature versions of Cat G, NE, and PR3. The triad of aminoacids consists of aspartate, histidine, and serine aminoacids which is required for the activities of all three of these proteases (Pham and Ley, 1999). These residues scattered at various locations in primary structure of each of three residues, but in the tertiary structure, they are brought together in an active site area (Korkmaz et al., 2010). Serine proteases have two main modes of action: intracellular, where microbial proteins break down inside the phagosome while extracellularly, they control immunological functions and facilitate the breakdown of ECM constituents (Pham and Ley, 1999). The majority of research on the function of neutrophil serine proteases in the lung has mainly concentrated on NE; however, PR3 and Cat G should not be discounted because they are present in significant amounts in the sputum of CF patients and also inside bronchial alveolar lavage fluid (Witko-Sarsat et.al., 1999; Sepper et al., 1995).

Tuberculosis It has been demonstrated that the activated macrophages’ cells death caused by Mycobacterium tuberculosis is due to overexpression of Cat G, not of NE. Cat G and NE each have a different level of substrate specificity. The Cat G cleaves only the C-terminal end of aromatic amino acids while NE cleaves C-terminal of hydrophobic amino acids (Korkmaz et al., 2008; Reece et al., 2010). The proteolysis of undiscovered target sequences caused by Cat G increases the necrosis inside the infected macrophages. It has been demonstrated that SerpinB3a inhibition of Cat G is required to stop the necrosis brought out by IFN-c in macrophages infected with M. tuberculosis (Reece et al., 2010). MMPs play a crucial role in mediating the tissue-destructive response in TB (Elkington et al., 2011a; Walker et al., 2012). MMPs are capable of cleaving ECM components (Elkington et al., 2011b). In humans, MMPs-1 breaks the type I and II collagen (fibrillar components) of ECM. In comparison to control participants, the respiratory secretions of TB patients have higher levels of MMPs-III which further activates metallo matrix protein I (Walker et al., 2012; Seddon et al., 2013).

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Asthma Asthma is a chronic inflammatory disorder of lungs which is now becoming more common. By 2025, it is predicted that there will be 100 million asthmatics worldwide (Kim et al., 2010). Asthma affects the lung to produce mucus and results in elevated IgE levels, remodeling of the airways, and hyperactivity of the airways by triggering the activation of adaptive immunity (Braman, 2006). Asthma manifestation is represented by an acute inflammatory response and the obstruction of the airway (Barnes, 2008). Proinflammatory cells, such as macrophages cells, mast, and neutrophils, invade the tissues of lungs in both acute and chronic asthma (Holgate, 2008; Agrawal and Shao 2010). Serine proteases and MMPs are significant extracellular proteases secreted by proinflammatory cells because they play significant roles in the pathogenesis of asthma (Broide, 2008; Cataldo et al., 2000; Corry et al., 2002; Greenlee et al., 2006; Greenlee et al., 2007; McMillan et al., 2004; Tsai et al., 2016). The plasminogen can turn into active enzyme plasmin (u-PA) by tissue-type plasminogen activator (PA). Both the breakdown of ECM components and the disintegration of fibrin are correlated with tissue PA and u-PA (Rijken and Sakharov, 2001). The significant inhibition of tissue-type PA and u-PA is brought about by PAI-1 that helps to promote formation of matrix by halting the breakdown of matrix. Mast cells are responsible for starting allergy via inflammation in airways of asthma patients (Pesci et al., 1993). The mast cells and the bronchial epithelial cells are the primary source of PAI. Interaction between bronchial epithelium cells and mast cells is very important for maintaining inflammation and alteration in their structure in asthmatic condition (Cho et al., 2000). The inflammation mediated by IgE is responsible for pathogenesis of asthma. The increase in the PAI-1 production has been known to play a crucial role in fibrosis development (Cho et al., 2015). Drugs that block the activation of MCs, such as anti-IgE, could potentially be helpful in avoiding asthmatic airway remodeling.

Physiological functions and applications of serine proteases Proteases carry out a wide range of intricate physiological processes. Their presence in all types of living organisms is the evidence of their significance in carrying out the necessary metabolic and regulatory functions. Protein catabolism, blood coagulation, cell growth and migration, tissue organization, morphogenesis during development, inflammation, tumor growth, and metastasis are just a few of the physiological and pathological processes that proteases are essential for. They also activate zymogens, release hormones and pharmacologically active peptides from precursor proteins, and transport secretory proteins across membranes.

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While internal proteases are essential for the control of metabolism, external proteases often catalyze the breakdown of bigger protein molecules into smaller ones for subsequent cell absorption. Proteases participate in a number of important processes, including protein turnover, sporulation and conidial discharge, regulation of gene expression, feeding, and germination as well.

Applications of serine proteases in other fields Protease enzymes are used in a variety of medical procedures. Proteases have been successfully exploited by scientists for a variety of applications in the medical field (Davidenko, 1999). Immobilized alkaline protease, an enzyme obtained from bacillus species, is used to produce soft gel-based pharmaceutical formulations, the composition of ointments, nonwoven tissues, and innovative bandage materials. Organization of protease enzymes from the fungus Aspergillus oryzae was used as a diagnostic tool for the treatment of several lytic enzyme-deficient diseases (Rao, et al., 1998). According to reports, alkaline-fibrinolytic protease selectively breaks down fibrin, pointing to potential uses for the enzyme in thrombolytic therapy and anticancer medications (Mukherjee and Rai, 2011). Collagenases are gradually utilized for medicinal purposes in the creation of slowrelease dosage forms with the aid of alkaline protease activity. A brand-new, very active semialkaline protease with strong collagenolytic activity was produced from Aspergillus niger LCF9. Alkaline proteases do not release any amino acids while hydrolyzing different forms of collagen or liberate low molecular weight peptides that may have therapeutic uses (Barthomeuf, et al., 1992). Proteases are particularly useful in procedures for sterilizing nonprotein products made from ugly or slightly deceptive extracts, such as blood-line carbohydrate gums and muco-polysaccharides. Proteases can be used to solubilize keratin components, which are then changed into surplus items like feathers to protein concentrates for evaluation as appealing feeds with the assistance of proteases. An alkaline protease has good keratolytic reactivity as well. Proteases are also useful in gelatin-containing photographic movie theaters with X-ray screens and in the liquefaction of technological and ethnic organic waste. Proteases can be used by both humans and animals as digestive assistance as well.

Conclusion and future perspective The lung is equipped with a variety of antiproteases and anti-inflammatory agents to combat infections. The equilibrium between proteases of host and the endogenous inhibitors secreted by them shifts in the favor of the proteases in respiratory illnesses

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like COPD and asthma. It is known that protease enzymes like cathepsins, MMPs, and NSPs work in conjunction with bacterial proteases and are crucial for the development of various lung illnesses. Therefore, it is crucial to manage severe inflammations inside lungs with substances that maintain the lung protease-antiprotease balance by the endogenous upregulating inhibitors of proteases or the downregulation of host protease activity. The proteolytic inhibition of proteases by inhibitors has been reported in inflammatory cells which were having abundance of oxidants and proteases. Although they were broken and rendered inactive by the protease enzymes such as cathepsins at their N-terminal end, this does not alter the inhibitory effect of SLPI and trappin-2/ elafin (Taggart et al., 2001). The antiprotease action of elafin, however, may be rendered inactive by P. aeruginosa proteases (Quinn et al., 2010). Protease inhibitors have been encapsulated into liposomes to prevent the unintended proteolysis of antiproteases. Aerosol administration of antiproteases contained in liposomes may be advantageous due to its several advantages, including prolonged release and relatively high loading capacities. Since NE is a well-known serine protease and has been linked to a number of lung disorders, including CF, it has been identified as a therapeutic target by synthetic drugs (Delacourt et al., 2002). After the phase 1 clinical trial, DX-890, a small protein inhibitor of NE, was found to be acceptable in rats and people (Dunlevy et al., 2012). It has been established that this substance helps prevent the neutrophil transmigration through the epithelial barrier and is implicated in the release of IL-8 from CF neutrophils. The results of phase 3 clinical trials will ensure whether DX-890 is effective as a treatment for various lung disorders. In another study, the dual MMP9/MMP12 inhibitor AZ11557272 was discovered to be protective against smoke-induced emphysema in a guinea pig model system (Churg et al., 2007). The enzyme proteases have attracted wider attention globally in an effort to take advantage of their physiological and biotechnological implications. Proteolytic enzymes are valued for their relevance like laboratory reagents for clinical purpose, importance in industries as well as their usage in a variety of medical therapies. Serine proteases are used in a variety of medical procedures as well. Serine proteases have the potential to be used therapeutically; they are effective in treating a large number of human disorders such as CF, asthma, allergies, tuberculosis, lung cancer, and lung fibrosis. Due to several uses, scientists have been adopting various protein engineering approaches to create novel enzymes that are pH and temperature stable. Thus, after analyzing all of the potential uses for serine proteases, we can conclude that these uses will likely to grow over the coming years, particularly in the field of medicine.

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Walker, N. F., Clark, S. O., Oni, T., Andreu, N., Tezera, L., Singh, S., & Elkington, P. T. (2012). Doxycycline and HIV infection suppress tuberculosis-induced matrix metalloproteinases. American Journal of Respiratory and Critical Care Medicine, 185(9), 989–997. Witko-Sarsat, V., Halbwachs–Mecarelli, L., Schuster, A., Nusbaum, P., Ueki, I., Canteloup, S., & Nadel, J. A. (1999). Proteinase 3, a potent secretagogue in airways, is present in cystic fibrosis sputum. American Journal of Respiratory Cell and Molecular Biology, 20(4), 729–736. Wolters, P. J., Pham, C. T., Muilenburg, D. J., Ley, T. J., & Caughey, G. H. (2001). Dipeptidyl peptidase I is essential for activation of mast cell chymases, but not tryptases, mice. Journal of Biological Chemistry, 276(21), 18551–18556. Yewdell, J., & García-Sastre, A. (2002). Influenza virus still surprises. Current Opinion in Microbiology, 5(4), 414–418.

Biographical Sketch Name: Dr. Priti Education: M.Sc. & Ph.D. Biotechnology

Publications 1) Duhan P, Bansal P and Rani S: Isolation, identification and characterization of endophytic bacteria from medicinal plant Tinospora cordifolia. South African Journal of Botany 2020; 134: 43–49. 2) Priti and Rani S: Isolation of Endophytic fungi from Tinospora cordifolia. Research Journal of Biotechnology, 2021; 16(2): 5–15. 3) Bansal P, Tuli HS, Sharma V, Ranjan K, Mohapatra R K, Dhama K, Priti, Sharma A K: Targeting omicron (B.1.1.529) SARS CoV-2 spike protein with selected phytochemicals: an in-silico approach for identification of potential drug. Journal of Experimental biology and Agricultural Science; 2022.

Raman Kumar✶, Poonam Bansal, and Praveen Kumar

Chapter 3 Role of serine proteases in cancer progression and metastasis Abstract: Serine proteases are largest part of human-associated protease family of protein-cleaving enzymes wherein serine acts as the nucleophilic amino acid at the active site. The nucleophilicity of catalytic Ser is mainly dependent on the catalytic triad of Asp, His, and Ser, known as the charge relay system. Serine proteases play an essential role in co-ordination of diverse physiological functions like digestion, blood coagulation, immune response, and apoptosis. Deregulated expression and activity of serine proteases mediate several events related to fundamental processes of tumor development and metastasis. Cell-surface anchored serine proteases belonging to the Type II transmembrane serine proteases family like matriptase, matriptase-2, hepsin, prostasin, HAT, testisin, and TMPRSS2, 3 and 4, and secreted serine proteases like urokinase plasminogen activator, kallikrein-related peptidase, fibroblast activation protein-α, HtrA, and granzymes are found to be associated with cancer progression via extracellular matrix remodeling, intracellular signaling, immunosuppression, etc. Understanding the connection between serine protease functions and cancer outcomes provides insight into cancer development and the relevant information about the therapeutic target. Keywords: serine proteases, extracellular matrix, tumor development, metastasis

Introduction Proteases are the largest and diverse cluster of hydrolytic enzymes that are classified based on their site of action, structure of active site, and specific reaction mechanism. The term “proteases” states enzymes that digest proteins. The term was introduced in 1903 from University of Oxford by S.H. Vines (Professor of Botany). In addition, the term “peptidase” was introduced later by Petersen and Short. These enzymes generally cleaved peptides into amino acids. Enzymes initiate breakdown of proteins into smaller fragments through hydrolysis of the peptide bonds (Philipps-Wiemann, 2018).

✶ Corresponding author: Raman Kumar, Department of Biochemistry, Kurukshetra University, Kurukshetra 136119, Haryana, India, e-mail: [email protected] Poonam Bansal, Department of Bioscience and Technology, Maharishi Markandeshwar (Deemed to be University), Mullana, Ambala 133207, Haryana, India Praveen Kumar, Department of Biochemistry, Kurukshetra University, Kurukshetra 136119, Haryana, India

https://doi.org/10.1515/9783111325040-003

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Raman Kumar, Poonam Bansal, and Praveen Kumar

Proteases, also termed proteinases or peptidases, are enzymes that carry out proteolysis (hydrolytic reaction cleaves both proteins and peptides into smaller peptides or into amino acid residues). Proteolysis is considered one of the most important biological reactions in the living system. Proteases are widely distributed in nature and perform role in major biological processes (Mótyán et al., 2013). These enzymes were evolved to perform a variety of reactions through different mechanisms. Different protease classes may carry out the same reaction through a completely different catalytic process. Proteases regulate the fate, localization, and action of many proteins, which are involved in the processing of cellular information, regulate protein-protein interactions, generate novel bio-active molecules, and generate, transduce, and amplify molecular signals (López-Otín and Bond, 2008). In human body, proteases perform the regulation of all the biological reactions like differentiation of cellular functions, motility, cellular division, and cell death. These enzymes take part in intracellular protein degradation via several systems viz. lysosomal and proteasomal degradation processes (Ward, 2011). These enzymes regulate protein functions inside the cell along with cell apoptosis and also participate in other essential processes of human body (López-Otín and Bond, 2008). Mostly, proteases are originally produced as their inactive forms called zymogens and further activated in a tightly regulated extracellular/intracellular activation cascade, often commenced by external stimuli. Protease cascades have been well distinguished in many important physiological processes like complement system, plasminogen activation, blood coagulation, and digestive cascades (Craik et al., 2011).

Classification of proteases Proteases are classified on the basis of their mechanism of catalysis and total seven known distinct classes of proteases are identified, that is, serine, metallo, cysteine, aspartic, glutaic, asparagine, and threonine proteases (Dong et al., 2021). In all the proteases classes, serine proteases are considered as the largest proteolytic class, containing 13 clans and 40 families. More than one-third part of the identified proteases comprises serine proteases (Di Cera, 2009). Different types of proteases and their respective active site residues are depicted in Figure 1.

Chapter 3 Role of serine proteases in cancer progression and metastasis

Proteases

Mechanism [Active site residues]

Serine Proteases

Nucleophillic serine

Cysteine Proteases

Nucleophillic cysteine

Threonine Proteases

Threonine residue

Aspartic Proteases

Asparate

Glutamic Proteases

Glutamic acid

Metalloproteases

Metal

Aspargine Proteases

Aspargine

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Figure 1: Different proteases and their active site residues.

Serine proteases Serine proteases (EC 3.4.21) are a family of proteases, utilizing activated serine (Ser) in its substrate-binding site to catalytically hydrolyze peptide bonds. These enzymes contain three residues at their active site: Ser, histidine (His), and an aspartate (Asp). Nucleophilic serine (Ser) in its active site attacks the carbonyl carbon of peptide bond of substrate and forms an acyl-enzyme intermediate. The nucleophilicity of this Ser is mainly dependent on the catalytic triad of aspartate (Asp), histidine (His), and Ser, known as the charge relay system. This catalytic triad was first seen before 30 years ago in chymotrypsin (Page and Di Cera, 2008). In all serine proteases families, the S1 family of the PA clan superfamily has the largest number of serine proteases. It possessed largest group of homologous proteinases in the human genome responsible for various biological processes (Di Cera, 2009). Trypsin, chymotrypsin, and elastase-like serine proteases are part of serine proteases S1 family (Laskar et al., 2012; Table 1). The proteases activity of serine proteases is regulated at various levels, such as transcriptional, post-transcriptional, and post-translational. Serine proteases are synthesized as zymogen (inactive form) and the activation of zymogen starts when the proteases reach their desired sites of proteolytic action (Craik et al., 2011). Serine proteases are well-established and widespread and significantly play various roles in physiological and pathological processes of host. Serine proteases are known to participate in large number of biological processes as modeled enzymes for investigating different catalytic mechanisms. These enzymes are involved in regulation of distinct physiological functions of human body like digestion, immune response, cell signaling, blood coagulation, and the complement system (Neitzel, 2010; Laskar et al., 2012).

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Table 1: Different types of serine proteases and their substrate specificity. Enzymes types

Substrate specificity

Trypsin-like serine protease

Basic residues (lysine, arginine)

Chymotrypsin-like serine protease

Tyrosine, phenylalanine, and tryptophan

Elastase-like serine protease

Cleavage of carboxyl groups present on small hydrophobic amino acids such as glycine, alanine, and valine

Thrombin-like serine protease

Catalyzes the conversion of fibrinogen to fibrin

Subtilisin-like serine protease

Nucleophilic attack on the peptide (amide) bond

Serine proteases are divided into two main classes on the basis of their localization inside the extracellular matrix (ECM): cell-surface-anchored or membrane-anchored serine proteases and secreted serine proteases. Cell-surface-anchored serine proteases are unique subgroup of S1 serine proteases that are found to be directly attached to the plasma membrane via its carboxy- or amino-terminus domains (Poddar et al., 2017). These proteases are involved in different types of physiological processes viz. fertilization, embryo development, cell signaling, tissue morphogenesis, and maintenance of epithelial barrier (Szabo and Bugge, 2011). Based on their structural features, cell-surface-anchored serine proteases are divided into different subgroups and these enzymes are attached to the cell membrane by three modes: (i) carboxy-terminus transmembrane domain (type I transmembrane serine proteases), (ii) amino-terminus transmembrane domain having cytoplasmic extension (TTSPs or type II transmembrane serine proteases), and (iii) carboxyl-terminus transmembrane domain with glycosyl phosphatidylinositol (GPI) linkage that is added post-translationally. Tryptase γ1 (type I serine proteases) and both testisin and prostasin (GPI-linked serine proteases types) have a carboxyl-terminus hydrophobic extension which acts as transmembrane domain (approximately 310–370 amino acids). GPIlinkage has been well-known to alter carboxyl-terminus domain of testisin and prostatin post-transcriptionally (Antalis et al., 2010; Poddar et al., 2017). Catalytic domain of all cell-surface-anchored serine proteases is structurally conserved that belongs to S1 peptidase family. Serine proteases commonly present in their inactive form as zymogens and the autoactivation cleavage of enzymes takes place after basic residues of conserved activation motif form a two-chain structure having disulfide linkage between them, finally dividing into pro- and catalytic domain. This catalytic domain remains cell surface bound (Szabo and Bugge, 2011; Böttcher-Friebertshäuser, 2018). Out of 176 identified human serine proteases, there is a subset of 17 serine proteases called as TTSPs (Tanabe and List, 2017). This protease family is further divided into four subfamilies: (i) matriptase, (ii) hepsin/transmembrane protease/serine (TMPRSS), (iii) human airway trypsin-like (HAT)/differentially expressed in squamous cell carcinoma (DESC), and (iv) corin. These enzymes are synthesized as zymogens and activated after

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proteolytic cleavage. TTSPs like hepsin, matriptase, matriptase-2, TMPRSS2, 3, 4, and 13 have potential for autoactivation which suggests the basal level for their own zymogen form (Tanabe and List, 2017; Martin and List, 2019). This showed that many of the TTSPs could function as initiators of proteolytic cascades. Secreted serine proteases belong to serine proteases S1 family. These enzymes are generally released by secretory vesicles to extracellular space. Trypsin, thrombin, and chymotrypsin are the prototypes of the S1 family. Secreted serine proteases like urokinase plasminogen activators (uPA) and kallikreins are known to participate in pericellular proteolysis either by binding with the coreceptors or by stimulating zymogens of other substrates. These enzymes are involved in many physiological processes like nutrients uptake, tissue repair, and immune system (Poddar et al., 2017). Generally, proteases are involved in various processes of human body system such as tissue morphogenesis and remodeling, cellular proliferation and differentiation, angiogenesis, heat shock and unfolded protein responses, autophagy, apoptosis, senescence, necrosis, inflammation, and immunity. These enzymes are involved in the regulation of cell behavior, survival, and death, but alterations in proteolytic systems trigger further multiple pathological severities (López-Otín and Bond, 2008; Herszényi et al., 2014). Deregulation of extracellular and pericellular proteolytic events linked with the secreted and cell-surface-anchored serine proteases, respectively, are the major cause of initiation of several diseases. Proteases play a significant role in the reconstitution and breakdown of ECM in different physiological and pathological processes viz., wound repair, tissue remodeling, angiogenesis, protein turnover, destructive diseases, inflammatory disorders, tumor development, and metastasis (Herszényi et al., 2014). Studies revealed that proteases that are produced and released by stromal and cancerous cells play essential roles in modulation of tumor-stromal interactions. The functions of proteolytic enzymes in tumor are too complex and varied (Wilson and Singh, 2008). Several biomarkers of aggressive cancer are directly associated with altered proteolytic activity of these enzymes that led to tumorigenesis. Studies on different cancers suggested that there are multiple mechanistic events through which cancerous cells co-opt proteolytic system, including stromal interactions, overexpressed activated proteases, and even cellular uptake of proteases released from stromal cells (Mason and Joyce, 2011). Studies reported that abnormal expression and activities of stromal cells-derived serine proteases have been significantly linked with tumor initiation and metastasis (Tagirasa and Yoo, 2022).

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Role of cell-surface anchored serine proteases in cancer Many cell-surface anchored serine proteases are expressed abnormally in human cancers and considered as major biomarkers that indicate the disease state. Experimental studies on carcinogenic models revealed that cell surface-anchored serine proteases are overexpressed by tumor cells and are involved in promoting tumorigenesis (Antalis et al., 2010). Cell surface-anchored serine proteases led to occurrence of primary tumors (identified using genetic mouse models), primary tumor progression (identified through cell proliferation/apoptotic studies, cell-based assays, and xenograft models), migration/ invasion of cancerous cells (identified via in vitro invasion experimental studies), and metastatic lesions development (identified using studies based on metastasis in genetic engineered mouse models and xenograft models) (Martin and List, 2019). Today, many experimental studies are available that evaluated the expression of individual enzymes from tumor development cum progression and focused on to understand mechanistic insight into the role of serine proteases in proliferation of cancer cells and metastasis (Lee et al., 2021).

Matriptase Matriptase is an epithelial specific cell-surface anchored serine protease that has received wide attention because of its continuous abnormal expression in epithelial tumors (Zoratti et al., 2015). These enzymes have C-terminal trypsin-like serine protease domain of 855 amino acids, two tandem repeats of C1r/Cls, bone morphogenic protein 1 domains, urchin embryonic growth factor, four cysteine-rich domains similar to low-density lipoprotein receptor, a single sea urchin sperm protein, enterokinase, and agrin domain (Zuo et al., 2019). Matriptase has been reported to participate in ECM disruption and tissue remodeling as well (Uhland, 2006). Matriptase plays a very important functional role in several pathways of biological processes. Studies reported that matriptase is expressed by cells of epithelial origin and is overexpressed in many human cancers (Welman et al., 2012). It activates many oncogenic proteins like uPA, protease-activated receptor-2 (PAR-2), and hepatocyte growth factor (HGF). Induction of these substrates further activate matrix metalloproteases (MMPs) and plasmin and thus trigger various signaling pathways that are involved in the cancer development such as MMP-induced ECM degradation and tyrosine-protein kinase MET signaling (Zuo et al., 2019). Studies on prostate and breast cancers have revealed that this enzyme is expressed abnormally that is further linked to tumors of different stages and grade (Wu et al., 2010). HGF activator inhibitor-1 (HAI-1) has been reported as the cognate inhibitor of matriptase that regulates enzyme activity in cells by forming a noncovalent complex. The matriptase/HAI-1 ratio

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in cancerous cells is higher as compared to normal cells. Misbalance of enzyme and its inhibitor causes tumor development and progression (Zuo et al., 2019).

Hepsin Hepsin (HPN) is also known as TMPRSS1. Hepsin gene is located on human chromosome (19q13.11) and it encodes for 45 kDa protein having 417 amino acids. Physiologically this protein is involved in many cellular processes such as metabolism, blood coagulation, cell growth and morphology, membrane integrity, cleavage of extracellular substrates, and proteolytic processing of some growth factors. Elevated expression of hepsin has been reported at transcriptional and translational levels in several solid cancers viz. breast, kidney, prostate, and ovarian cancers (Lu et al., 2022). Hepsin is involved in the cleavage and activation of pro-HGF, pro-uPA, promacophage stimulating protein (MSP), and Laminin332. It activates pro-HGF and is suppressed by HGF activator inhibitor-1B (HAI-1B) and 2 (HAI-2). Hepsin activates different proteolytic events inducing several nonactive proteases which are further involved in the breakdown of ECM proteins (Poddar et al., 2017). In vivo and in vitro studies revealed that the dysregulated hepsin participates in cancer invasion and metastasis. High level of expression of hepsin in prostate cancer has also been reported (Willbold et al., 2019). In silico protein-protein interaction studies and other mechanistic assessments revealed the interconnection of hepsin with eight other oncogenic proteins, whose expressions were considerably linked with hepsin level in prostate cancer. This oncogenic nature of hepsin associated with its proteolytic properties has been reported to affect epithelium integrity and regulatory interaction with other genes to promote cellular proliferation, epithelial mesenchymal transition (EMT)/metastasis, tyrosine-kinase-signaling pathways, and inflammatory events (Lu et al., 2022). Amplifications of hepsin at genome level were also significantly linked to metastasis of prostate cancer (Pal et al., 2006). Hepsin has been observed as utmost upregulated gene in prostate cancer. Overexpression of hepsin is noticed in many cancers viz. lung, bladder, renal, breast, gastric, prostate, uterine, and ovarian cancers (Li et al., 2021).

Prostasin Prostasin (protease serine S1 family member 8) is a GPI-anchored extracellular serine protease (40 kDa) having trypsin-like cleavage specificity. It is encoded by serine protease 8 (PRSS8) gene located on chromosome 16p11.2. Prostasin is a regulator of epithelial sodium channels (ENaCs) present in epithelial tissues (Tamir et al., 2016; Bao et al., 2022). Prostasin is proteolytically activated by matriptases (Selzer-Plon et al., 2009). Expression of prostasin in epithelial cells has been observed which plays an important

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Raman Kumar, Poonam Bansal, and Praveen Kumar

role in the initiation of several tumors. Irregular expression of prostasin has been reported in many cancers including prostate, uterus, urinary bladder, and ovarian cancer when compared with the expression in corresponding normal tissues. Studies have shown that prostasin is involved in the cleavage of extracellular domain of epidermal growth factor of epithelial cells by virtue of which this receptor remains phosphorylated and thus triggers cancer progression. High level of secreted prostasin found in sera of early-stage ovarian cancer (OVC) patients has been considered as a screening biomarker for the detection of OVC at an early stage of cancer (Tamir et al., 2016).

Human airway trypsin-like protease Human airway trypsin-like protease (HAT) or serine 11D or TMPRSS11D is a type of cell-surface-anchored proteolytic enzyme. In humans, the molecular mass of HAT is approximately 46 kDa. While the coding gene is a human orthologue of a long splice variant of rat adrenal secretory serine protease (AsP), also called RAT1, that is, rat airway trypsin-like serine protease. Studies have reported that after cleavage of four major substrates of RAT1, that is, uPA receptor (uPAR), PAR-2, severe acute respiratory syndrome coronavirus spike proteins, and hemagglutinin of the influenza viruses; it triggers specific cell response in epithelial cells, within the extracellular and pericellular environment (Menou et al., 2017). Abnormal expression of HAT has been reported in cancerous tissues at different carcinogenic stages. Diminished level and even a complete protein loss were observed in the advanced stages of cancer. Expression of HAT in nonproliferating or differentiated cellular strata and its complete loss in dedifferentiated epithelial cells is a major feature of squamous cell carcinogenesis. This showed that HAT can be used as a biological marker for clinical grading and assessment of squamous cell carcinomas (Duhaime et al., 2016). Differential expression of HATL5 has been reported in cancers related to epithelial cells. Studies of esophageal and cervical cancer tissues showed that the squamous epithelial cells lose expression of HATL5 protein during malignancy (Miller et al., 2014).

Testisin Testisin, is also called as PRSS21 and ESP-1, which is highly expressed in various tumor cell lines and showed lower expression in normal tissues, except testicular germ cells. Overexpression of testisin provokes colony formation and induces malignant transformation in ovarian cancer cells. Testisin was observed to be overexpressed in cervical cancer as compared to corresponding normal cervical tissues (Yeom et al., 2010). Studies showed that testisin expression correlates with the stages

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of cancer (Conway et al., 2019). Experimental studies on metastatic serous papillary ovarian tumors showed that expression of testisin was decreased in metastatic ovarian tumor cells in contrast to primary carcinomas. This showed that expression of testisin mainly becomes upregulated in primary tumors than tumors of metastatic stage (Martin and List, 2019). Recently, increased expression of testisin was observed in ovarian tumors, with relatively mild expression in normal tissues. However, decreased protein levels were observed in metastatic ovarian serous carcinomas as compared to primary tumors (Conway et al., 2019).

TMPRSS (2, 3, and 4) TMPRSS2, TMPRSS3, and TMPRSS4 are membrane-anchored serine proteases mainly involved in the metastatic progression of tumors. Physiologically, TMPRSS2 is extremely expressed in colon, pancreas, kidney, and prostate tissues; however, small intestine, lung, and liver are known to exhibit weak expression (Tanabe and List, 2017). As localized on the surface of cancerous cells, TMPRSS2 mediates signal transduction between the cancer cell and ECM and is involved in the regulation of different cellular responses (Lam et al., 2015). Studies reported that TMPRSS2 is most probably involved in metastatic prostate cancer. Pro-HGF, matriptase, and ECM proteins (nidogen-1 and laminin β1) have been identified as substrates for TMPRSS2-induced prostate cancer progression (Ko et al., 2020). Studies showed that the TMPRSS2:ERG (v-ets avian erythroblastosis virus E26 oncogene) homolog gene fusion plays an important role in the development of prostate cancer via activation of ERG transcription factor. This TMPRSS2: ERG fusion is involved in activation of NOTCH signaling pathway and induce progenitor cells proliferation in the developing prostate. TMPRSS2: ERG fusion has been strongly associated with disease severity and biochemical recurrence (Mollica et al., 2020). TMPRSS3 is a transmembrane protease or serine 3 found to be highly expressed in pancreatic and breast cancers and considered as a prometastatic mediator in such cancers (Li et al., 2018). Elevated TMPRSS3 level has been also detected in ovarian cancer. TMPRSS3 is involved in the ovarian cancer cells proliferation, invasion, and migration through activation of ERK1/2 pathway (Zhang et al., 2016). TMPRSS3 has also been reported in gastric cancer progression. TMPRSS3 was also found to be involved in the activation of ERK1/2 and PI3K/Akt signaling in gastric cancerous cells (Li et al., 2018). TMPRSS4 also belongs to Type II transmembrane serine proteases family which was identified first in pancreatic cancer. Elevated TMPRSS4 level has been observed in many cancers such as colorectal, ovarian, breast, thyroid, gallbladder, cervical, gastric, and liver cancer (Tanabe and List, 2017). Studies reported that TMPRSS4 involved in the development of cancer by reducing E-cadherin-associated cell-cell adhesion

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and promoting EMT. In colon cancer, TMPRSS4 has been reported to be involved to promote the activation of FAK signaling which includes ERK1/2, FAK, Src, Akt, and Rac1 activation. This in turn further stimulates SIP1/ZEB2 (transcription factors), leading to loss of E-cadherin. It is a major TMPRSS4-mediated process observed in EMT. TMPRSS4 is also involved in the enhancement of cancer cells invasion by activation of uPA and by inducing pro-uPA (Poddar et al., 2017). Enzyme was reported to induce prostate cancer cells proliferation via Slug and cyclin D1 activation (Lee et al., 2016).

Role of secreted serine proteases in cancer Secreted serine proteases play a major role in different metabolic processes to maintain the tissue homeostasis. These enzymes participated in many physiological functions from cell signaling and growth to remodeling of tissues. But, abnormal level of these enzymes led to invasive and metastatic cancer (Poddar et al., 2017).

Urokinase plasminogen activator Urokinase-type plasminogen activator (uPA) is a major serine protease that converts plasminogen (zymogen form) into active plasmin (Mahmood et al., 2018). The enzyme plays a very important role in various physiological processes of human body such as embryogenesis, fibrinolysis, cell migration, activation, and differentiation of white blood cells, tissues remodeling, angiogenesis, immune system, and inflammation (Poddar et al., 2017). After its synthesis, uPA is released as polypeptide glycosylated chain known as pro-uPA having 411 amino acids. It has three domains: (i) growth factor domain (GFD) having similarity with EGF, (ii) serine protease domain, and (iii) kringle domain. Active plasmin produced by uPA was found to be involved in several metastatic cascades (Mahmood et al., 2018). uPA system is active mostly in all tumors. This system controls ECM degradation by activation of ubiquitous protease plasmin. The main key components of uPA system are uPA, uPAR, and plasminogen activator inhibitor-1 and -2 (PAI-1, PAI-2). Together, all of them play an important role in ECM remodeling. Many in vitro and in vivo studies have shown that the overexpression of these components of uPA system (uPA, uPAR, and PAI-1) is involved in the enhancement of tumor cell invasion and metastatic event. These components were considered as prognostic biomarkers and therapeutic targets in many cancer malignancies (Madunić, 2018). uPA in combination with uPAR promotes several proteolytic cascade events that further induce tumor growth through metastatic process of cancerous cells. Cell signaling by uPAR leads to the activation of Tyr kinases, serine kinase FAK, Raf, and Src, and ERK/MAPK pathways and thus trigger the modulation of cell-cell interactions, cell proliferation, and

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metastasis (Poddar et al., 2017). uPA system is strongly involved in pathophysiology of pancreatic ductal adenocarcinoma (PDAC). More than 90% of pancreatic cancers have been reported to be initiated by irregular proteolytic cascade of this system. It was also reported that overexpression of uPA and uPAR is the first step of induction of metastatic process of cancerous cells through generation of serine protease plasmin from cell surface localized plasminogen, thus triggers many downstream processes in cells that induce tumor cell migration and invasion (Kumar et al., 2022). An elevated uPA level in tumor tissue of breast cancer has been confirmed as a strong prognostic factor (Banys-Paluchowski et al., 2019). uPA, uPAR, and also PAI-1 have been reported to be constitutively expressed in human breast cancer. Elevated levels of uPA and uPAR enhanced ECM degradation, adhesion, migration, and invasion in many cancers especially in breast cancer (Poddar et al., 2017). Studies revealed that uPA system is a major prognostic factor in many human cancers such as lung, breast, bladder, intestine, stomach, uterus, thyroid, kidney, head, and soft tissues (Madunić, 2018).

Kallikrein-related peptidase Kallikrein-related peptidases (KLKS) or Kallikrein belongs to a family of secreted serine proteases and plays a very important role in several physiological processes such as ECM modification, epidermal homeostasis, angiogenesis, innate immunity, tooth enamel formation, male reproduction, and neural development. In total, 15 secreted KLKs were found in humans. KLK1-2, 4–6, 8, and 10–15 have trypsin-like, while KLK3, 7, and 9 have chymotrypsin-like properties (Tagirasa and Yoo, 2022). Abnormal expression, secretion, and function of KLKs were found to be associated with many pathological conditions predominantly in endocrine-related breast, ovary, and prostate malignancies (Avgeris et al., 2012). KLK-mediated ECM proteins degradation considered as major critical part of KLKs role in occurrence of cancer (Stefanini et al., 2015). Studies showed that PSA/KLK3, 2, and 4 play a major regulatory role in prostate cancer progression. These KLKs induce tumor growth via stimulation of insulin-like growth factor axis and protease-activated receptors activation. These KLKs also promote ECM degradation via activation of uPA- and MMP-related cascades and induce EMT in prostate cancer. This leads to aggressive invasive tumor and distant metastasis (Avgeris et al., 2012). Higher level of KLKs was reported in ovarian cancer. Overexpression of KLKs is responsible for irregular degradation of ECM and thus promotes invasive tumor. Genomic instability in KLK chromosomal locus was also observed in ovarian cancer patients (Dong et al., 2014). Increased KLK6 level is common in various human malignancies including colon cancer. Aberrant KLK6 expression in cell lines (colon cancer) and its connection to PAR2 receptor signaling activates PAR2 and thus induced ERK1/2 phosphorylation and cell proliferation (Bouzid et al., 2022).

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Fibroblast activation protein-α Fibroblast activation protein-α (FAP) is generally produced by activated fibroblasts. Cancer-associated fibroblasts (CAFs) were reported to induce irregulated expression of FAP in tumor stroma and thus promote immunosuppression, tumor development, invasion, and metastasis (Bughda et al., 2021). Low or undetectable level of FAP expression was observed in normal tissues. However, its expression was aberrant in many tumors. FAP is overexpressed in breast, pancreatic, lung, colorectal, intrahepatic bile duct, brain, and ovarian cancers. High level of FAP expression was also observed in nonepithelial tissue-derived tumors, such as myeloma and melanoma (Xin et al., 2021). Modified tumor microenvironment or inflammation initiated by FAP+ CAFs promotes FAP expression via stimulation of TNF-α and TGF-β1 (Tagirasa and Yoo, 2022). Feig et al. (2013) reported that FAP+CAFs produced chemokine ligand 12, stimulating immune suppression activities and, thus, leading to failure of T-cell checkpoint inhibitors in pancreatic ductal adenocarcinoma. FAP protein binds to Enolase1 and promote NF-κB pathway to induce metastatic cascade in colorectal cancer cells (Yuan et al., 2021). Phosphatidylinositol-3-kinase (PI3K) signaling pathway-mediated proliferation of breast cancer cells by FAP was also observed in earlier studies reported in literature. FAP also induces the growth and migration of lung cancer cells by the activation of PI3K and sonic-hedgehog signaling (Jia et al., 2018). Studies reported that FAP also promotes tumor development and progression through its nonenzymatic activities. FAP induces tumor growth and cancer cells invasion in breast via nonenzymatic functions (Fitzgerald and Weiner, 2020). It was reported that transfection of breast cancer cell lines with FAP of low enzymatic activity exhibited rapid ECM degradation and tumor growth in vivo as compared to nontransfected cell lines (Xin et al., 2021). This showed that the enzyme might be able to induce ECM remodeling, cancer development, and progression.

HtrA Human HtrA (high temperature requirement) belongs to serine proteases family having four members viz. HtrA 1, 2, 3, and 4 (Chien et al., 2009). HtrA is involved in several biological processes of human body such as mitochondrial functions, cell signaling, and apoptosis. Dysregulation of its functioning leads to many clinical manifestations including cancer (Skorko-Glonek et al., 2013; Poddar et al., 2017). In humans, HtrA1 has been reported as first member of this protein family isolated from fibroblast. Downregulation of HtrA1 in many cancers such as glioma, melanoma, ovarian tumors, lung, and endometrial cancer has been reported earlier (Poddar et al., 2017). HtrA2 functions as a tumor-promoting factor and is highly expressed in many cancers. HtrA2 expression was

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found to be increased in thyroid malignant tumors as compared to corresponding normal tissues (Wu et al., 2021). HtrA2 promotes the growth of hepatocellular carcinoma very aggressively. HtrA2 level was reported to be high in hepatocellular carcinoma patients (Wang et al., 2018). Downregulation of HtrA2 was reported in chronic lymphocytic leukemia which corresponds to normal B-cells. Downregulated HtrA2 was also observed in ovarian tumor. Thus, studies revealed that HtrA2 expression is variable. It is upregulated in many tumors and downregulated in others. Tumor specific variation was also observed in HtrA3 expression. Upregulated expression of HtrA3 was found in pancreatic adenocarcinoma, esophageal adenocarcinoma, and seminoma, as compared to normal controls (Chien et al., 2009). However, its downregulation has also been observed in some primary tumors compared to normal controls. Reduced expression of HtrA3 was observed in breast, ovarian, endometrial and lung cancers (Skorko-Glonek et al., 2013; Wu et al., 2021). Like HtrA3, variable expression of HtrA4 was also reported in some cancers. Upregulated HtrA4 was observed in breast carcinoma as compared to normal breast samples and its downregulation was observed in hormone-refractory prostate cancer metastatic stage as compared to primary prostate carcinoma (Chien et al., 2009).

Granule-associated enzymes Granule-associated enzymes (Gzm) are a type of secreted serine proteases having structural relatedness with chymotrypsin. These enzymes have conserved residues and disulfide bridges. Gzms are classified based on their cleavage specificity. There are total five human GZMs, viz. GzmA, B, H, K, and M. Being proinflammatory, these enzymes participate in the initiation and progression of various cancers (Pączek et al., 2022). GzmB has been reported in human primary breast carcinomas, primary bladder cancers, urothelial carcinoma, and pancreatic carcinoma cells (Wang et al., 2015; Tagirasa and Yoo, 2022). GzmM was also reported to be involved in EMT, promoting tumor growth and metastatic cascade through induction of STAT3 signaling pathway (Wang et al., 2015).

Concluding remarks and future prospective Proteases participate in a wide range of physiological functioning of human body. Moreover, these enzymes also contribute to abnormal physiological functioning and disease states. Serine proteases are well-known, widespread, and significantly play various roles in physiology and disease pathology in humans. Serine proteases are considered as predominant enzymes involved in cancer pathogenesis as well. During tumor development and malignancy, several serine proteases act as key players at the tumorstromal interface. Both cell-surface-anchored and secreted serine proteases work in a

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multidirectional mode by interaction with each other or other class of proteases and also with major cell signaling pathways and thus lead to the activation/inactivation of chemokines, growth factors, cytokines, and kinases to promote cancer progression and malignancies. Studies revealed that abnormal expression of serine proteases in cancer contributed to formation of invasive tumor and metastasis through varied enzyme mechanisms. Aberrant expression of these proteases is a hallmark of several cancers. Serine proteases are considered as promising targets for diagnosis and therapeutic development against cancer. Therefore, more experimental studies are needed to understand the proteases network through which their dysregulation occur at the transcriptional and translational level, and how these enzymes are impetuous at different stages of tumorigenesis. Elucidation of mechanistic insight of procancerous properties of serine proteases will help to understand their roles in cell physiology and cancer.

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Welman, A., Sproul, D., Mullen, P., Muir, M., Kinnaird, A. R., Harrison, D. J., Faratian, D., Brunton, V. G., & Frame, M. C. (2012). Diversity of matriptase expression level and function in breast cancer. PloS One, 7(4), e34182. Willbold, R., Wirth, K., Martini, T., Sültmann, H., Bolenz, C., & Wittig, R. (2019). Excess hepsin proteolytic activity limits oncogenic signaling and induces ER stress and autophagy in prostate cancer cells. Cell Death & Disease, 10(8), 1–14. Wilson, T. J., & Singh, R. K. (2008). Proteases as modulators of tumor–stromal interaction: Primary tumors to bone metastases. Biochimica et Biophysica Acta (Bba)–reviews on Cancer, 1785(2), 85–95. Wu, L., Li, X., Li, Z., Cheng, Y., Wu, F., Lv, C., Zhang, W., & Tang, W. (2021). HTRA serine proteases in cancers: A target of interest for cancer therapy. Biomedicine & Pharmacotherapy, 139, 111603. Wu, S. R., Cheng, T. S., Chen, W. C., Shyu, H. Y., Ko, C. J., Huang, H. P., Teng, C. H., Lin, C. H., Johnson, M. D., Lin, C. Y., & Lee, M. S. (2010). Matriptase is involved in ErbB-2–induced prostate cancer cell invasion. The American Journal of Pathology, 177(6), 3145–3158. Xin, L., Gao, J., Zheng, Z., Chen, Y., Lv, S., Zhao, Z., Yu, C., Yang, X., & Zhang, R. (2021). Fibroblast activation protein-α as a target in the bench-to–bedside diagnosis and treatment of tumors: A narrative review. Frontiers in Oncology, 11, 3187. Yeom, S. Y., Jang, H. L., Lee, S. J., Kim, E., Son, H. J., Kim, B. G., & Park, C. (2010). Interaction of testisin with maspin and its impact on invasion and cell death resistance of cervical cancer cells. FEBS Letters, 584(8), 1469–1475. Yuan, Z., Hu, H., Zhu, Y., Zhang, W., Fang, Q., Qiao, T., Ma, T., Wang, M., Huang, R., Tang, Q., Gao, F., Zou, C., Gao, X., Wang, G., & Wang, X. (2021). Colorectal cancer cell intrinsic fibroblast activation protein alpha binds to Enolase1 and activates NF-κB pathway to promote metastasis. Cell Death & Disease, 12(6), 1–15. Zhang, D., Qiu, S., Wang, Q., & Zheng, J. (2016). TMPRSS3 modulates ovarian cancer cell proliferation, invasion and metastasis. Oncology Reports, 35(1), 81–88. Zoratti, G. L., Tanabe, L. M., Varela, F. A., Murray, A. S., Bergum, C., Colombo, É., Lang, J. E., Molinolo, A. A., Leduc, R., Marsault, E., Boerner, J., & List, K. (2015). Targeting matriptase in breast cancer abrogates tumour progression via impairment of stromal-epithelial growth factor signalling. Nature Communications, 6(1), 1–13. Zuo, K., Qi, Y., Yuan, C., Jiang, L., Xu, P., Hu, J., Huang, M., & Li, J. (2019). Specifically targeting cancer proliferation and metastasis processes: The development of matriptase inhibitors. Cancer and Metastasis Reviews, 38(3), 507–524.

Biographical sketch Name: Raman Kumar Affiliation: Kurukshetra University, Kurukshetra Education: M.Sc. & Ph.D. Biochemistry

Research and Professional Experience – –

Experienced in supervision and perform Microbiology laboratory operations (Research Methodologies/Pathogen Testing/Data analysis/Review Data) Worked on metabolic profiling of Pediococcus acidilactici NCDC 252 to explore the beneficial effects of bacterial active components/metabolites and to establish this strain as a useful probiotic strain.

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Publications Raman Kumar, Poonam Bansal, Jasbir Singh, Suman Dhanda* (2020). Purification, partial structural characterization and health benefits of exopolysaccharides from potential probiotic Pediococcus acidilactici NCDC 252. Process Biochemistry, 99: 79–86. Raman Kumar, Poonam Bansal, Jasbir Singh, Suman Dhanda* and Jitender Kumar Bhardwaj (2020). Aggregation, adhesion and efficacy studies of probiotic candidate Pediococcus acidilactici NCDC 252: A strain of dairy origin. World Journal of Microbiology and Biotechnology, 36: 10. Poonam Bansal, Raman Kumar and Suman Dhanda (2022). Characterization of starter cultures and nutritional properties of Pediococcus acidilactici NCDC 252: A potential probiotic of dairy origin. Journal of Food Processing and Preservation, DOI: https://doi.org/10.1111/jfpp.16817.

Raman Kumar✶, Poonam Bansal, and Praveen Kumar

Chapter 4 Role of serine proteases in lung diseases: a view from acute and chronic lung infection complications Abstract: Proteases correspond to a large and different group of hydrolytic enzymes that cleave proteins into smaller fragments and classified based on their site of action, enzyme active site structure, and specific reaction mechanisms. Dysregulated protease activity and protease/antiprotease imbalance have long been a concern in the pathogenesis of lung infections. Among various classes of proteases, serine proteases are the most important proteases responsible for the impairment observed in the lungs. Evidences suggested that neutrophil serine protease (NSP; neutrophil elastase, proteinase 3, and cathepsin G), transmembrane serine protease (TMPRSS2, TMPRSS4, and TMPRSS11D), and HtrA are major pathogenic determinants exposed to be linked with multiple lung diseases including pulmonary hypertension, cystic fibrosis, chronic obstructive pulmonary disease, viral pathogenesis, inflammation, and cancer. This chapter presents the overview and mechanistic insights of serine protease-driven complications in the pathogenesis of lung diseases. Keywords: serine proteases, protease/antiprotease imbalance, lung infections, cancer

Introduction Proteases (or proteinases) are ubiquitous in all living organisms that cleave both proteins and peptides (Bansal et al., 2021b). The term “proteases” states enzymes that digest proteins. It was introduced in 1903 from University of Oxford by S. H. Vines (Professor of Botany). In addition, the term “peptidase” was introduced after some time by Petersen and Short. These enzymes generally cleave peptides into amino acids. Enzymes initiate breakdown of proteins into smaller fragments through hydrolysis of the peptide bonds (Philipps-Wiemann, 2018). As per Enzyme Commission clas-

✶ Corresponding author: Raman Kumar, Department of Biochemistry, Kurukshetra University, Kurukshetra 136119, Haryana, India, e-mail: [email protected] Praveen Kumar, Department of Biochemistry, Kurukshetra University, Kurukshetra 136119, Haryana, India Poonam Bansal, Department of Biosciences and Technology, Maharishi Markandeshwar (Deemed to be University), Mullana, Ambala 133207, Haryana, India

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sification, these enzymes belong to group 3, that is, hydrolases that hydrolyze peptide bonds (subgroup 4) (Sumantha et al., 2006). Proteases are a large cluster of different types of hydrolytic enzymes. These enzymes are classified based on their target site, configuration of their active site, active pH range, the type of substrate, unique, and specific reaction mechanism (Solanki et al., 2021). Proteases are ubiquitous in nature and perform different types of important biological functions in living system such as growth, development, regulation, adaptation, germination, protein turnover, disease, and death. These enzymes are involved in the regulation of different biological events in humans viz. cellular differentiation, cell division, motility, and death (Ward, 2011). Such enzymes are known to be highly regulated at various levels, that is, at transcriptional, translational, and post-translational. Upon their release as active proteins, these enzymes are placed to specific tissues, organs, and subcellular sites either by circulatory systems viz. lymphatic or vascular or by proteinprotein interactions. Mostly, proteases are originally produced as inactive forms called zymogens which are involved in highly regulated extracellular or intracellular activation cascades, initiated by external stimuli (Craik et al., 2011). Enzymatic activity of these proteases is tightly regulated by their antagonists, that is, protease inhibitors (Rawlings et al., 2004). Proteases represent nearly 2% of human genome having 565 enzyme members. Based on their structural pattern and catalytic properties, proteases are classified into seven distinct types, that is, serine, cysteine, metallo, aspartic, glutaic, asparagine, and threonine proteases (Taggart et al., 2017; Dong et al., 2021).

Serine proteases: catalytic mechanism, classification, and types Serine proteases represent almost one-third of overall identified proteases (BöttcherFriebertshäuser, 2018). Serine proteases have nucleophilic serine (Ser) in its active site. It attacks the carbonyl carbon of peptide bond of substrate and forms an acyl-enzyme intermediate. The nucleophilicity of this Ser is mainly dependent on the catalytic triad of aspartate (Asp), histidine (His), and Ser, known as the charge relay system (Page and Di Cera, 2008). Amid all protease classes, serine proteases are considered as the largest proteolytic class, which contains 13 clans and 40 families. Amongst all the serine proteases families, the S1 family of the PA clan superfamily has the largest number of serine proteases, mostly exhibiting trypsin-like activities. However, some of them also have elastase-like and chymotrypsin-like specificities (Di Cera, 2009; Laskar et al., 2012). Serine proteases are found extracellularly on the surface of cell membrane or intracellularly in subcellular structures such as mitochondria or lysosomes, in the nucleus or in the cytoplasm. Mostly serine proteases are of secreted type, but some are receptor-bound having a transmembrane domain to get anchored to the cytoplasmic membrane (Taggart et al.,

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2017; Menou et al., 2017). Serine proteases are classified into two main classes on the basis of their localization within the extracellular matrix (ECM): (i) cell-surface anchored serine proteases (membrane-anchored serine proteases) and (ii) secreted serine proteases. Cellsurface-anchored serine proteases are distinctive subgroup of S1 serine proteases attached to the cell membrane by its carboxy- or amino-terminus domains (Poddar et al., 2017). Cell-surface-anchored serine proteases are divided into different subgroups based on their structural characteristics. Enzymes are attached to the cell membrane by three modes: (i) carboxy-terminus transmembrane domain (type I transmembrane serine proteases), (ii) amino-terminus transmembrane domain having cytoplasmic extension (TTSPs or type II transmembrane serine proteases), and (iii) carboxyl-terminus transmembrane domain with glycosyl phosphatidylinositol (GPI) linkage that is added post-translationally (Antalis et al., 2010; Poddar et al., 2017). Secreted serine proteases are the well-established serine proteases S1 family members which are commonly formed from secretory vesicles into the extracellular space (Poddar et al., 2017; Table 1). Table 1: Classification of cell-surface-anchored and secreted serine proteases. Cell-surface-anchored serine proteases Type I transmembrane serine proteases

Type II transmembrane serine proteases (TTSPs)

Tryptase gamma  () HAT/DESC (human airway trypsin-like protease/differentially expressed in squamous cell carcinoma) subfamily: HAT, DESC, TMPRSSA, HAT-like , HAT-like , HAT-like , and HAT-like  () Hepsin/TMPRSS (transmembrane protease/serine) subfamily: hepsin, TMPRSS, TMPRSS, TMPRSS, TMPRSS/ spinesin, MSPL (mosaic serine protease large-form), and enteropeptidase () Matriptase subfamily: matriptase, matriptase-, matriptase, and polyserase- () Corin subfamily: corin

Secreted serine proteases GPI-linked serine proteases Prostasin, testisin

Urokinase plasminogen activator, kallikrein-related peptidase, fibroblast activation protein-α, HtrA, granuleassociated enzymes

The protease activity of serine proteases is regulated at transcriptional, post-transcriptional, and translational levels. Serine proteases are synthesized as inactive zymogens form and then converted to its active form when the enzyme reaches its desired sites of proteolytic action (Craik et al., 2011). Serine proteases are participated in the regulation

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of various physiological functions of human body like digestion, blood coagulation, immune response, cell and cytokines signaling, and ECM remodeling (Neitzel, 2010; Laskar et al., 2012).

Proteases and lung diseases Proteases perform a major role in maintenance of lungs health and initiation of lung infection (Woods et al., 2020). In normal lungs, these enzymes participate in the balancing of homeostasis that regulate lungs regeneration and repair (Chakraborti et al., 2017). Protease activities are regulated well both at the transcriptional and translational level. Enzyme activities are also regulated by modulatory factors (pH), inhibitory prodomains, and antiproteases at the protein level in healthy cells and tissues (McKelvey et al., 2021). Protease activity needs to be tightly regulated for the normal functioning of lungs. Excessive protease levels, and uncontrolled enzymes activity, have been considered as hallmarks in chronic lung diseases (CLDs). Aberrant expression of proteases at genomic level, elevated proteases activities, and proteases-antiproteases imbalance actively contributes to initiation of various lung infections and pulmonary diseases, including cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), pulmonary hypertension, viral pathogenesis, inflammation, and cancer (Taggart et al., 2017; McKelvey et al., 2020). Among various classes of proteases, serine proteases are major proteases responsible for the impairment observed in the lungs (Twigg et al., 2015) (Figure 1). From the past

Normal Lung

Lung Cancer

Protease Inhibitor

Protease Inhibitor

Serine proteases

Serine proteases

Figure 1: Imbalance of serine proteases and proteases inhibitors in lung cancer.

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few years, scientists have shown keen interest in serine proteases to identify the essential roles of these enzymes in various pathophysiological processes. Studies revealed that serine proteases are major pathogenic determinants that are found to be linked with many lung diseases (Meyer and Jaspers, 2015).

Serine proteases and cystic fibrosis Loss of functional mutation in CFTR gene (cystic fibrosis transmembrane conductance regulator) caused autosomal recessive genetic disorder known as CF. Pathogenesis of CF was associated with its consequence on the respiratory system (Chen et al., 2021). Impaired CFTR channels in CF patients stop the functioning of sodium-chloride ions across epithelial membranes and lead to higher levels of dehydrated mucus secretions in lungs and impaired mucociliary clearance, promoting high mucus plugging, chronic inflammation, and polymicrobial infection (Chakraborti et al., 2017). Serine proteases are reported to be playing a major role in CF pathogenesis (McKelvey et al., 2020). Studies have shown that sputum of CF patients contains high neutrophils level and subsequently elevated levels of NSPs. NSPs are major proteases responsible for the damage observed in CF-associated lungs. This includes neutrophil elastase (NE), cathepsin G (Cat G), and proteinase 3 (PR3) (Twigg et al., 2015). Recently, another type of neutrophil-derived protease, that is, NSP4, was also expressed in lungs, associated with CF (Taggart et al., 2017). Extracellular NSPs have been reported to be involved in the immune cells recruitment at inflammation site using cytokines-mediated signaling process by IL-1 family members and IL-8, prompting the secretion of damage-associated molecular patterns like HMGB1 (high mobility group box protein1), which is considered as a major biomarker of CF pathogenesis. Activation of altered neutrophil chemotaxis process increased the inflammatory cell infiltration, further prompting the vicious cycle of CF inflammation. NSPs are also involved in the regulation of other proteases including matrix metalloproteinases (MMPs). NSPs-activated MMP-9 and 12, and NSPs, promote ECM remodeling and abnormal widening of the bronchi or their branches (bronchiectasis) that are characteristics of CF (McKelvey et al., 2020). Endogenous antiproteases in tissues serve to regulate NSPs activities. Inability of these inhibitors such as elafin, alpha-1 antitrypsin (AAT), and secretory leukocyte proteinase inhibitor (SLPI) to perform their function properly is also associated with CF prognosis (Twigg et al., 2015). NE is considered as a key mediator protease that is involved in CF pathogenesis. NE was reported to induce mucins secretions in epithelial cells of airway and increased mucus plugging in the CF-associated lung. This enzyme was also reported to diminish the ciliary beat frequency and damaged epithelium airway, which contributes to weaken mucociliary clearance and promotes mucus accumulation in lungs (Voynow and Shinbashi, 2021).

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Serine proteases and pulmonary hypertension Pulmonary arterial hypertension (PAH) is associated with the vascular system of lungs, mainly damaging the pulmonary arterioles (Lai et al., 2014). PAH causes thickening and blockage of distal arteries in lungs due to vascular cell dysfunction and perivascular inflammation (Taylor et al., 2018). This leads to the increase of pulmonary arterial pressure and cause failure of right ventricle leading to death if untreated (Lan et al., 2018). Perivascular inflammation has been reported in all PAH patients (Rabinovitch et al., 2014). Perivascular inflammation is associated with the production of cytokines by inflammatory and vascular cells and proteases-mediated ECM degradation. Increased cytokines levels and peptides induce stimulation and recruitment of circulating immune cells including neutrophils. These neutrophils release proteolytic enzymes, mostly NE, that cause vascular injury (Hu et al., 2020). NE has been considered as a major proteolytic enzyme that causes pathogenesis of PAH. NE is involved in the degradation of all ECM components including elastin, collagen, fibronectin, and laminin. Degraded ECM releases growth factors (fibroblast and epidermal growth factor) and bioactive peptides that have mitogenic effect on smooth muscle cells (SMCs) and fibroblasts (Taylor et al., 2018). Higher levels of NE also activate MMPs and promote ECM degradation, followed by tenascin C production. Tenascin C upregulates proliferation of SMCs and various growth factor receptors (Burgstaller et al., 2017; Taylor et al., 2018).

Chronic obstructive pulmonary disease COPD is a progressive disorder of lower respiratory tract causing severe emphysema and chronic bronchitis. Emphysema is due to the destruction of alveoli and lung tissue that leads to reduced gaseous exchange in the lower airways. Chronic bronchitis is due to hypersecretion of mucus by goblet cells that causes thickness of mucus layer (Dey et al., 2018). The protease:antiprotease imbalance is considered as the major cause of pathogenesis of COPD (Lomas, 2016). Serine proteases such as NE, cat G and C, dipeptidyl peptidase 4 (DPP IV), proteinase 3 (PR3), tryptase, and chymase of mast cells cause COPD pathogenesis (Burster et al., 2021). Uemasu et al. (2020) reported that imbalance of serine protease in small airways causes the development of centrilobular emphysema in COPD. Elevated levels of NSPs (about 20-fold higher than basal) from neutrophils in acute and chronic inflammation overcome antiprotease effects and thus accelerated uncontrolled proteolytic activities and later the lung pathogenesis. Destruction of elastin by NE causes pulmonary emphysema. NE also induces mucin secretion, activates MMPs, and reduces the level of tissue inhibitors of metalloproteinases (TIMPs) (Chakraborti et al., 2017). Peroxisome proliferator-activated receptor gamma (PPARγ) served as a potent inhibitor of transcription of proinflammatory cytokines including IL-8 and thus controlled inflammation and tissue damaging. Studies showed that NE partially degraded PPARγ and increased

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the production of IL-8 in bronchial epithelium to provoke COPD pathogenesis (Kwak et al., 2021). Elevated level of PR3 and Cat G at inflammatory sites has also been associated with proinflammation through stimulation of various mechanisms (Chakraborti et al., 2017). Studies revealed that PR3 degraded ECM proteins (fibronectin, elastin, collagen, vitronectin, and laminin) at their GXXPG site of β-fold conformation. This causes structural damage through ECM degradation and damage connective tissues inside lung interstitium leading to emphysema. PR3 also induces mucus secretion in airways by serous cells of submucosal gland. This causes impaired mucus clearance and promotes chronic bronchitis (Crisford et al., 2018). Cat G is also involved in chemoattraction and ECM disruption (Gao et al., 2018). DPP IV acts as a neutrophil chemorepellent. Reduced serum level of DPP IV was observed in pathogenesis of COPD. Tryptases are of serine proteases type, released by mast cells. Abnormal expression of tryptases has also been observed in smoking-related CLDs. Tryptase activities were found to be 3.4 times higher in severe COPD patients in contrast to mild COPD stage. Chymases are mast cells derived serine proteases that possessed cat G-like specificity. Elevated chymase level (because of excessive mast cell degranulation) leads to ECM degradation, production of active MMPs from zymogens as well as activation of TGF-β/Smad signaling and stimulation of proinflammatory cytokines (IL-1β, IL-18) and thus, promotes COPD. Chymase activity was also observed in PAH, pulmonary fibrosis, and asthma (Dey et al., 2018). Increased uPA activities in epithelial airway and alveolar cells, causing damage of small airways and alveolus of the lung, and thus, were also found to be associated with COPD pathogenesis (Chakraborti et al., 2017).

Serine proteases and viral pathogenesis Influenza is a highly contagious respiratory illness caused by influenza viruses that affects millions of people each year. Out of influenza viruses A, B, and C genera, influenza A and B viruses are the most dangerous ones to the community health (BöttcherFriebertshäuser et al., 2013). Haemagglutinin (HA) protein has been considered as a major virulence factor to cause influenza virus-mediated pathogenicity. It binds to sialic acid of cell surface receptor and separates into HA1 and HA2 subunits by host protease(s). Fusion of HA subunits with cell membrane of host starts the infection process (Chakraborti et al., 2017). Cleavage of HA protein is mediated extracellularly by trypsin, plasmin, and tryptase from mast and bronchiolar epithelial cells and by some bacterial proteases (Bahgat et al., 2011). Although some other proteases particularly serine proteases from lungs have also been involved in the spread of influenza virus (Laporte and Naesens, 2017), TMPRSS2 and human airway trypsin-like proteases mediate HA cleavage of influenza viruses H1N1 (A/Memphis/14/96), H2N9 (A/Mallard/Alberta/205/98), and H3N2 (A/ Texas/6/96). This promotes the higher pathogenicity of such viruses in lungs.

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Granzymes (Gzm) viz. GzmA, B, and E, TMPRSS2 and TMPRSS4 are also identified to be involved in cleavage of 1918 H1N1 HA and leads to the progression of the influenza disease (Chakraborti et al., 2017). Some other serine proteases like urokinase, plasma kallikrein, kallikrein 5, and kallikrein 12 are also known to proteolytically activate influenza virus infectivity (Kido et al., 2019).

Serine proteases and SARS-CoV-2 virus pathogenesis COVID-19 outbreak by infection of SARS-CoV-2 has been the underlying cause of serious respiratory illness. The virus has unique, enveloped RNA and evolutionary resemblance to SARS-CoV; that’s why called as coronavirus 2 (SARS-CoV-2). Disease severity causes serious respiratory infection resulting into death due to permanent alveolar damage and failure of respiratory system (Bansal et al., 2021a, 2022). The major virulence factor of SARS-CoV-2 that causes virus-mediated pathogenicity is attributed to S-glycoprotein (fusion viral protein) having S1 and S2 subunits. S-glycoprotein plays an important role in the entry of virus into host cell after its binding with ACE-2 (angiotensin-converting enzyme-2 receptor) (Bansal et al., 2021b). Entry of virus particle inside host cells needs the cleavage of S protein via host proteases. Cell surface transmembrane proteases have been reported to involve in cleavage of S protein (Rahbar Saadat et al., 2021). The TMPRSS2-mediated SARS-CoV-2 entry was reported with significant changes in ACE2 (Shulla et al., 2011; Murza et al., 2020). After S-glycoprotein binding with ACE2, TMPRSS2 proteolytically degraded S protein into its S1 and S2 subunits (Dessie and Malik, 2021).

Serine proteases and lung cancer Cancer is a cluster of diseases distinguished by the uncontrolled cell growth and spread of abnormal cells (Kumar and Dhanda, 2017). Lung cancer is a major cause of cancerassociated deaths in the whole world due to its late diagnosis and limited treatment interventions. Lung cancer is divided into two main histological groups, that is, non-small cell lung carcinoma and small cell lung carcinoma (Inamura, 2017). Serine proteases represent major protease enzymes involved in tumorigenesis (Tagirasa and Yoo, 2022). Elevated serine proteases level promotes modification of tumor microenvironment by directly cleaving the ECM and activates different growth factors and stimulates proinflammatory cytokines which further induces proliferation and invasion of cancer cells (Murray et al., 2016). Deregulated expression and activity of many secreted and cell membrane-associated serine proteases are known to mediate a series of events relevant to fundamental processes of tumor development and metastasis (Ma et al., 2019). Transmembrane serine protease 2 is considered as a prognostic factor for lung adenocarcinoma (Schneider et al., 2022). Upregulation of both TMPRSS4 and matriptase has been

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observed in lung cancer (Murray et al., 2016). According to Tsai et al. (2014) HAI-2 inhibits prostate cancer cells invasion and metastasis by matriptase regulation. This indicates that activity of matriptase is mainly controlled by HAI-2 expression in human prostate cancer. However, matriptase/inhibitor imbalance led to matriptase activation, and thus increasing cell invasion, tumorigenicity, and metastasis. uPA has also been reported and acts as diagnostic marker in lung cancer (Poddar et al., 2017). Human kallikrein-related peptidases 5 and 7 are also associated with stimulation of malignant processes of lung cancer (Ma et al., 2019). HtrA proteases play a major role in tumor development and progression. Studies have shown that HtrA1 present in normal human organs and tissues has been associated with initiation of tumorigenesis (Wu et al., 2021). Downregulation of HtrA1 was also observed in lung cancers. However, mild HtrA1 expression has been found in tumors of primary stage and lymph node metastasis (Chakraborti et al., 2017).

Conclusion and future prospective Proteases contribute to several physiological functions of human body. These enzymes play a vital role in both health and pathogenesis of the lung. Serine proteases are considered as the largest proteolytic class which is well-characterized and widespread which significantly play different roles in physiological and pathological processes. Serine proteases help to maintain normal homeostatic functions of human body. Enzymes also participate in regeneration and repair of lungs. Experimental evidences suggested the importance of serine proteases and their inhibitors in lung diseases. Dysregulated protease activity and protease/antiprotease imbalance are associated with manifestation of different types of lung infections including pulmonary hypertension, CF, COPD, viral pathogenesis, lung inflammation, and cancer. Chronic inflammatory lung pathologies are increased with elevated serine protease activities. Serine proteases particularly NSPs and transmembrane serine proteases induced inflammatory environment in the lung airways mediated by proinflammatory signaling, chemoattraction, and perpetuating their own proteolytic activities, causing respiratory tissue damage. Serine proteases are considered as promising targets for diagnosis and therapeutic intervention due to their abnormal expression and activities in lung diseases. Antiprotease therapy (either by artificial or mutated endogenous antiproteases) could be considered as a therapeutic option to decrease the inflammatory tissue damage and the treatment of different lung diseases. Investigation of mechanistic insight of proinflammatory and elevated proteolytic events of serine proteases will facilitate the understanding of their roles in pathogenesis of lung infections.

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Rahbar Saadat, Y., Hosseiniyan Khatibi, S. M., Zununi Vahed, S., & Ardalan, M. (2021). Host serine proteases: A potential targeted therapy for COVID-19 and influenza. Frontiers in Molecular Biosciences, 816. Rawlings, N. D., Tolle, D. P., & Barrett, A. J. (2004). Evolutionary families of peptidase inhibitors. Biochemical Journal, 378(3), 705–716. Schneider, M. A., Richtmann, S., Gründing, A. R., Wrenger, S., Welte, T., Meister, M., Kriegsmann, M., Winter, H., Muley, T., & Janciauskiene, S. (2022). Transmembrane serine protease 2 is a prognostic factor for lung adenocarcinoma. International Journal of Oncology, 60(4), 1–12. Shulla, A., Heald-Sargent, T., Subramanya, G., Zhao, J., Perlman, S., & Gallagher, T. (2011). A transmembrane serine protease is linked to the severe acute respiratory syndrome coronavirus receptor and activates virus entry. Journal of Virology, 85(2), 873–882. Solanki, P., Putatunda, C., Kumar, A., Bhatia, R., & Walia, A. (2021). Microbial proteases: Ubiquitous enzymes with innumerable uses. 3 Biotech, 11(10), 1–25. Sumantha, A., Larroche, C., & Pandey, A. (2006). Microbiology and industrial biotechnology of food-grade proteases: A perspective. Journal of Chemical Technology & Biotechnology, 44(2), 211–220. Taggart, C., Mall, M. A., Lalmanach, G., Cataldo, D., Ludwig, A., Janciauskiene, S., Heath, N., Meiners, S., Overall, C. M., Schultz, C., Turk, B., & Borensztajn, K. S. (2017). Protean proteases: At the cutting edge of lung diseases. European Respiratory Journal, 49(2). Tagirasa, R., & Yoo, E. (2022). Role of Serine Proteases at the Tumor-Stroma Interface. Frontiers in Immunology, 13. Taylor, S., Dirir, O., Zamanian, R. T., Rabinovitch, M., & Thompson, A. R. (2018). The role of neutrophils and neutrophil elastase in pulmonary arterial hypertension. Frontiers in Medicine, 5, 217. Tsai, C. H., Teng, C. H., Tu, Y. T., Cheng, T. S., Wu, S. R., Ko, C. J., Shyu, H. Y., Lan, S. W., Huang, H. P., Tzeng, S. F., Johnson, M. D., Lin, C. Y., Hsiao, P. W., & Lee, M. S. (2014). HAI-2 suppresses the invasive growth and metastasis of prostate cancer through regulation of matriptase. Oncogene, 33(38), 4643–4652. Twigg, M. S., Brockbank, S., Lowry, P., FitzGerald, S. P., Taggart, C., & Weldon, S. (2015). The role of serine proteases and antiproteases in the cystic fibrosis lung. Mediators of Inflammation, 2015. Uemasu, K., Tanabe, N., Tanimura, K., Hasegawa, K., Mizutani, T., Hamakawa, Y., Sato, S., Ogawa, E., Thomas, M. J., Ikegami, M., Muro, S., Hirai, T., & Sato, A. (2020). Serine protease imbalance in the small airways and development of centrilobular emphysema in chronic obstructive pulmonary disease. American Journal of Respiratory Cell and Molecular Biology, 63(1), 67–78. Voynow, J. A., & Shinbashi, M. (2021). Neutrophil elastase and chronic lung disease. Biomolecules, 11(8), 1065. Ward, O. P. (2011). Proteases. Comprehensive Biotechnology, 3, 571. Woods, A., Andrian, T., Sharp, G., Bicer, E. M., Vandera, K. K. A., Patel, A., Mudway, I., Dailey, L. A., & Forbes, B. (2020). Development of new in vitro models of lung protease activity for investigating stability of inhaled biological therapies and drug delivery systems. European Journal of Pharmaceutics and Biopharmaceutics, 146, 64–72. Wu, L., Li, X., Li, Z., Cheng, Y., Wu, F., Lv, C., Zhang, W., & Tang, W. (2021). HTRA serine proteases in cancers: A target of interest for cancer therapy. Biomedicine & Pharmacotherapy, 139, 111603.

Biographical sketch Name: Raman Kumar Affiliation: Kurukshetra University, Kurukshetra Education: M.Sc. & Ph.D. Biochemistry

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Research and Professional Experience – –

Experienced in supervision and perform Microbiology laboratory operations (Research Methodologies/Pathogen Testing/Data analysis/Review Data) Worked on metabolic profiling of Pediococcus acidilactici NCDC 252 to explore the beneficial effects of bacterial active components/metabolites and to establish this strain as a useful probiotic strain.

Publications Raman Kumar, Poonam Bansal, Jasbir Singh, Suman Dhanda✶ (2020). Purification, partial structural characterization and health benefits of exopolysaccharides from potential probiotic Pediococcus acidilactici NCDC 252. Process Biochemistry, 99: 79–86. Raman Kumar, Poonam Bansal, Jasbir Singh, Suman Dhanda✶ and Jitender Kumar Bhardwaj (2020). Aggregation, adhesion and efficacy studies of probiotic candidate Pediococcus acidilactici NCDC 252: A strain of dairy origin. World Journal of Microbiology and Biotechnology, 36: 10. Poonam Bansal, Raman Kumar and Suman Dhanda (2022). Characterization of starter cultures and nutritional properties of Pediococcus acidilactici NCDC 252: A potential probiotic of dairy origin. Journal of Food Processing and Preservation, DOI: https://doi.org/10.1111/jfpp.16817.

Semim Akhtar Ahmed, Anuj Kumar Borah, and Jagat C. Borah✶

Chapter 5 Inhibition of the serine exopeptidase DPP-IV as a means of mitigation of diabetic nephropathy Abstract: Diabetic nephropathy (DN) is a serious consequence of diabetes that leads to chronic kidney disease (CKD), end stage renal disease (ESRD), and cardiovascular damage. It is recognized as a major microvascular complication of diabetes which is characterized by increase in urine albumin excretion (albuminuria), glomerular lesions along with tubulointerstitial lesions, and tubular atrophy and decline in glomerular filtration rate (GFR). Current treatment regimen for DN targets regulation of high blood glucose and hypertension. Dipeptidyl peptidase-IV (DPP-IV) inhibitors (gliptins) are a major class of antidiabetic medications which cover almost 50% of the market distribution of the oral hypoglycemic medicines. The serine exopeptidase, DPP-IV or adenosine deaminase complexing protein 2 or CD26, is a form of transmembrane type II glycoprotein, expressed in different cell types including smooth muscles, adipocytes, endothelial cells, renal proximal tubular cells, and podocytes. DPP-IV enzyme rapidly cleaves and inactivates incretins like glucagon-like peptide-1 (GLP-1) and glucosedependent insulinotropic polypeptide (GIP). Incretins, released from enteroendocrine cells, enhance insulin secretion and decrease glucagon levels. DPP-IV inhibitors increase the levels of active GLP-1 and GIP by inhibiting DPP-IV enzymatic activity and improve hyperglycaemic conditions. Since the introduction of sitagliptin in 2006, as the first DPP-IV inhibitors, a number of such gliptins have been in picture including saxagliptin, linagliptin, and alogliptin (US, FDA) and anagliptin, teneligliptin, trelagliptin, and omarigliptin (approved in Japan). Many of these DPP-IV inhibitors have also been in use along with metformin. Unfortunately, these drugs can slow the process but do not prevent disease progression and physical and financial costs associated with the disease. Long-term safety, prognosis, and bioavailability are also questionable while using these synthetic leads. Therefore, there has been a pressing need for

✶

Corresponding author: Jagat C. Borah, Laboratory of Chemical Biology, Life Sciences Division, Institute of Advanced Study in Science and Technology, Guwahati 781035, Assam, India; Academy of Scientific and Innovative Research Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India, e-mail: [email protected] Semim Akhtar Ahmed, Chemical Biology Lab, Institute of Advanced Study in Science and Technology (IASST), Paschim Boragaon, Guwahati 35, Assam, India; Academy of Scientific and Innovative Research Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India Anuj Kumar Borah, Department of Biochemistry, Royal School of Bio-Sciences, The Assam Royal Global University, Betkuchi, NH-37, Guwahati, Assam, India

https://doi.org/10.1515/9783111325040-005

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novel and potent natural therapeutic agents that are effective with fewer or no side effects. Extensive search is going on to evaluate the properties of medicinal plants and their phytocompounds as potent DPP-IV inhibitors. Numerous plant-derived phytocompounds like alkaloids, flavonoids, terpenoids, phenols, and glycosides are reported to be inhibitors of DPP-IV. Berberine, resveratrol, luteolin, quercetin, coumarin, galangin, apigenin, naringenin, and so on are proved to be highly effective DPP-IV inhibitors. This chapter highlights latest research on the role of bioactive compounds or natural plant derived products as alternative to available synthetic DPP-IV inhibitors. The article also summarizes the important molecular mechanisms involved to expand this research area for potential draggability. Keywords: diabetic nephropathy, DPP-IV inhibitors, serine exopeptidase, phytocompounds

Introduction Diabetic nephropathy (DN) is one of the main consequences of diabetes, being firmly related with increased mortality rate, cardiovascular damage, and end-stage renal disorders (Tuttle et al., 2014). The major and earliest clinical sign of DN is albuminuria which is mainly caused by glomerular leakage, a key marker of kidney damage. Along with albuminuria, DN is characterized by increase in glomerular lesions along with tubulointerstitial lesions and tubular atrophy and decline in glomerular filtration rate (GFR). Reduction of albuminuria and GFR should be considered together as significant markers for the mitigation of DN (Sulaiman, 2019). Dipeptidyl peptidase-IV (DPP-IV) inhibitors are novel avenues for the management of diabetes and its related complications. DPP-IV inhibitors control hyperglycemic levels including Favourable effects on body weight, maintain glycated hemoglobin levels, and do not cause hypoglycemia (Green et al., 2006; Salvatore et al., 2009). DPP-IV, which is a type of serine exopeptidase, was first explored by Hopsu-Havu and Glenner in 1966 (Hopsu-Havu & Glenner, 1966). The said group reported that this new enzyme can hydrolyze a synthesized chromogenic substrate, glycyl-prolyl-βnaphthylamide in liver and kidney of rat (Hopsu-Havu & Glenner, 1966). The biological activity of DPP-IV is mainly accredited to its protease actions which can degrade numerous substrates acting as cell surface coreceptor with different biological activities such as growth factors, vasoactive peptides, chemokines, and neuropeptides and also interact with extracellular matrix elements like collagen and fibronectin (Green et al., 2006; Lin et al., 2019). DPP-IV as a therapeutic goal for treatment of type-2-diabetes was brought into the focus after the approval of the DPP-IV inhibitors (Mulvihill & Drucker, 2014). DPP-IV is regarded as a type of new adipokine and a pleiotropic enzyme that is accountable for the circulating DPP-IV activity in the plasma (Akoumianakis & Antoniades, 2017). DPP-IV inhibitors help to protect glucagon-like peptide-1 (GLP-1), gastric inhibitory peptide (GIP), gut hormonal degradation which reduce postprandial glucagon secretion,

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Release of active GLP-1 & GIP in response to an increased glucose concentration in the digestive tract lumen GLP-1 GIP GLP-1

GLP-1 GIP

+

DPP-IV

maintain satiety, and slow down gastric emptying. Therefore, the DPP-IV inhibitorbased medications regulate actions of GLP-1 and GIP which could play an important role in metabolism (Figure 1) (Salvatore et al., 2009; Singh et al., 2017). DPP-IV rapidly degrades incretins GLP-1

GIP

GLP-1 GLP-1 GIP

+

DPP-IV

GI tract

Increase blood glucose level

GLP-1 GIP

Food intake

GIP

DPP-IV inhibitor blocks incretin degradation

GIP

GLP-1 GLP-1

GIP

Incretin stimulates insulin secretion and lower blood glucose levels

GIP

DPP-IV inhibitor

Figure 1: Schematic mechanism of action of DPP-IV inhibitors in diabetes and diabetic nephropathy.

DPP-IV inhibitors are considered as the second-line therapy after the metformin treatment fails as they have no integral hypoglycemic risk and body weight also remains neutral. During hyperglycemic conditions, they also help in reducing glucagon secretion (Kumar, 2012). Fixed dose combinations of DPP-IV inhibitors with metformin are widely used in diabetic patients with no pre-existing cardiovascular disorder. DPP-IV inhibitors are also commonly used in DN treatment and related renal problems because of their efficacy and safety profile in patients (Ahren, 2008). Besides, DPP-IV inhibitors can also be administered in combination with either sodium-glucose cotransporter-2 (SGLT-2) inhibitor and metformin or insulin and metformin (Gallwitz, 2019).

Structure and functions of DPP-IV DPP-IV is an 88 kDa serine exopeptidase, also known as adenosine deaminase complexing protein 2. It contains a cytoplasmic domain (1–6 amino acids) together with transmembrane domain (7–28 amino acids) and an extracellular domain (29–766 amino acids) coupled with the main catalytic unit (Lin et al., 2019). It acts as type II transmembrane serine protease, with a catalytic triad of serine, histidine, and aspartic acid, which are located in the C-terminal region within the last 140 residues (Aertgeerts et al., 2004). DPP-IV is expressed in the surfaces of many cells and has been associated with signal transduction, immune regulation, and apoptosis. Human DPP-IV is a bound neuraminic acid which consists of two identical subunits of glycoprotein (MW 120,000 Da).

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DPP-IV plays multifunctional role in highly expressed liver, kidney, placenta, intestine, endothelial cells, and lymphocytes. It belongs to αβ-hydrolyze domain and is an anchored serine (Aertgeerts et al., 2004; Singh et al., 2017). DPP-IV exists in two forms: the soluble form (sDPP-IV) and the plasma membrane-anchored form (mDPP-IV) (Lin et al., 2019). sDPP-IV secreted from lymphocytes circulates in the blood and found in higher amount in kidney. sDPP-IV performs multiple roles in regulating homeostasis, muscle activity, chemotaxis, and immunocyte activation (Casrouge et al., 2018; Hasan & Hocher, 2017). It increased arteriolar diameter of skeletal muscle by reducing vasoconstriction and also functions as myokine by stimulating smooth muscles inflammation from blood vessel via acting on protease-activated receptor 2 (PAR2)/ERK/NF-κB signaling cascade to promote smooth muscle cell proliferation (Neidert et al., 2018; Wronkowitz et al., 2014). However, Romacho et al. (2016) reported that sDPP-IV-induced smooth muscle inflammation might cause microvascular endothelial dysfunction, which ultimately leads to chronic kidney disease. sDPP-IV levels in the serum or other body fluids can be monitored as a biomarker of different disorders such as chronic obstructive pulmonary disease progression, and prognosis of malignant pleural mesothelioma (Fujimoto et al., 2014; Somborac-Bačura et al., 2012), whereas the mDPP-IV is mainly found in the kidney, gastrointestinal tract, T lymphocytes, and reproductive organs. mDPP-IV, also known as CD26, is a type of T-cell receptor that responds to antigen-presenting cells and its main biological roles include the regulation of blood vessel activity and immune response via acting on both epithelial and endothelial cells. It also acts as an important target in the treatment of autoimmune diseases, transplantation, and hypersensitivity (Lin et al., 2019; Mulvihill & Drucker, 2014; Uhlén et al., 2015; Wang et al., 2018). DPP-IV enzyme particularly separates dipeptides from the N-terminal segment of peptide substrates having a proline or alanine residues in the penultimate region which consists of average 30 residues. In addition, a slow release of the dipeptides with residues X-Gly or X-Ser has been observed (Bongers et al., 1992; Hinke et al., 2000; Lambeir et al., 2002). DPP-IV was also known to recognize physiological peptides containing the specificity conformation at their cleavage site such as neuropeptide Y, paracrine chemokines like RANTES, circulating peptide hormones like peptide YY, GLP-1, and -2, GIP (Aertgeerts et al., 2004; Mentlein et al., 1993). Several studies showed that substrate selectivity of DPP-IV is mainly determined by the residues surrounding the scissile bond. However, it is also reported that nonconserved residues throughout the peptide also play a function in substrate binding and catalysis in long-range interactions (Zhu et al., 2003).

Role of DPP-IV inhibitors in diabetic nephropathy DPP-IV is a new type of adipokine usually gets secreted from adipose tissue that has been considered as a major link between metabolic syndrome and insulin resistance

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as it maintained endocrine function during obesity. It gets expressed in brush border layer of human kidney proximal tubules, epithelial layer of descending limb of Henle’s loop, and glomerular podocytes (Bae, 2016; Lamers et al., 2011). As reported, diabetes induced by streptozotocin (STZ) and high fat diet resulted in upregulation of DPP-IV expression in the renal tubular cells of rat kidneys (Yang et al., 2007) whereas immunofluorescence staining study showed the occurrence of DPP-IV in the glomerular podocytes of DN patients, but it is absent in healthy kidneys (Sharkovska et al., 2014). DPP-IV inhibitors are known to perform cell protective functions against numerous diabetic complications affecting organs such as heart, kidneys, retina, liver, and neurons. DPP-IV inhibitors were examined in various experimental models of acute and chronic kidney damage. Sitagliptin was the first approved DPP-IV inhibitor, examined against Zucker diabetic fatty (ZDF) rat DN model to determine its role on metabolic functioning and kidney damage (Mega et al., 2011). The results showed that sitagliptin administration improved kidney injury and decreased renal oxidative damage. Liu et al. (2012) demonstrated that vildagliptin could decrease kidney damage by improving histological parameters, albuminuria, and creatinine levels in diabetic rats induced with STZ. They reported that vildagliptin resulted in reno-protection through GLP-1 receptor activation and cyclic adenosine monophosphate (cAMP) modulation guided decrease in oxidative damage and transforming growth factor beta 1 (TGFβ1) expression. However, Sharkovska et al. (2014) found that in a type 2 diabetic db/db mice, Linagliptin improved diabetic kidney injury without any effect on blood pressure and glucose homeostasis. Linagliptin also decreased advanced glycation end products (AGEs) levels and RAGE receptor expression, 8-hydroxy-2′-deoxyguanosine, lymphocyte infiltration, and mRNA levels of intercellular adhesion molecule-1 (ICAM-1) and a proinflammatory peptide expression in the renal tissue of STZ-induced diabetic rats. Linagliptin inhibited endothelial-to-mesenchymal transition induced by TGFβ2 to improve the renal fibrosis by abolishing DPP-IV and integrin beta 1 levels. Linagliptin administered DPP-IV inhibition also lowered the DN condition in CD-1 diabetic mice by ameliorating urinary albumin-to-creatinine ratio, plasma cystatin C, and histopathological profile of the renal tissue (Kanasaki et al., 2014; Shi et al., 2015). Furthermore, another DPP-IV inhibitor, gemigliptin, normalized oxidative stress, mesangial expansion, albuminuria, and podocyte cell death in the kidneys of diabetic mice (Jung et al., 2015; Moon et al., 2016). Furthermore, a new study demonstrated that Linagliptin also reduced albuminuria in Akita mice (Glp1r+/+ diabetic-prone) but not in Akita mice (Glp1r‒/‒– diabetic-prone); nevertheless, Linagliptin also improved the kidney histopathology in both type of mice. These results conferred that DPP-IV inhibition might play kidney-protective functions via glucagon-like peptide-1 receptor (GLP-1R) and also through various substrates like stromal derived factor-1 alpha (Takashima et al., 2016). These studies suggested that DPP-IV inhibition has a lot of promising targets that might be engaged in the DN pathogenesis.

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Synthetic DPP-IV inhibitors Inhibitors of DPP-IV are a type of incretin-based oral antihyperglycaemic drugs for the treatment of diabetes which have been in use for a long time. DPP-IV inhibitors do not result in hypoglycaemia and can be used during renal complications with dose regulation (Green et al., 2006). Sitagliptin was the first class of DPP-IV inhibitor to become accessible for clinical use in 2006 which is also used together with the SGLT-2 inhibitors and metformin for blood glucose control. Sitagliptin was followed by the introduction of structurally similar vildagliptin (Green et al., 2006). Other types of DPP-IV inhibitors, such as vildagliptin, litagliptin, and saxagliptin were approved by US FDA to control glucose level along with beneficial effects in diabetic patients with renal and cardiovascular risk (Singh et al., 2017). Gemigliptin was approved in Korea whereas anagliptin and teneligliptin were approved in Japan in the year 2012 (Sharma et al., 2016; Watanabe et al., 2015). Trelagliptin and omarigliptin are another two DPPIV inhibitors approved in Japan in 2015 (Suzuki et al., 2018). Alogliptin was approved by FDA in 2013 followed by evogliptin and gosogliptin approval in South Korea and Russia, respectively (Rameshrad et al., 2019). The patients taking sulfonylurea for treatment together with DPP-IV drug, an increased risk of low blood sugar has been speculated. Safety and efficacy of DPP-IV inhibitors have been favorable and advantageous in relation to other antidiabetic agents and they do not have an intrinsic possibility of hypoglycemia and bodyweight loss. But some adverse effects like headache, nausea, heart failure, hypersensitivity, and nasopharyngitis, skin reactions can be seen in some patients (Salvo et al., 2016). The FDA also issued warning for DPP-IV inhibitors approved by them which may cause joint pain and heart failure (Mascolo et al., 2016). A study in 2018 reported developing of inflammatory bowel disease (such as ulcerative colitis) that reaches to the peak level after about three to four years of use followed by decreased incidence after more than four years of use (Zheng et al., 2018). However, the reliability and durable safety profile of DPP-IV inhibitors are yet to be validated. Alternatively, DPP-IV inhibitors based on phytocompounds are safer to use as compared to synthetic drugs. Several novel antidiabetic agents and compounds are coming up in clinical progression by utilizing the mechanism of DPP-IV inhibition.

Phytocompounds as DPP-IV inhibitors Phytocompounds due to their greater benefit to risk ratio, cost-effectiveness, and multifaceted-effects have been largely investigated as potential natural alternatives to synthetic ones which are comparatively hazardous (synthetic inhibitors of DPP-IV). Cinnamomum verum J. Presl. (Sali et al., 2018), Commiphora mukul (Hook. ex Stocks) Engl. (Borde et al., 2016), Allium sativum L. (Kalhotra et al., 2020), Phyllanthus emblica

Chapter 5 Inhibition of the serine exopeptidase DPP-IV

Figure 2: Structures of some plant-derived molecules with reported use in inhibition of DPP-IV.

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L. (Borde et al., 2016), Syzygium cumini (L.) Skeels (Kosaraju et al., 2014), Hibiscus sabdariffa L. (Peng et al., 2014), Momordica charantia L. (Baek et al., 2018), Rosmarinus officinalis L. (Bower et al., 2014), etc. are some of the plants whose preparations/compounds have been reported to harbor DPP-IV inhibitory activities. Phytopreparation of Chinese rhubarb, Ginger, Arjun tree, and Grapes has demonstrated DPP-IV inhibitory action in animal models as well (Borah et al., 2022). Investigations focusing on herb-drug interaction, toxicological assessment, in vivo effectiveness, and bioavailability are yet to be made with majority of these phytopreparations. Identification of the bioactive principle of these profound DPP-IV inhibitory herbal preparations is also a goal yet to be achieved. Nonetheless, a vast array of phytocompounds have been investigated for their DPP-IV inhibitory efficacy in in silico screening methods and direct screening methods such as in vitro cell-based assay, enzyme assay, and in vivo animal models (Figure 2) (Lin et al., 2019). Extensive literature reports that 6-gingerol, apigenin, magniferin, berberine, curcumin, emodin, and naringenin are some of the phytochemicals having demonstrated potent in vivo DPP-IV inhibitory effect in various animal models. On the other hand, DPP-IV inhibitory activity of natural plantderived compounds like calebin A, epigallocatechin-3-gallate, chrysin, lupeol, kaempferol, rugosin A and rugosin B, and resveratrol have been reported in in vitro studies. Molecular docking studies indicate that luteolin, calenduloside E, and α-bergamotene can be the drug leads of future, once in vitro and in vivo studies are made suitable and appropriate models (Table 1). Table 1: Potential of different phytocompounds in DPP-IV inhibition. Phytocompound name

Remarks/findings

Level of investigation

-Gingerol Type of compound: Phenol Source: Zingiber officinale

Resulted in inhibition of plasma DPP-IV in Type  diabetic mice, Leprdb/db (Samad et al., ).

In vivo

Apigenin Type of compound: Flavone Source: Chamomilla matricaria L., Apium graveolens, Cynara cardunculus, Spinacia oleracea, etc.

Lowered DPP-IV enzyme activity in plasma In vivo and hippocampal homogenate of albino Wister rats treated/untreated with high fat diet (HFD) (Jagan et al., ).

Berberine Type of compound: Alkaloid Sources: Plants belonging to the Berberis genus

In vitro and In silico studies demonstrated that in vivo berberine can inhibit DPP-IV in vitro (Mohanty & Suman, ) with IC values of . µM), and in vivo in STZ-induced diabetic rat model (Wang et al., ).

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Table 1 (continued) Phytocompound name

Remarks/findings

Level of investigation

Calebin A Type of compound: Phenol Source: Curcuma longa

Calebin A induced a dose-dependent inhibition of DPP-IV activity in vitro (Oliveira et al., ). Calebin A could interact with catalytic site of DPP-IV and further inhibited in vitro DPP-IV enzymatic activity (Chalichem et al., ).

In silico and in vitro studies

Calenduloside E Type of compound: Triterpenoid saponin Source: Aralia elata, Alternanthera

Binds active site of DPP-IV (Kalhotra et al., ).

Molecular docking studies

Chrysin Type of compound: Flavone Sources: Passiflora caerulea, Passiflora incarnata, Oroxylum indicum

Chrysin inhibited DPP-IV in a doseIn vitro dependent manner in vitro (Kalhotra et al., ).

Coumarins Type of compound: Coumarin Sources: Dipteryx odorata, Melilotus officinalis, Cinnamomum verum, Prunus armeniaca, Hierochloe odorata

Coumarin binds active site of DPP-IV (docking) and inhibits DPP-IV (with IC value of . nmol/mL; Singh et al., ).

Molecular docking studies and in vitro

Curcumin Type of compound: Phenol Sources: Curcuma longa

Curcumin exerted dose-dependent and time-dependent DPP-IV inhibitory activity (Huang et al., ); it repressed DPP-IV activity in high fat, high sugar diet (HFSD) fed mice (Cao et al., ).

In vitro and in vivo

Ellagic acid Type of compound: Polyphenol Sources: Punica granatum, Rubus sp., Fragaria ananassa, Juglans regia

Ellagic acid interacted with DPP-IV and inhibited DPP-IV activity in vitro (with IC value of  µM) (Mohanty et al., ).

In silico and in vitro studies

Emodin Type of compound: Anthraquinone Source: Acalypha australis, Polygonum cuspidatum, Kalimeris indica, Frangula alnus, Cassia occidentalis

Emodin interacted with glutamic acid residue of DPP-IV; it inhibited DPP-IV in vitro with IC value of . µM. In in vivo experiment, emodin lowered DPPIV activity in both Ob/Ob (‒/‒) mice and Balb/c mice in a dose-dependent manner (Wang et al., ).

Molecular docking, in vitro, and in vivo studies

Ephedrine Type of compound: Alkaloids Source: Ephedra aurantiaca, Ephedra alata, Ephedra americana, Ephedra altissima

Ephedrine and its derivative inhibited DPP-IV in computational studies and in in vitro experiments with an IC value ranging from  µM to  mM (OjedaMontes et al., ).

In vitro

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Semim Akhtar Ahmed, Anuj Kumar Borah, and Jagat C. Borah

Table 1 (continued) Phytocompound name

Remarks/findings

Level of investigation

Epigallocatechin--gallate Type of compound: Polyphenol Sources: Camellia sinensis, Vaccinium sp., Fragaria ananassa, Actinidia deliciosa

Epigallocatechin--gallate acid interacted with DPP-IV and inhibited DPP-IV activity in vitro (with IC value of . µM) (Hou et al., )

In silico and in vitro studies

Galangin Type of compound: Flavonol Sources: Alpinia galanga, Alpinia officinarum, Helichrysum aureonitens

Galangin showed inhibition potential against DPP-IV in docking studies and in vitro model in a dose-dependent manner (Kalhotra et al., ).

Molecular docking studies and in vitro

Gallic acid Type of compound: Phenolic acid Sources: Sumac, oak bark, tea leaves, witch hazel, gallnuts

Gallic acid interacted with DPP-IV and inhibited DPP-IV activity in vitro (with IC value of . µM) (Mohanty et al., ).

In silico and in vitro studies

Kaempferol Type of compound: Flavonol Sources: Tilia spp., Sophora japonica, Equisetum spp., Ginkgo biloba, Moringa oleifera, genus Berberis, Delphinium, Citrus, Camellia, etc.

Kaempferol interacted and inhibited DPPIV activity in vitro (with IC value of . ± . µM) (Fan et al., ).

In silico and in vitro studies

Lupeol Type of compound: Triterpenoid Sources: Mangifera indica, Camellia japonica, Acacia visco, Abronia villosa

Lupeol inhibited DPP-IV in vitro (IC value of . µM) (Saleem et al., ).

In vitro

Luteolin Type of compound: Flavone Source: Apium graveolens, Brassica oleracea, Cynara cardunculus, Petroselinum crispum

Luteolin interacts and binds with active site of DPP-IV (Davella & Mamidala, ; Fan et al., ) and inhibited DPP-IV activity in vitro (Fan et al., ).

Molecular docking studies

Mangiferin Type of compound: Xanthonoid Source: Mangifera indica, Iris unguicularis, Bombax ceiba leaves, Anemarrhena asphodeloides

Mangiferin-mediated in vitro DPP-IV In vitro and in vivo inhibition was comparable to that of sitagliptin and vildagliptin; mangiferin also inhibited serum DPP-IV activity in high fat diet-induced diabetic rats (Suman et al., ).

Myricetin Type of compound: Flavone Source: Camellia sinensis, Syzygium cumini

Myricetin inhibited DPP-IV in vitro and in vivo in HFD + STZ-induced diabetic mice (Lalitha et al., ).

In vitro and in vivo

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Table 1 (continued) Phytocompound name

Remarks/findings

Level of investigation

Naringenin Type of compound: Flavanone Source: Citrus species, grapefruits, tomatoes, grapes, Smyrna figs

Naringenin interacted with DPP-IV (docking study); inhibited DPP-IV (enzyme assay) more efficiently than sitagliptin; lowered DPP-IV activity in serum sample of mice (Fan et al., ; Parmar et al., ).

In silico studies, in vitro, and in vivo studies

Quercetin Type of compound: Flavonoid Sources: Sambucus canadensis, Ginkgo biloba, Camellia sinensis, Hypericum perforatum

Quercetin actively bound to DPP-IV (docking study) and inhibited DPP-IV activity in vitro (with IC value of . nmol/mL; Singh et al., ).

Molecular docking studies and in vitro

Resveratrol Type of compound: Polyphenol Sources: Veratrum grandiflorum, Polygonum cuspidatum, plants in Paeoniaceae, Dipterocarpaceae, Leguminosae, Polygonaceae, Poaceae, Vitaceae, Gnetaceae, Cyperaceae, Gramineae families

Resveratrol bound at the active site of DPP-IV and inhibited DPP-IV activity in vitro (with IC value of . ± . µM) (Fan et al., ).

In silico and in vitro studies

Rugosin A and Rugosin B Type of compound: Tannins Sources: Quercus suber, Coriaria japonica, Rosa gallica

DPP-IV inhibitory activity of Rugosin A and In vitro Rugosin B was dose-dependent and were comparable to that of diprotein (Kato et al., ).

Rutin Type of compound: Flavonoid Source: Passiflora incarnata, Camellia sinensis, Ruta graveolens

Dose dependent inhibitory effect of rutin in STC- cells (Lee et al., ).

In vitro

α-Bergamotene Type of compound: Sesquiterpene Source: Daucus carota, bergamot, kumquat, cottonseed, Citrus sp.

Inhibited DPP-IV in molecular docking studies (Sali et al., ).

Molecular docking studies

Conclusion and future prospective Currently, DPP-IV is considered as one of the most promising therapeutic targets to treat diabetes and diabetic nephropathy. A large number of DPP-IV inhibitors already approved were found to be well tolerated, with a low incidence of adverse effects, such as hypoglycaemia, weight gain, and gastrointestinal complications. Furthermore,

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DPP-IV inhibitors also enhanced the glycemic control either as monotherapy or as a combination therapy with other antidiabetic drugs. However, long-term clinical studies about the adverse effects of DPP-IV inhibitors are limited in literature. It is also not clear how DPP-IV inhibitors play a role in the prediabetic condition and in the progression of diabetes. Therefore, further extensive research will be required to understand DPP-IV as an important antidiabetic and nephroprotective target. Moreover, it is also evident that most of the potent DPP-IV inhibitors from natural sources have greater activity in nanomolar range. Standard drugs like Saxagliptin, sitagliptin, and vildagliptin have complex molecular structures as compared to the natural phenolic and flavone compounds. Flavonoids and phenolic compounds also have their additive benefits as they are present in a number of foods and possess antioxidant characteristics. So, the scientists working on the new chemical entity tracking for the DN treatment can also explore in depth about these natural plant-derived compounds for lead generation.

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Biographical sketch Name: Dr. Jagat C Borah Affiliation: Institute of Advanced Study in Science and Technology (An Autonomous Institute under DST, Govt. of India) Paschim Boragaon, Garchuk, Guwahati-35, Assam, India

Education – – – –

Postdoc from Icahn School of Medicine at Mount Sinai, New York City, USA (2006–2008) Ph.D. from CSIR NEIST Jothat (Total Synthesis of Bioactive Natural Products), (2006) M.Sc. (Organic Chemistry) from Dibrugarh University, Assam (2001) B.Sc. (Chemistry Hons.) from Govt. Science College, Jorhat, Assam (1998)

Research and Professional Experience – – –

Associate Professor, IASST Guwahati (October 2018 to till date) Principal Scientist, CSIR-NEIST Jorhat, Assam (March 2016 to October 2018) Scientist-C, Institute of Bioresources and Sustainable Development, Takyelpat, Imphal, Manipur (October 2012 – March 2016)

Publications (last three years) 1. Bhaswati Kashyap, Kangkon Saikia, Suman Kumar Samanta, Debajit Thakur, Sanjay Kumar Banerjee, Jagat Chandra Borah, Narayan Chandra Talukdar (2022). Kaempferol 3-O-rutinoside from Antidesma acidum Retz. stimulates glucose uptake through SIRT1 overexpression followed by GLUT4 translocation in skeletal muscle L6 cells. Journal of Ethnopharmacology, 301, 115788. https://doi.org/10.1016/j.jep.2022. 115788 2. Pranamika Sarma, Simanta Bharadwaj, Deepsikha Swargiary, Semim Akhtar Ahmed, Yunus Sheikh, Sagar Ramrao Barge, Prasenjit Manna, Narayan Chandra Talukdar, Jayanta Bora, Jagat Chandra Borah (2022). Iridoid glycoside isolated from Wendlandia glabrata and the role of its enriched fraction in regulating AMPK/PEPCK/G6Pase signaling pathway of hepatic gluconeogenesis. New J Chem. 46, 13167–13177 https://doi.org/10.1039/D1NJ05856H. 3. Simanta Bharadwaj, Gurumayum Shalini Devi, Pranamika Sarma, Barsha Deka, Sagar Ramrao Barge, Bhaswati Kashyap, Prasenjit Manna, Jagat C. Borah, Narayan Chandra Talukdar (2022). Prophylactic role

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of Premna herbacea, a dietary leafy vegetable in managing hepatic steatosis via regulating AMPK/SREBP1/ ACC/HMGCR signaling pathway. Food Bioscience. 101720, https://doi.org/10.1016/j.fbio.2022.101720. 4. Barsha Deka, Prasenjit Manna, Jagat Chandra Borah, Narayan Chandra Talukdar (2022). A review on phytochemical, pharmacological attributes and therapeutic uses of Allium hookeri. Phytomedicine Plus, 100262. https://doi.org/10.1016/j.phyplu.2022.100262 5. AK Borah, Semim A Ahmed and JC Borah (2022). Phytomedicine as a source of SGLT2 inhibitors, GLP-1 secretagogues and DPP-IV inhibitors for mitigation of Diabetic Nephropathy. Phytomedicine Plus, 100225. https://doi.org/10.1016/j.phyplu.2022.100225 6. Sagar Bargea,b, Dhananjay Jadec, Selvaraj Ayyamperumalc, Prasenjit Mannaa, Jagat Boraha, Chandrasekar Moola Joghee Nanjanc, Moola Joghee Nanjand and Narayan Chandra Talukdara (2021). Potential inhibitors for FKBP51: an in silico study using virtual screening, molecular docking and molecular dynamics simulation. Journal of Biomolecular Structure and Dynamics, https://doi.org/10.1080/07391102.2021.1994877 7. Sagar Barge, Barsha Deka, Bhaswati Kashyap, Simanta Bharadwaj, Raghuram Kandimalla, Aparajita Ghosh, Partha Pratim Dutta, Suman Kumar Samanta, Prasenjit Manna, Jagat C. Borah✶, Narayan Chandra Talukdar✶ (2021). Astragalin mediates the pharmacological effects of Lysimachia candida Lindl on 2 adipogenesis via downregulating PPARG and FKBP51 signaling cascade. Phytotherapy Research 2021, 1–14. 8. Bhaswati Kashyap, Sagar Ramrao Barge, Simanta Bharadwaj, Barsha Deka, Seydur Rahman, Aparajita Ghosh, Prasenjit Manna, Partha Pratim Dutta, Yunus Sheikh, Raghuram Kandimalla, Suman Kumar Samanta, Joshodeep Boruwa, Shilpi Saikia, Deepsikha Swargiary, Parul Kamboj, Deepika Tuli, Uttam Pal, Jagat C. Borah✶, Sanjay Kumar Banerjee✶, Narayan Chandra Talukdar✶ (2021). Evaluation of therapeutic effect of Premna herbacea in diabetic rat and isoverbascoside against insulin resistance in L6 muscle cells through bioenergetics and stimulation of JNK and AKT/mTOR signaling cascade. Phytomedicine 93, 153761. 9. Anuj Kumar Borah, Pranamika Sharma, Archana Singh, Kangkan Jyoti Kalita, Sougata Saha, and Jagat Chandra Borah✶ (2021). Adipose and non-adipose perspectives of plant derived natural compounds for mitigation of obesity. Journal of Ethnopharmacology. 280, 114410. 10. Sagar Barge, Dhananjay Jade, Gokul Gosavi, Narayan C Talukdar, Jagat C Borah✶ (2021). In-silico screening for identification of potential inhibitors against SARS-CoV-2 transmembrane serine protease 2 (TMPRSS2). European Journal of Pharmaceutical Sciences, 162, 105820. 11. Barsha Deka, Sagar Barge, Simanta Bharadwaj, Bhaswati Kashyap, Prasenjit Manna, Jagat C Borah, Narayan Chandra Talukdar (2021). Beneficial effect of the methanolic leaf extract of Allium hookeri on stimulating glutathione biosynthesis and preventing impaired glucose metabolism in type 2 diabetes. Archives of Biochemistry and Biophysics, 708, 108961. 12. Parul Kamboj, Soumalya Sarkar, Sonu Kumar Gupta, Neema Bisht, Deepika Kumari, Sagar Barge, Bhaswati Kashyap, Barsha Deka, Simanta Bharadwaj, Seydur Rahman, Partha Pratim Dutta, Jagat C Borah, Narayan Chandra Talukdar, Sanjay Kumar Banerjee, Yashwant Kumar (2021). Methanolic extract of Lysimachia candida Lindl. prevents high-fat-high-fructose-induced fatty liver in rats: Understanding the molecular mechanism through untargeted metabolomics study. Frontiers in Pharmacology, 12, Article 653872.

Preeti Chanalia✶, Poonam Bansal, and Dimpi Gandhi

Chapter 6 Serine proteases in arthritis Abstract: Serine proteases along with other metalloproteinases constitute about twothird of all proteinases. They are typically extracellular proteinases. Some of the important physiological functions played by serine proteinases include wound healing, digestion, blood coagulation, and fertility. Despite these functions, serine proteases are also active in many pathological as well as physiological processes within the bone tissue. Although they are essential for the adequate maintenance of bone and cartilage their inappropriate expression can lead to exacerbation of inflammation as well as destruction of bone and cartilage tissue. There are multiple pathways through which serine proteinases exert their effects like interactions with signaling molecules such as transforming growth factor β (TGFβ), binding to protease-activated receptors (PARs), and direct proteolysis of extracellular matrix (ECM) proteins. In certain cases, they also work synergistically with matrix metalloproteases for remodeling the bone tissues. The effective results of such interactions are not yet clear, but it has been found that there exists some important relationship between certain serine proteases and arthropathies as well as metastatic bone invasion. In arthritis the proteolysis of cartilage is predominantly driven by metalloproteinase but serine proteinases also perform certain crucial functions including direct ECM degradation, proMMP matrix metalloproteinase activation, receptor activation, and cytokine regulation. By understanding the crucial function and contribution of each serine proteinases in the disease process, the effective treatment based on inhibitors or agonists can be developed. Serine protease inhibitors have been shown to act as promising factors in reducing the severity of arthritis; however, a greater specificity is essentially required to avoid any undesired systemic effects. Further improvement in the understanding of this enzyme class will be helpful in the identification of more novel pharmacological targets that can be used to prevent the destruction of cartilage in arthritic patients. Keywords: serine proteases, protease-activated receptors, inflammation, metalloproteinase, transforming growth factor β

✶

Corresponding author: Preeti Chanalia, Research and Innovation Department, Shri Krishna AYUSH University Kurukshetra, Haryana, India, e-mail: [email protected] Poonam Bansal, Department of Biosciences and Technology, Maharishi Markandeshwar (Deemed to be University), Mullana, Ambala, 133207, Haryana, India Dimpi Gandhi, Research Associate I, National Brain Research Centre, Gurgaon, Haryana, India https://doi.org/10.1515/9783111325040-006

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Introduction Serine proteases (SPs) exhibit significant roles in extracellular proteolysis. The catalytic triad consists of serine, aspartate, and histidine and they are optimally active at neutral pH. Their major roles are in blood coagulation, digestion, fertility, and wound healing. These proteases have the potential to cleave broad spectrum of proteins including extracellular matrix (ECM), proinflammatory mediators, growth factors, surface receptors, and bacterial cell-wall proteins as evident from several studies in literature. Osteoarthritis (OA) and rheumatoid arthritis (RA) are most prevalent as well as most studied among arthritis. Both these diseases are clinically different but both lead to the destruction of articular cartilage as well as exposure of the related bone. Cartilage consists of ECM that is devoid of vascularization and capable of harboring only chondrocytes. In arthritis, the proteolytic degradation of cartilage is mainly derived by metalloproteinases but serine proteinases play some significantly essential functions such as direct degradation of ECM, activation of proMMP (matrix metalloproteinase), regulation of cytokines, and activation of receptor. In this chapter the importance and functions of serine proteinases in different systems viz. complement system, plasminogen-plasmin system, immune cells, proprotein convertases, type II transmembrane serine proteinases (TTSP), and high temperature requirement proteinases have been discussed. Furthermore, the functions of serine proteinases as well as proteinase-activated receptors (PARs) in the breakdown of cartilage in arthritis are also discussed (Wilkinson et al., 2017b).

Cartilage ECM changes in arthritis due to serine proteases Serine proteinases from immune cells The major role in inflammatory arthropathies is played by immune cell infiltration. Different types of SP from leukocytes are known for their contribution toward the disease. The SP of neutrophils (NSPs) are proteinase-3 (PR3), cathepsin G (CTSG), and neutrophil elastase (NE). These proteases are kept stored in granules of polymorphonuclear leukocytes viz. basophils, neutrophils, and eosinophils. These enzymes are released during degranulation. In inflammatory arthropathies, the roles of NSPs have been well described, like significantly elevated NE levels have been reported in several studies (Huet et al., 1992; Elsaid et al., 2003). During the progression of RA, NSPs are involved in direct ECM degradation and they also have broad substrate specificity. For example, in a study of RA in a mice model degranulation of neutrophils resulted in degradation of cartilage and inhibition of NE had effectively reduced articular cartilage destruction (Janusz and Durham, 1997). In some other study involvement of NE in the degradation of tissue inhibitor of metalloproteinase (TIMP)1 has also been reported, which further

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marks the possibility of interactions of serine and metalloproteinase activities (Nagase et al., 1997). Granzymes are also a type of serine proteinases that are stored in the natural killer cell granules of cytotoxic T-lymphocytes. They are responsible for inducing apoptosis in target cells. Granzyme A and granzyme B both have been detected in synovial fluids in case of RA. The levels of these granzymes have been found associated with joint erosion in case of patients with early RA and these proteinases are capable of inducing direct degradation of components of cartilage, for example, aggrecan (Ronday et al., 2001; Froelich et al., 1993). These proteinases are expressed by leukocytes as well as by chondrocytes (Horiuchi et al., 2003). A study on collagen-induced arthritis (CIA) model depicts that a mouse, deficient with granzyme A exhibited reduction in bone erosion, inflammatory cytokine production, and joint damage in comparison to controls. It has also been reported that granzyme A can induce osteoclastogenesis that can lead to arthritis (Santiago et al., 2017). Mast cells, a type of leukocytes, play important role in immune surveillance as well as in case of allergic responses (Espinosa and Valitutti, 2017). SPs of mast cells include chymase and tryptase that exhibit chymotrypsin-like and trypsin-like activities, respectively (Caughey, 2016). In RA, chymase and tryptase both have the potential to activate several proMMPs (Milner et al., 2008) and the ability of tryptase to cleave various ECM components have also been mentioned in literature (Milner et al., 2008). In a study, mouse administered with tryptase inhibitor, when induced with arthritis showed alteration in the inflammation parameters but no change in the levels of joint destruction was observed (Denadai-Souza et al., 2017). This observation further supports the role of tryptase in RA. Tryptase has also been reported to possess the potential to activate PAR2 signaling which suggests the progression of inflammatory arthritis through this signaling mechanism (Palmer et al., 2007). Serine proteinases NE, CG, and PR3 of circulating leukocytes don’t exhibit transcriptional regulation. These are synthesized by bone marrow precursors as inactive precursors and are stored in millimolar concentrations within the granules of polymorphonuclear leukocytes (PMNs) and monocytes. The inactive forms of these enzymes are converted to active forms by cathepsin C, which is also present within the same granules. The contents of NE, CG, and PR3 are homogeneous in PMNs; however, circulating monocytes contain high amount of NE, CG, and PR3. These monocytes contain a PMN-like proinflammatory (P) phenotype. After maturation of these P monocytes into macrophages, their SPs are replaced by MMPs. However, the macrophages can bind and internalize SP, which are released by PMNs. The serine proteinases are released when macrophages come under hypoxic conditions. After activation by proinflammatory mediators and immune complexes, PMNs and P monocytes can freely release the NE, CG, and PR3 by degranulation. After getting released these active serine proteinases can bind to the plasma membranes of PMNs and monocytes. NE, CG, and PR3 in membrane-bound form on activated PMNs and monocytes have same spectrum of catalytic activity as that of soluble forms of the proteinases.

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Plasminogen/plasminogen activators Plasminogen activators (PAs) can convert plasminogen to its active form plasmin. PAs are of two types: viz. tissue-type activators and urokinase-type PAs (tPA and uPA) and these PAs are synthesized in the form of proenzymes. The synthesis of tissue-type PA occurs in endothelial cells, and uPA is produced by inflammatory cells, endothelial cells, and fibroblasts. Urokinase-type PA is found stored within PMN granules (80% in the specific granules and 20% in the gelatinase granules), but it is synthesized and secreted by activated mononuclear phagocytes, endothelial cells, and fibroblasts. A cell-surface form of uPA is also expressed by the lung-resident cells as well as inflammatory cells. This cell-surface bound form can bind to their respective receptors, that is, high-affinity uPA receptors (uPARs) present on their plasma membrane. uPA has two types of domains: growth-factor-like domain and catalytic domain. Growth factor like domain is present besides N-terminus of uPA can bind to uPAR on lung-resident cells and leukocytes; however, the catalytic domain remains available for plasminogen. Plasmin has a broad spectrum of substrate specificity and it plays numerous functions. However, its major role is described in fibrinolysis, which is a significant process of blood clotting (Draxler and Medcalf, 2015). These plasminogen/PA components have been shown to have their presence in the joints, for example, uPA has been detected in synovial fluids of arthritis patients (OA and RA), and their levels increase in patients of RA (Busso et al., 1997). Although the plasminogen mRNA has not been found expressed in articular cartilage but still plasmin has been determined in the synovial fluids (Caughey and Highton, 1967). Plasmin can cause direct degradation of ECM by cleaving components of ECM like glycoproteins, fibronectin as well as proteoglycans. Plasmin has the potential to activate a number of proMMPs (Milner et al., 2008). A study of cartilage explant culture, the administration of a uPA inhibitor, resulted in the protection of cytokine-stimulated collagen release from cartilage. However, the addition of plasminogen to the cartilage explant culture causes induction of collagen release. These observations suggest that PAs can cause activation of plasmin which further leads to the activation of proMMP (Milner et al., 2001). Some conflicting results have also been observed by some studies on animal models because of differences in the progression of disease and disease initiation between the different models. In a study, the absence of plasminogen as well as uPA protected mice from the CIA and inflammatory arthritis. On the contrary, the studied mice exhibited significant exacerbation of disease followed by antigen-induced arthritis (Li et al., 2005a; Li et al., 2005b; Cook et al., 2010; Busso et al., 1998). Due to such observations, it is demonstrated that the effect of fibrinolytic proteinases on the disease development is crucial in determining the severity of the disease (Li et al., 2005a; De Nardo et al., 2010). In another study of murine model of inflammatory arthritis, induced by tumor necrosis factor (TNF) α, a mouse deficient with plasminogen exhibited protection of knee joints from arthritis. But there was exacerbation of development of arthritis in

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paw joints. These observations further marked the significance of microenvironment of joints on the progression of disease (Keffer et al., 1991; Raghu et al., 2014).

Serine proteinases of complement cascade In innate immunity, the complement cascade when gets activated plays significant roles in the clearance of pathogens (Thurman et al., 2017). Major crucial constituents of this cascade system are the serine proteinases C1r, C1s, C2, Factor I, Factor D, and Factor B. After detection of a foreign antigen in the body, the cascade system of proteolytic events gets triggered for the formation of membrane attack complex (MAC) that ultimately leads to cell lysis (Harris, 2018). This complement cascade is a normal and crucial component of immune response; however, dysregulated activation of this complement system has been reported in a number of inflammation-related disorders including RA. For example, the components of complement system have been reported in synovium, but the levels of complement components are markedly higher in RA (Gulati et al., 1994). The complement components are also expressed in chondrocytes and this expression can be regulated by proinflammatory cytokines (Bradley et al., 1996). In a study of RA patients, C1s in its active form have been found to be associated to the degradation of articular cartilage (Nakagawa et al., 1999). It has also been reported that C1s can degrade collagen types I and II; however this degradation does not result in the generation of the “three quarter and one quarter” fragments as in classical collagenase cleavage. It suggests that in arthritis C1s is unlikely to be main collagenase (Yamaguchi et al., 1990; Woolley et al., 1975). After the proteolytic degradation of cartilage, the components of cartilage matrix are released and they can initiate the activation of complement system. This activated complement system can further activate various components within the complement cascade. Such type of activation of complement factors can be used as targets having therapeutic potential for RA (Happonen et al., 2012; Thurman et al., 2017). The crucial role of complement system in the progression of pathogenesis of OA has been determined by Wang et al. (2011), which supports the paradigm describing significance of inflammation in OA. Several studies based on proteomics and genomics approaches determined that in synovial fluid and membranes of OA patients, there is an increased activation and expression of complement components as compared to nondiseased tissue. In mice model, C5 or C6 complement components (not SP, but downstream) deficient mice exhibited protection from breakdown of cartilage following meniscectomy. However, in a CD59a- (an inhibitor of MAC) deficient mice, the cartilage damage had increased. It was demonstrated by immunohistochemistry that MAC are present on the cell surface OA chondrocytes that have the potential to induce proinflammatory cytokines as well as matrix-degrading enzymes.

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Activated protein C (APC) Activated protein C (APC), a serine proteinase, is converted to its activated form from its precursor, that is, protein C by thrombin. It plays a significant role as an antithrombotic in the blood coagulation pathways, mainly of inactivation of factors Xa and VIIIa. Certain other functions of APC have also been reported like antiinflammatory, proregenerative, and antiapoptotic functions (Griffin et al., 2015). In a study on OA and RA patients, APC was found in the synovial fluid and there was enhancement of cartilage breakdown induced by cytokines. Such breakdown is correlated with increase in the activation of proMMP2 and proMMP9 (BuissonLegendre et al., 2004; Jackson et al., 2009). It has also been reported that MMP2 and APC are colocalized in the synovial cells and endothelial linings in the joints of RA patients (Buisson-Legendre et al., 2004). Some studies have demonstrated that APC have the potential to reduce MMP9 expression by binding to protein c receptor of endothelial lining of synovial fibroblasts. More so, APC can increase the expression of MMP2 in the synovial fibroblasts (Xue et al., 2007). APC also have the potential to induce breakdown of cartilage in case of human OA patients even without stimulating proinflammatory cytokines. This breakdown is dependent on the metalloproteinase activity. However, interestingly it was also reported that the expression of main metalloproteinases in chondrocytes of OA patients is not affected by APC and APC cannot directly activate proMMP13, but it was suggested that APC can interact with other metalloproteinases of proteolytic cascade that lead to destruction of cartilage (Jackson et al., 2014a). Importantly, it has been determined that APC does not affect metalloproteinases expression in OA chondrocytes. APCs are unable to directly stimulate cartilage degradation but they interact with other metalloproteinases in the proteolytic cascades leading to cartilage destruction (Jackson et al., 2009).

Type II transmembrane serine proteinases In osteoarthritic chondrocytes, the initiation of degradation of cartilage occurs in the pericellular matrix, which suggests the potential of membrane-bound serine proteinases in the disease initiation (Hollander et al., 1995). The elevated levels of TTSP matriptase (suppressor of tumourigenicity 14) have been described in the cartilage of OA patients in comparison to healthy tissues (Milner et al., 2010). Matriptase, a SP, is a prime enzyme playing the role of activator of many proMMPs, for example, proMMP3 (Jin et al., 2006). proMMP1 is a collagenase crucial for cartilage destruction and matriptase can directly activate proMMP1. In a study of OA, after addition of matriptase, proMMP1 and proMMP3 expressed significantly in cartilageelevated release of proteoglycan and collagen. This process depends on PAR2 and metalloproteinases, which act as matriptase substrates (Milner et al., 2010; Wilkinson et al., 2017b). In destabilization of the medial meniscus (DMM) OA model, dose-

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dependent administration of matriptase inhibitors resulted in cartilage protection (Glasson et al., 2007; Wilkinson et al., 2017b). Interestingly, it has been determined that matriptase has the potential to initiate proteolytic cascades of cartilage breakdown because it can get autoactivated (Qiu et al., 2007). These findings suggest the role of matriptase in cartilage destruction. TTSP and hepsin, somewhat related to matriptase, were also reported to exhibit induction of cartilage degradation when added to OA cartilage cultures of humans. However, their administration releases low levels of collagen as compared to the release by matriptase, which is due to their reduced capacity for PAR2 activation (Wilkinson et al., 2017a). These observations suggest that related proteinases can have similar but somewhat different substrate repertoires (Qiu et al., 2007).

Proprotein convertases Subtilisin and kexin-type proprotein convertases (PCSK) belong to a unique family composed of nine calcium-dependent serine proteinases (PCSK1-9). These proteinases cause specific cleavage after the sequence which is highly basic in nature (RXR/KR↓). These are responsible for activation and processing of several proteinases as well as growth factors. Furin (PCSK3) is studied most extensively among all the PCSKs. Several PCSKs including furin possess transmembrane domains due to which they can carry out intracellular processing of their substrates present in their trans-golgi network (Milner et al., 2008). Several PCSK activities have been reported in various diseases and their inhibitors are still under clinical studies (Klein-Szanto and Bassi, 2017). In a study on ex vivo model of bovine cartilage going through cartilage breakdown, the administration of Dec-RVKK-CH2Cl which is an inhibitor of furin-like proteinases resulted in protection from collagen breakdown (Milner et al., 2003). The administration of the same inhibitor also resulted in the reduction of levels of collagenases. These observations mark the involvement of these proteinases in the pathways of collagenase activation. In such studies reduction in the levels of active MMP2 was also determined (Milner et al., 2003; Milner et al., 2008). Furin has also been reported to be responsible for the activation of proMMP13 which is a key collagenase involved in the pathways of destruction of type II collagen (Sato et al., 1996; Knauper et al., 1996). The inhibitor Dec-RVKK-CH2Cl has also been reported to give partial protection against proteolytic breakdown. This breakdown process involves ADAMTS4 and ADAMTS5, which are reported to possess activation motifs as that of furin (Longpre et al., 2009). Although the presence of furin is determined in cartilage and the levels of furin get elevated in OA, it has not been clear yet that which one is the major PCSK responsible in cartilage breakdown (Moldovan et al., 2000). In contrast, a study of murine model of CIA exhibited that after administration of furin inhibitor the score of arthritis increased involving destruction of joints and expression of MMP, while the administration of furin resulted in the reduction of these

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parameters (Lin et al., 2012). It has also been hypothesized that furin might be playing an immunomodulatory role by mobilizing the Treg cells, although the mechanism is not yet clear. Moreover, it has also been reported that silencing of furin gene may result in increased invasiveness and growth of human RA synoviocytes and also in increased production of TNFα and IL-1β (Wu et al., 2017). Synovial inflammation is markedly increased in RA, which shows the possibility of different roles of furin in the pathology of joint diseases in different cells and tissues. The main PCSK involved in the activation of aggrecanase in the cartilage of humans is PACE4, that is, paired amino acid cleaving enzyme 4. The level of PACE4 is elevated in OA. In a study of bovine and human OA cartilages, PACE4 silencing resulted in the reduction of proteoglycan release as well as neoepitopes of aggrecan generated by aggrecanse (Malfait et al., 2008). In another study it was determined that single-nucleotide polymorphisms related to PCSK6 gene resulted in protection from knee pain in human OA, while pcsk6 null mice resulted in protection from pain (Malfait et al., 2012).

Proteinase-activated receptors (PAR) The PAR family plays crucial roles in vascular physiology, progression, and development of cancer and in inflammation as well. Due to these roles, PAR family has emerged out as a potential therapeutic target. PARs are activated by a proteolytic mechanism in which N-terminus is cleaved and a ligand domain is exposed. In this way, PARs provide a link between cellular responses and extracellular proteinases (Yau et al., 2013). During tissue damage, a number of PAR-activating serine proteinases are generated; hence PARs provide their contribution to the processes of inflammation and repair. There are four family members of PAR (PAR1-4); out of these PAR2 is mainly associated with arthritis. SP tryptase, matriptase, or trypsin can activate the PAR2 (Yau et al., 2013). The presence of PAR2 is reported in particular chondrocytes, and its expression increases in cartilage of OA (Xiang et al., 2006). Some studies on animal models exhibited the potential of PAR2 to be used as a target for the prevention of joint destruction. In a study of chronic inflammatory arthritis, PAR2-deficient mice exhibited reduction in joint inflammation as well as protection of cartilage integrity (Ferrell et al., 2003). Some other studies on murine models indicated that deficiency of PAR2 resulted in delayed maturation of osteophytes (Huesa et al., 2016) that also supports the observation of delayed callus formation in the repairing of fractured bone (O’Neill et al., 2012; Georgy et al., 2012). These observations suggest the involvement of PAR2 as a driving factor in the differentiation and/or maturation of pathogenic chondrocytes. It has been reported that prevention of PAR2 activation in musculoskeletal diseases could be helpful for repairing of cartilage delaying the progression of OA (Yau et al., 2013). Further exploration of PAR2 activation in ECMs will lead to better understanding of new targets that can be used for treating the pathological joint destruction.

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A summary of the key proteinases has been represented in Figure 1 and the effect of serine proteinase gene deficiency is summarized in Table 1.

3

TTSP Tryptase Other SPs

Plasminogen

PAR

APC

proMP

Active MP

TIMP

1

PCSKs

Granzymes Serpins HtrA1

2

Chondrocyte or synovial fibroblast

1 HNE CTSG Tryptase Plasmin TTSP

Other catabolic/proinflammatory mediators

proMP

uPA tPA

Sub-lytic MAC

ECM degradation 3 Complement cascade

Figure 1: Schematic representation of degradation of cartilage by serine proteinases through different mechanisms. ECM of arthritic cartilage is mainly degraded by metalloproteinases (MP) such as MMP. However, the significant roles of serine proteinases regulation of catabolic process like proenzymes activation are as follows: (1) SPs, such as immune cell-derived SPs and plasmin, are involved in direct degradation of the ECM, HtrA1, and complement system also contributes toward pathology; (2) SPs can also induce expression of metalloproteinases by activation of PAR and complement system (Wilkinson et al., 2019).

Conclusion and future prospective The available therapies of OA can only provide the relief from pain, and there are no effective therapies available that can be used to alter the progression of disease. Therefore, new antiarthritic drugs are needed to be developed so as to prevent the cartilage loss from the surface of joints. Although the turnover of ECM in arthritis is mainly driven by metalloproteinases, serine proteinases exhibit significant roles in regulating MMPs activities. Due to these roles of SP, they are emerged out as significant targets in the development of novel therapeutic drugs.

NE inhibitor (orally) NE inhibitor (i.p.)

NE

WT

CIA – – – –

– – – –

NE–/– (Elane) CTSG–/– x NE–/– WT Collagen Antibody

–

(Ctsg)

Less inflammatory cells in the tissue of subsynovial space, joint space exhibited lesser exudate, and fibrin-like deposition Prevention of proteoglycan content destruction Resistance from arthritis induction Subsynovial space having lesser inflammatory cells Joint space has lesser or no exudate as well as no Proteoglycan loss Joint swelling is less severe Cartilage as well as bone destruction is inhibited Pannus formation is inhibited Reduction in severity and incidence of disease, infiltration of leukocytes of joint cavity, and formation of pannus and cartilage destruction

Arthritis having low arthritic score

CTSG –/–

–

CTSG Antitype IIinduced arthritis

Effects

Genotype Model

Treatment

Serine proteinase/ proteinase substrate

Table 1: Summary of studies of animal models of arthritis showing the effect of serine proteinase gene deficiency.

Kakimoto et al.,

Janusz and Durham, 

Adkison et al., 

References

92 Preeti Chanalia, Poonam Bansal, and Dimpi Gandhi

PAR – / – (Frl)

–

Protease-activated receptor  (PAR)

Inhibitor (i.p.) WT

WT

Inhibitor (IA)

Matriptase

GzmA–/–

(Gzma)

–

Recombinant WT protein (i.p.) Inhibitor (i.p.)

(GzmA)

Granzyme A

Furin

ACL DMM

DMM

DMM

CIA

CIA

Reduction in cartilage damage Antibody immunostaining is less in ADAMTS-cleaved and MMP-cleaved aggrecan, and less collagenase-cleaved type-II collagen in the damaged sites

– –

– – – –

– –

Delay in the maturation of osteophytes Weight bearing is altered on affected joints, which suggests reduction in pain perception Less osteosclerosis scores and cartilage damage Cartilage damage is reduced Reduction in OA pathology Reduced osteosclerosis scores and cartilage damage

Majorly normal joints with mild pannus Formation No erosion of bone and cartilage

– –

Reduction in clinical scores, levels of serum proinflammatory cytokines, and reduction of osteoclasts number

Arthritis score is reduction, thickness of synovial pannus, reduced destruction of bone as well as cartilage, reduced expression, and activity of MMP, concentration of proinflammatory cytokines Increase in the local concentration of anti-inflammatory cytokines Increase in the arthritis score, thickness of synovial pannus, invasion into the joints, expression of MMP, and increase in the local concentration of proinflammatory cytokines Reduction of anti-inflammatory cytokines concentrations Loss of bone is not affected

–

– –

– –

–

(continued)

Huesa et al., 

Wilkinson et al., b

Santiago et al., 

Lin et al., 

Chapter 6 Serine proteases in arthritis

93

WT

Plg–/– (Plg)

–

Inhibitor (IA)

–

SERPINE  (PAI-)

Tryptase

Plasminogen (Plg)

PAI- – / – (Serpine)

Recombinant WT protein (i.p.)

SERPINA  (α-AT)

CAIA

AIA

mBSA/IL-β (IA)

AIA

CIA

Genotype Model

Treatment

Serine proteinase/ proteinase substrate

Table 1 (continued)

– – – – –

– – – –

Arthritic levels are comparable at early stages with wild-type, however levels are exacerbated in later stages Prolongation in synovial thickness as well as joint inflammation Elevated levels of fibrin accumulation and bone erosion No signs of inflammation are reported in any mice Peripheral synovial tissues showed normal morphology of joints, no inflammation, and destruction of tissue No infiltration of neutrophils is reported. Fewer resting macrophages Followed by injection of saline or CII (LIA), arthritis is developed in mice No tissue destruction or inflammation was reported Inflammation as well as tissue damage is developed in mice after giving plasminogen injection intravenously

Reduction in tryptase-like activity Formation of oedema and production of cytokines No change in neutrophil infiltration, degeneration of hyaline cartilage, and erosion of subchondral bone

– – – –

Reduction of inflammatory response Reduced synovial infiltration, fibrin levels, and proteoglycan loss Increased activity of plasminogen activator and fibrinolysis

– – –

Li et al., a, b

Busso et al., 

Li et al., a

DenadaiSouza et al., 

Van Ness et al., 

Grimstein et al., 

– Delayed onset of arthritis, reduction in disease progression as well as incidence of severe arthritis

References

Effects

94 Preeti Chanalia, Poonam Bansal, and Dimpi Gandhi

–

uPA-–/– (Plau)

CAIA K/BxN

CIA

AIA

– – – – –

– – – – – – – – – Inflammation of joints Increase in thickness of synovial areas Increase in synovial fibrosis of some mice Reduction of proteoglycan content Fibrin accumulation and bone erosion are more pronounced Reduction in severity as well as incidence of arthritis Delay in onset of disease Severity of CIA is less in 75% mice Reduction in gene expression of MMP9, IL-1β, TNF, MCP-1, IL-6, u-PAR, u-PA, MMP3, ADAMTS4, MMP13, and t-PA No change in the levels of ADAMTS5 mRNA Only mild arthritis was developed in 43% of studied mice Mild arthritis was developed in 60% of studied mice Mild differences in development of arthritis with relatively normal joints Development of arthritis in mice followed by IA injection of saline De Nardo et al., 

Cook et al., 

Li et al., b

De Nardo et al., 

Busso et al., 

AIA, antigen-induced arthritis; ACL anterior cruciate ligament; CAIA, CII antibody-induced arthritis anterior cruciate ligament transection; IA, intraarticular; mBSA/IL1β, methylated BSA/IL-1β-induced arthritis; LIA, local injection-induced arthritis; STIA, K/BxN serum transfer-induced arthritis.

Urokinase type plasminogen activator (uPA)

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References Adkison, A. M., Raptis, S. Z., Kelley, D. G., & Pham, C. T. (2002). Dipeptidyl peptidase I activates neutrophilderived serine proteases and regulates the development of acute experimental arthritis. Journal of Clinical Investigation, 109, 363–371. Bradley, K., North, J., Saunders, D., Schwaeble, W., Jeziorska, M., Woolley, D. E., & Whaley, K. (1996). Synthesis of classical pathway complement components by chondrocytes. Immunology, 88, 648–656. Buisson-Legendre, N., Smith, S., March, L., & Jackson, C. (2004). Elevation of activated protein C in synovial joints in rheumatoid arthritis and its correlation with matrix metalloproteinase 2. Arthritis and Rheumatology, 50, 2151–2156. Busso, N., Peclat, V., Van Ness, K., Kolodziesczyk, E., Degen, J., Bugge, T., & So, A. (1998). Exacerbation of antigen-induced arthritis in urokinase-deficient mice. Journal of Clinical Investigation, 102, 41–50. Busso, N., Peclat, V., So, A., & Sappino, A. P. (1997). Plasminogen activation in synovial tissues: Differences between normal, osteoarthritis, and rheumatoid arthritis joints. Annals of the Rheumatic Diseases, 56, 550–557. Caughey, G. H. (2016). Mast cell proteases as pharmacological targets. European Journal of Pharmacology, 778, 44–55. Caughey, D. E., & Highton, T. C. (1967). Components of the fibrinolytic system in synovial joints. Normal bovine compared with normal and abnormal human synovial joints. Annals of the Rheumatic Diseases, 26, 297–305. Cook, A. D., De Nardo, C. M., Braine, E. L., Turner, A. L., Vlahos, R., Way, K. J., Beckman, S. K., Lenzo, J. C., & Hamilton, J. A. (2010). Urokinase-type plasminogen activator and arthritis progression: Role in systemic disease with immune complex involvement. Arthritis Research & Therapy, 12, R37. De Nardo, C. M., Lenzo, J. C., Pobjoy, J., Hamilton, J. A., & Cook, A. D. (2010). Urokinase-type plasminogen activator and arthritis progression: Contrasting roles in systemic and monoarticular arthritis models. Arthritis Research & Therapy, 12, R199. Denadai-Souza, A., Ribeiro, C. M., Rolland, C., Thouard, A., Deraison, C., Scavone, C., Gonzalez-Dunia, D., Vergnolle, N., & Avellar, M. C. W. (2017). Effect of tryptase inhibition on joint inflammation: A pharmacological and lentivirus-mediated gene transfer study. Arthritis Research & Therapy, 19, 124. Draxler, D. F., & Medcalf, R. L. (2015). The fibrinolytic system-more than fibrinolysis?. Transfusion Medicine Reviews, 29, 102–109. Elsaid, K. A., Jay, G. D., & Chichester, C. O. (2003). Detection of collagen type II and proteoglycans in the synovial fluids of patients diagnosed with non-infectious knee joint synovitis indicates early damage to the articular cartilage matrix. Osteoarthritis Cartilage, 11, 673–680. Espinosa, E., & Valitutti, S. (2017). New roles and controls of mast cells. Current Opinion in Immunology, 50, 39–47. Ferrell, W. R., Lockhart, J. C., Kelso, E. B., Dunning, L., Plevin, R., Meek, S. E., Smith, A. J. H., Hunter, G. D., McLean, J. S., McGarry, F., Ramage, R., Jiang, L., Kanke, T., & Kawagoe, J. (2003). Essential role for proteinase-activated receptor-2 in arthritis. Journal of Clinical Investigation, 111, 35–41. Froelich, C. J., Zhang, X., Turbov, J., Hudig, D., Winkler, U., & Hanna, W. L. (1993). Human granzyme B degrades aggrecan proteoglycan in matrix synthesized by chondrocytes. Journal of Immunology, 151, 7161–7171. Glasson, S. S., Blanchet, T. J., & Morris, E. A. (2007). The surgical destabilization of the medial meniscus (DMM) model of osteoarthritis in the 129/SvEv mouse. Osteoarthritis Cartilage, 15, 1061–1069. Goldbach-Mansky, R., Suson, S., Wesley, R., Hack, C. E., El-Gabalawy, H. S., & Tak, P. P. (2005). Raised granzyme B levels are associated with erosions in patients with early rheumatoid factor positive rheumatoid arthritis. Annals of the Rheumatic Diseases, 64, 715–721. Griffin, J. H., Zlokovic, B. V., & Mosnier, L. O. (2015). Activated protein C: Biased for translation. Blood, 125, 2898–2907.

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Grimstein, C., Choi, Y. K., Wasserfall, C. H., Satoh, M., Atkinson, M. A., Brantly, M. L., Campbell-Thompson, M., & Song, S. (2011). Alpha-1 antitrypsin protein and gene therapies decrease autoimmunity and delay arthritis development in mouse model. Journal of Translational Medicine, 9, 21. Gulati, P., Guc, D., Lemercier, C., Lappin, D., & Whaley, K. (1994). Expression of the components and regulatory proteins of the classical pathway of complement in normal and diseased synovium. Rheumatology International, 14, 13–19. Happonen, K. E., Heinegard, D., Saxne, T., & Blom, A. M. (2012). Interactions of the complement system with molecules of extracellular matrix: Relevance for joint diseases. Immunobiology, 217, 1088–1096. Harris, C. L. (2018). Expanding horizons in complement drug discovery: Challenges and emerging strategies. Semin Immunopathol, 40, 125–140. Hollander, A. P., Pidoux, I., Reiner, A., Rorabeck, C., Bourne, R., & Poole, A. R. (1995). Damage to type II collagen in aging and osteoarthritis starts at the articular surface, originates around chondrocytes, and extends into the cartilage with progressive degeneration. Journal of Clinical Investigation, 96, 2859–2869. Horiuchi, K., Saito, S., Sasaki, R., Tomatsu, T., & Toyama, Y. (2003). Expression of granzyme B in human articular chondrocytes. Journal of Rheumatology, 30, 1799–1810. Huesa, C., Ortiz, A. C., Dunning, L., Mcgavin, L., Bennett, L., Mcintosh, K., Crilly, A., Kurowska-Stolarska, M., Plevin, R., van’t Hof, R. J., Rowan, A. D., McInnes, I. B., Goodyear, C. S., Lockhart, J. C., & Ferrell, W. R. (2016). Proteinase-activated receptor 2 modulates OA-related pain, cartilage and bone pathology. Annals of the Rheumatic Diseases, 75, 1989–1997. Georgy, S. R., Pagel, C. N., Ghasem-Zadeh, A., Zebaze, R. M., Pike, R. N., Sims, N. A., & Mackie, J. (2012). Proteinase-activated receptor-2 is required for normal osteoblast and osteoclast differentiation during skeletal growth and repair. Bone, 50, 704–712. O’Neill, K. R., Stutz, C. M., Mignemi, N. A., Cole, H., Murry, M. R., Nyman, J. S., Hamm, H., & Schoenecker, J. G. (2012). Fracture healing in protease-activated receptor-2 deficient mice. Journal of Orthopaedic Research, 30, 1271–1276. Huet, G., Flipo, R. M., Richet, C., Thiebaut, C., Demeyer, D., Balduyck, M., Duquesnoy, B., & Degand, P. (1992). Measurement of elastase and cysteine proteinases in synovial fluid of patients with rheumatoid arthritis, sero-negative spondylarthropathies, and osteoarthritis. Clinical Chemistry, 38, 1694–1697. Jackson, M. T., Moradi, B., Smith, M. M., Jackson, C. J., & Little, C. B. (2014). Activation of matrix metalloproteinases 2, 9, and 13 by activated protein C in human osteoarthritic cartilage chondrocytes. Arthritis and Rheumatology, 66, 1525–1536. Jackson, M. T., Smith, M. M., Smith, S. M., Jackson, C. J., Xue, M., & Little, C. B. (2009). Activation of cartilage matrix metalloproteinases by activated protein C. Arthritis and Rheumatology, 60, 780–791. Janusz, M. J., & Durham, S. L. (1997). Inhibition of cartilage degradation in rat collagen-induced arthritis but not adjuvant arthritis by the neutrophil elastase inhibitor MDL 101,146. Inflammation Research, 46, 503–508. Jin, X., Yagi, M., Akiyama, N., Hirosaki, T., Higashi, S., Lin, C. Y., Dickson, R. B., Kitamura, H., & Miyazaki, K. (2006). Matriptase activates stromelysin (MMP-3) and promotes tumor growth and angiogenesis. Cancer Science, 97, 1327–1334. Kakimoto, K., Matsukawa, A., Yoshinaga, M., & Nakamura, H. (1995). Suppressive effect of a neutrophil elastase inhibitor on the development of collagen-induced arthritis. Cellular Immunology, 165, 26–32. Keffer, J., Probert, L., Cazlaris, H., Georgopoulos, S., Kaslaris, E., Kioussis, D., & Kollias, G. (1991). Transgenic mice expressing human tumour necrosis factor: A predictive genetic model of arthritis. EMBO Journal, 10, 4025–4031. Klein-Szanto, A. J. & Bassi, D. E. (2017). Proprotein convertase inhibition: Paralyzing the cell’s master switches. Biochemical Pharmacology, 140, 8–15.

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Knauper, V., Lopez-Otin, C., Smith, B., Knight, G., & Murphy, G. (1996). Biochemical characterization of human collagenase-3. Journal of Biological Chemistry, 271, 1544–1550. Li, J., Guo, Y., Holmdahl, R., & Ny, T. (2005a). Contrasting roles of plasminogen deficiency in different rheumatoid arthritis models. Arthritis and Rheumatology, 52, 2541–2548. Li, J., Ny, A., Leonardsson, G., Nandakumar, K. S., Holmdahl, R., & Ny, T. (2005b). The plasminogen activator/plasmin system is essential for development of the joint inflammatory phase of collagen type II- induced arthritis. American Journal of Pathology, 166, 783–792. Lin, H., Ah Kioon, M. D., Lalou, C., Larghero, J., Launay, J. M., Khatib, A. M., & Cohen-Solal, M. (2012). Protective role of systemic furin in immune response-induced arthritis. Arthritis and Rheumatology, 64, 2878–2886. Longpre, J. M., Mcculloch, D. R., Koo, B. H., Alexander, J. P., Apte, S. S., & Leduc, R. (2009). Characterization of proADAMTS5 processing by proprotein convertases. International Journal of Biochemistry and Cell Biology, 41, 1116–1126. Malfait, A. M., Arner, E. C., Song, R. H., Alston, J. T., Markosyan, S., Staten, N., Yang, Z., Griggs, D. W., & Tortorella, M. D. (2008). Proprotein convertase activation of aggrecanases in cartilage in situ. Archives of Biochemistry and Biophysics, 478, 43–51. Malfait, A. M., Seymour, A. B., Gao, F., Tortorella, M. D., Le Graverand-Gastineau, M. P., Wood, L. S., Doherty, M., Doherty, S., Zhang, W., Arden, N. K., Vaughn, F. L., Leaverton, P. E., Spector, T. D., Hart, D. J., Maciewicz, R. A., Muir, K. R., Das, R., Sorge, R. E., Sotocinal, S. G., Schorscher-Petcu, A., Valdes, A. M., & Mogil, J. S. (2012). A role for PACE4 in osteoarthritis pain: Evidence from human genetic association and null mutant phenotype. Annals of the Rheumatic Diseases, 71, 1042–1048. Milner, J. M., Elliott, S. F., & Cawston, T. E. (2001). Activation of procollagenases is a key control point in cartilage collagen degradation: Interaction of serine and metalloproteinase pathways. Arthritis and Rheumatology, 44, 2084–2096. Milner, J. M., Patel, A., Davidson, R. K., Swingler, T. E., Desilets, A., Young, D. A., Kelso, E. B., Donell, S. T., Cawston, T. E., Clark, I. M., Ferrell, W. R., Plevin, R., Lockhart, J. C., Leduc, R., & Rowan, A. D. (2010). Matriptase is a novel initiator of cartilage matrix degradation in osteoarthritis. Arthritis and Rheumatology, 62, 1955–1966. Milner, J. M., Patel, A., & Rowan, A. D. (2008). Emerging roles of serine proteinases in tissue turnover in arthritis. Arthritis and Rheumatology, 58, 3644–3656. Milner, J. M., Rowan, A. D., Elliott, S. F., & Cawston, T. E. (2003). Inhibition of furin-like enzymes blocks interleukin-1alpha/oncostatin Mstimulated cartilage degradation. Arthritis and Rheumatology, 48, 1057–1066. Moldovan, F., Pelletier, J. P., Mineau, F., Dupuis, M., Cloutier, J. M., & Martel-Pelletier, J. (2000). Modulation of collagenase 3 in human osteoarthritic cartilage by activation of extracellular transforming growth factor beta: Role of furin convertase. Arthritis and Rheumatology, 43, 2100–2109. Nagase, H., Suzuki, K., Cawston, T. E., & Brew, K. (1997). Involvement of a region near valine-69 of tissue inhibitor of metalloproteinases (TIMP)-1 in the interaction with matrix metalloproteinase 3 (stromelysin 1). Biochemical Journal, 325, 163–167. Nakagawa, K., Sakiyama, H., Tsuchida, T., Yamaguchi, K., Toyoguchi, T., Masuda, R., & Moriya, H. (1999). Complement C1s activation in degenerating articular cartilage of rheumatoid arthritis patients: Immunohistochemical studies with an active form specific antibody. Annals of the Rheumatic Diseases, 58, 175–181. Palmer, H. S., Kelso, E. B., Lockhart, J. C., Sommerhoff, C. P., Plevin, R., Goh, F. G., & Ferrell, W. R. (2007). Protease-activated receptor 2 mediates the proinflammatory effects of synovial mast cells. Arthritis and Rheumatology, 56, 3532–3540. Qiu, D., Owen, K., Gray, K., Bass, R., & Ellis, V. (2007). Roles and regulation of membrane-associated serine proteases. Biochemical Society Transactions, 35, 583–587.

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Raghu, H., Jone, A., Cruz, C., Rewerts, C. L., Frederick, M. D., Thornton, S., Degen, J. L., & Flick, M. J. (2014). Plasminogen is a joint-specific positive or negative determinant of arthritis pathogenesis in mice. Arthritis and Rheumatology, 66, 1504–1516. Ronday, H. K., Van Der Laan, W. H., Tak, P. P., De Roos, J. A., Bank, R. A., Tekoppele, J. M., Froelich, C. J., Hack, C. E., Hogendoorn, P. C., Breedveld, F. C., & Verheijen, J. H. (2001). Human granzyme B mediates cartilage proteoglycan degradation and is expressed at the invasive front of the synovium in rheumatoid arthritis. Rheumatology (Oxford), 40, 55–61. Santiago, L., Menaa, C., Arias, M., Martin, P., Jaime-Sanchez, P., Metkar, S., Comas, L., Erill, N., GonzalezRumayor, V., Esser, E., Galvez, E. M., Raja, S., Simon, M. M., Sprague, S. M., Gabay, C., MartinezLostao, L., Pardo, J., & Froelich, C. J. (2017). Granzyme A contributes to inflammatory arthritis in mice through stimulation of osteoclastogenesis. Arthritis and Rheumatology, 69, 320–334. Sato, H., Kinoshita, T., Takino, T., Nakayama, K., & Seiki, M. (1996). Activation of a recombinant membrane type 1-matrix metalloproteinase (MT1-MMP) by furin and its interaction with tissue inhibitor of metalloproteinases (TIMP)-2. FEBS Letters, 393, 101–104. Thurman, J. M., Frazer-Abel, A., & Holers, V. M. (2017). The evolving landscape for complement therapeutics in rheumatic and autoimmune diseases. Arthritis and Rheumatology, 69, 2102–2113. Van Ness, K., Chobaz-Peclat, V., CastelluccI, M., So, A., & Busso, N. (2002). Plasminogen activator inhibitor type-1 deficiency attenuates murine antigen-induced arthritis. Rheumatology (Oxford), 41, 136–141. Wang, Q., Rozelle, A. L., Lepus, C. M., Scanzello, C. R., Song, J. J., Larsen, D. M., Crish, J. F., Bebek, G., Ritter, S. Y., Lindstrom, T. M., Hwang, I., Wong, H. H., Punzi, L., Encarnacion, A., Shamloo, M., Goodman, S. B., Wyss-Coray, T., Goldring, S. R., Banda, N. K., Thurman, J. M., Gobezie, R., Crow, M. K., Holers, V. M., Lee, D. M., & Robinson, W. H. (2011). Identification of a central role for complement in osteoarthritis. Nature Medicine, 17, 1674–1679. Wilkinson, D. J., Desilets, A., Lin, H., Charlton, S., Arques, M. C., Falconer, A., Bullock, C., Hsu, Y. C., Birchall, K., Hawkins, A., Thompson, P., Ferrell, W. R., Lockhart, J., Plevin, R., Zhang, Y., Blain, E., Lin, S. W., Leduc, R., Milner, J. M., & Rowan, A. D. (2017a). The serine proteinase hepsin is an activator of promatrix metalloproteinases: Molecular mechanisms and implications for extracellular matrix turnover. Scientific Reports, 7, 16693. Wilkinson, D. J., Arques, M. D. C., Huesa, C., & Rowan, A. D. (2019). Serine proteinases in the turnover of the cartilage extracellular matrix in the joint: Implications for therapeutics. British Journal of Pharmacology, 176, 38–51. Wilkinson, D. J., Habgood, A., Lamb, H. K., Thompson, P., Hawkins, A. R., Desilets, A., Leduc, R., Steinmetzer, T., Hammami, M., Lee, M. S., Craik, C. S., Watson, S., Lin, H., Milner, J. M., & Rowan, A. D. (2017b). Matriptase induction of metalloproteinase dependent aggrecanolysis in vitro and in vivo: Promotion of osteoarthritic cartilage damage by multiple mechanisms. Arthritis and Rheumatology, 69, 1601–1611. Woolley, D. E., Lindberg, K. A., Glanville, R. W., & Evanson, J. M. (1975). Action of rheumatoid synovial collagenase on cartilage collagen different susceptibilities of cartilage and tendon collagen to collagenase attack. European Journal of Biochemistry, 50, 437–444. Wu, C., Song, Z., Liu, H., Pan, J., Jiang, H., Liu, C., Yan, Z., Feng, H., & Sun, S. (2017). Inhibition of furin results in increased growth, invasiveness and cytokine production of synoviocytes from patients with rheumatoid arthritis. Joint Bone Spine, 84, 433–439. Xiang, Y., Masuko-Hongo, K., Sekine, T., Nakamura, H., Yudoh, K., Nishioka, K., & Kato, T. (2006). Expression of proteinase-activated receptors (PAR)-2 in articular chondrocytes is modulated by IL1beta, TNF-alpha and TGF- beta. Osteoarthritis Cartilage, 14, 1163–1173. Xue, M., March, L., Sambrook, P. N., Fukudome, K., & Jackson, C. J. (2007). Endothelial protein C receptor is overexpressed in rheumatoid arthritic (RA) synovium and mediates the anti-inflammatory effects of activated protein C in RA monocytes. Annals of the Rheumatic Diseases, 66, 1574–1580.

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Yamaguchi, K., Sakiyama, H., Matsumoto, M., Moriya, H., & Sakiyama, S. (1990). Degradation of type I and II collagen by human activated C1-s. FEBS Letters, 268, 206–208. Yau, M. K., Liu, L., & Fairlie, D. P. (2013). Toward drugs for protease-activated receptor 2 (PAR2). Journal of Medicinal Chemistry, 56, 7477–7497.

Biographical sketch Name: Preeti Chanalia Affiliation: Research Assistant, Research & Innovation Department, Shri Krishna AYUSH University, Kurukshetra, Haryana Education: MSc. & Ph.D. (Biochemistry)

Business Address Shri Krishna AYUSH University, Umri Road, Sector 8, Kurukshetra, Haryana, PIN 136118

Research and Professional Experience – – –

Research Assistant position at Shri Krishna AYUSH University, Kurukshetra, Haryana since 2021. Guest faculty at Department of Biochemistry, Kurukshetra University, Kurukshetra in 2020. Assistant Professor at Softvision college, Indore, Madhya Pradesh in 2016-2017.

Honors University Research Scholarship for PhD (2010) Rajiv Gandhi national Fellowship for PhD (2012-2017)

Publications from last 3 Years –

Gandhi, D., Chanalia, P., Bansal, P., Dhanda, S. 2020. “Peptidoglycan Hydrolases of Probiotic Pediococcus acidilactici NCDC 252: Isolation, Physicochemical and In Silico Characterization”. Int J Pept Res Ther 26(4): DOI: 10.1007/s10989-019-10008-3.

Kapil Singh Narayan✶, Reenu Kashyap, Raman Kumar, Anil K. Sharma, Anil Panwar, and Varruchi Sharma

Chapter 7 Serine protease HtrA: a promising therapeutic target to develop antimicrobial therapy Abstract: Serine protease is present in all life forms (prokaryotic and eukaryotic) and has evolved as a most functional diverse group. A serine protease family known as high-temperature requirement A (HtrA) protein is closely associated with bacterial fitness and is involved in several infectious diseases. This HtrA protein complex helps bacteria to survive against antibiotics and other stressful environments by changing the function of E-cadherin (cell adhesion protein) and other membrane proteins such as proteoglycans and fibronectin. Although HtrA serine protease has not been defined in all pathogens, it has always been considered a remarkable target for several potential drugs to treat bacterial infections. Due to the growing bacterial adaptation and/or resistance acquisition against antimicrobial agents, the research focusing on HtrA serine protease can be a suitable target to cure bacterial infections. Keywords: high-temperature requirement, E-cadherin, serine proteases, inhibitors

Introduction HtrA (high-temperature-requirement protein A) protein originated from MEROPS peptidase family S1; subfamily S1C is mainly found in mammals, plants, and bacteria. In bacteria, HtrA proteins exist in different forms such as DegP, MucD, DegQ, YkdA, DegQ, and ✶

Corresponding author: Kapil Singh Narayan, Department of Clinical Studies, School of Veterinary Medicine, New Bolton Center, University of Pennsylvania, Kennett Square, PA 19348, USA, e-mail: [email protected] Reenu Kashyap, 382 West Street Road, CAHP Building, New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, PA 19348 Raman Kumar, Department of Biochemistry, Kurukshetra University, Kurukshetra 136119, Haryana, India Anil K. Sharma, Department of Biosciences and Technology, Maharishi Markandeshwar (Deemed to be University), Mullana, Ambala 133207, Haryana, India Anil Panwar, Department of Bioinformatics and Computational Biology College of Biotechnology, CCS Haryana Agricultural University Hisar, Haryana, 125004 Varruchi Sharma, Department of Biotechnology and Bioinformatics, Sri Guru Gobind Singh College Sector-26, Chandigarh (UT), India https://doi.org/10.1515/9783111325040-007

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protease DO. All these HtrA proteins have a unique sequence in their protein domain, including various other specific features exhibiting specific activities. Structurally, it comprises a trypsin-like protease domain with a conserved sequence of His-Asp-Ser and one or two postsynaptic density protein (PDZ) domains involved in substrate binding, recognition and oligomerization (Skórko-Glonek et al., 1995) (Figure 1). In addition to the variable N-terminus, another regions called Sec signal peptide, trans-membrane domains, or selfprocessing domains are also present that are closely related to their subcellular localization. The N-terminus contains essential activity for HtrA protease that is required for the stability of the homotrimer. DegP/HtrA is the first protease identified in Escherichia coli that helps in high-temperature-survivability (Strauch et al., 1989). Subsequently, many studies have also shown that the HtrA protein was essential for the survivability and colonization of pathogens under extreme conditions or invasion of host defense mechanisms. However, it also helps increase the pathogenicity of virulence factors by rupturing the host epithelial cell barrier (Boehm et al., 2012). Many studies showed that it promotes or inhibits biofilm formation through potential mechanisms depending on the bacterial species (Biswas et al., 2005). Therefore, the overall role of the HtrA family protease in bacterial pathogenicity is multifaceted and can be further studied. N-

C-

SP

TM

TPD

PDZ

PDZ

Substrate binding Figure 1: Domain assembly of HtrA protein. SP stands for signal sequence, TM stands for transmembrane region, TDP stands for trypsin-like serine protease domain, and PDZ stands for PDZ domains.

Moreover, newer bactericidal/bacteriostatic treatments can be developed (Wessler et al., 2017). Although it may also produce adverse effects, its protease domain may cause cross-reactions in low doses. It may also inhibit biofilm or reverse activation, leading to chronic infections (Skorko-Glonek et al., 2013). HtrA family proteases can be used in antibacterial drug development against bacterial infections (Skorko-Glonek et al., 2013).

Role of HtrA family proteases under stress In extreme conditions, such as stress, oxidative stress or heat, and acidity, cellular proteins aggregate and eventually lead to bacterial cell damage. Therefore, HtrA family proteins help to eventually restore cellular homeostasis (Clausen et al., 2002). It provides resistance based on the activities of protease and chaperone in a tempera-

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ture-dependent manner. In Helicobacter pylori, the HtrA protein helps bacteria adapt to harsh environmental conditions of the human stomach (Hoy et al., 2010). A new mutant N6 strain was discovered to sense and responded in extreme conditions. In brief, Cpx and sE envelope proteins are present in many Gram-negative bacteria that respond during stress (Duguay et al., 2004). Upon activation, the HtrA protein is upregulated and localized within periplasmic space through an N-terminal signal peptide that leads to refolding or degradation of misfolded proteins (Zhang et al., 2019). Similarly, in Pseudomonas aeruginosa, MucD and DegP proteins are present that regulate the transcription of sigma factors, that is, RpoE, during stress conditions. The mucD mutant P. aeruginosa strain showed higher sensitivity to oxidative stress, and the HtrA protein protects against environmental stress (Yu et al., 1995). This showed that the HtrA family protease has a remarkable role in bacteria survival under stress.

HtrA family protease enhances bacterial aggressiveness by disrupting the host epithelial barrier HtrA family protease also helps cleavage the E-cadherin protein, which allows invasion during bacterial infection. E-cadherin is a crucial core protein of the epithelial membrane and acts as an adhesive connection between cells. It also acts as a messenger to regulate cell survival and polarity (Mehta et al., 2015). E-cadherin also played an essential role in protection against many infectious agents, including H. pylori, Enteropathogenic E. coli, Clostridium jejuni, and Salmonella enterica subsp. enterica (Abfalter et al., 2016). HtrA protein was secreted in outer membrane vesicles, and when it came in association with host cells, it cleaved E-cadherin. Another cleavage sequence pattern (VITA)(VITA)-x-x-D-(DN) was also defined that was cleaved by HtrA of H. pylori (Schmidt et al., 2016). HtrA is also involved in the cleavage of other epithelial barrier proteins, such as fibronectin, occludin, and claudin-8 (Backert et al., 2018). Therefore, a pathogen, in this way, can penetrate between cells and translocate into deeper tissues and organs. HtrA protein in Acinetobacter baumannii, called PKF, facilitates the translocation of bacteria by lysing the adhesion proteins and migrates extracellularly through intestinal epithelium (King et al., 2013). Similarly, P. aeruginosa causes intestinal sepsis through the degradation of mucin protein by MucD protease (Hayashi et al., 2013). This showed that the HtrA family protease is an essential protein for the invasion of bacteria.

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HtrA family protease as a virulence factor Many studies have also suggested that HtrA protein may also be involved in bacterial pathogenesis that has direct or auxiliary virulence influence in many bacteria such as H. pylori, Bacillus anthracis, P. aeruginosa, C. jejuni, Listeria monocytogenes, Clostridium difficile, Haemophilus parasuis, Streptococcus pneumoniae, and Borrelia burgdorferi (Bakker et al., 2014). If HtrA protease is absent, it reduces bacterial invasion and decreases pathogenic toxins secretion. HtrA protein pathogenicity in B. anthracis depends on the catalytic domain. MucD protease in P. aeruginosa is present extracellularly and protects bacteria against many cytokines and chemokines, including IL-1β, MIP-2, KC, and IL-8. PKF may also save A. baumannii via serum resistance from the innate immune system (Mochizuki et al., 2014). In addition, CagA protein helps in the translocation and phosphorylation of H. pylori, and expression of HtrA protein in H. pylori may be able to cleave the adhesion junction protein and expose integrin a5b1 site for CagA attachment (Harrer et al., 2017). Hence, the HtrA protein promotes bacterial virulence by different potential mechanisms.

HtrA family protease in biofilm formation Bacterial biofilms consist of a complex mixture of microbes that stick to each other, mainly to a surface. The formation of bacterial biofilms consists of two sequential processes: (i) primary attachment of the bacterial cells to a surface and (ii) growth-based formation of a multilayer of bacteria involving intra- and intergenic cell-to-cell interactions and adhesions (Biswas & Biswas, 2005). Bacterial biofilms by microorganisms increase their resistance to stressful environments and also from antibiotics (Ramírez-Larrota, & Eckhard, 2022). HtrA protease also plays a vital role in bacterial biofilm formation. The role of HtrA in the expression of surface proteins and biofilm formation by Streptococcus mutants has been reported (Biswas & Biswas, 2005) and similarly reported that htrA mutation in S. mutans altered the expression of several surface factors and proper biofilm formation (Backert et al., 2018). Therefore, HtrA inhibitors might be helpful to clear pathogenic bacteria infection by destroying its biofilm phenotype (Xue et al., 2021).

HtrA inhibitor’s role in drug development The HtrA protein is versatile and can be used as a target in many antibacterial therapies. It also has a vital role in the extreme environment as it helps bacteria to invade and survive in a stress-resistant climate (Rai et al., 2018). All pathogenic bacteria and commensals in the microbiota express HtrA proteases that function as potent virulence

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or pathogenic factors to initiate bacterial pathogenesis. This attracted scientists to develop a suitable inhibitor against HtrA proteins. Targeting HtrA proteins offers several advantages, that is, it is secreted extracellularly and presented on the cell surface of bacteria and thus easily accessible to drug molecules; it shows distinct enzymatic active site and substrate recognition pattern; it cleaved proteoglycans, E-cadherin, and fibronectin as host factors that lead to bacterial pathogenesis. These characteristics make HtrA a potentially attractive candidate for novel therapeutic approaches to treat bacterial pathogenesis (Wessler et al., 2017). Helicobacter HtrA inhibitor (HHI) was the first reported molecular compound inhibiting H. pylori HtrA. This inhibitor blocked HtrA-mediated E-cadherin cleavage and subsequent bacterial transmigration across the epithelial layer (Wessler et al., 2017, Hoy et al., 2010). Several other inhibitors are also found that can inhibit H. pylori translocation and proliferation. Recently, a potent serine protease inhibitor (JO146) is also identified against C. trachomatis HtrA (CtHtrA), which was fatal when added to cultures during the mid-replicative phase (Ong et al., 2013). Inhibitors of HtrA proteins are summarized in Table 1. Table 1: Inhibitor of HtrA proteins. HtrA inhibitors Helicobacter pylori HtrA Inhibitor (HHI): HHI

HHI 

Inhibiton targets

HHI effectively blocks the lysis of Ecadherin on gastric epithelial cells by specifically binding to H. pylori HtrA inhibit the proliferation and migration of H. pylori

Xue et al., ; Song et al., 

HHI

HHI

HHI

References

Prevention of E-cadherin cleavage and selective migration across model gastric epithelium by specific binding to H. pylori HtrA

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Table 1 (continued) HtrA inhibitors Listeria monocytogenes HtrA Inhibitors: HHI

Chlamydia trachomatis HtrA Inhibitors: JO (Boc-Val-Pro-ValP (OPh) )

Inhibiton targets

References

Inhibition of protease activity of Listeria monocytogenes HtrA

Xue et al., ; Song et al., 

Targeting protease activity of C. trachomatis HtrA prevents development of infectious Chlamydia progeny

Xue et al., ; Song et al., 

Conclusion and future prospective New strategies are urgently needed to combat bacterial infections. At first glance, targeting a widespread bacterial enzyme is not straightforward. However, considering the HtrA-mediated host cell factor processing as a significant step in the pathogenesis of many infectious bacteria opens up a new perspective. Inhibiting extracellular HtrA by compounds that do not penetrate the bacterial membrane will not affect the colonization and survival of commensals, most likely. Thus, the sole interference of pathogens with their individual virulence/pathogenic factors with the epithelium will be limited. Potent HtrA inhibitors penetrating the periplasm of H. pylori might pave the way towards a targeted anti-H. Pylori treatment because H. pylori physiology essentially requires functional HtrA activity. Many antibiotics have also been developed that can inhibit these pathogens. However, due to the increasing problem of drug resistance in these bacteria, emerging new agents are needed to inhibit these bacteria more efficiently. Pathogen-selective HtrA inhibitors represent an excellent potential for drug discovery opportunities.

References Abfalter, C. M., Schubert, M., Götz, C., Schmidt, T. P., Posselt, G., & Wessler, S. (2016). HtrA-mediated Ecadherin cleavage is limited to DegP and DegQ homologs expressed by Gram-negative pathogens. Cell Communication and Signaling, 14(1), 1–12. Backert, S., Bernegger, S., Skórko‐Glonek, J., & Wessler, S. (2018). Extracellular HtrA serine proteases: An emerging new strategy in bacterial pathogenesis. Cellular Microbiology, 20(6), e12845.

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Bakker, D., Buckley, A. M., de Jong, A., van Winden, V. J., Verhoeks, J. P., Kuipers, O. P., & Corver, J. (2014). The HtrA–like protease CD3284 modulates virulence of Clostridium difficile. Infection and Immunity, 82 (10), 4222–4232. Biswas, S., & Biswas, I. (2005). Role of HtrA in surface protein expression and biofilm formation by Streptococcus mutans. Infection and Immunity, 73(10), 6923–6934. Boehm, M., Hoy, B., Rohde, M., Tegtmeyer, N., Bæk, K. T., Oyarzabal, O. A., & Backert, S. (2012). Rapid paracellular transmigration of Campylobacter jejuni across polarized epithelial cells without affecting TER: Role of proteolytic-active HtrA cleaving E-cadherin but not fibronectin. Gut Pathogens, 4(1), 1–12. Clausen, T., Southan, C., & Ehrmann, M. (2002). The HtrA family of proteases: Implications for protein composition and cell fate. Molecular Cell, 10(3), 443–455. Duguay, A. R., & Silhavy, T. J. (2004). Quality control in the bacterial periplasm. Biochimica Et Biophysica Acta (BBA)-molecular Cell Research, 1694(1–3), 121–134. Harrer, A., Boehm, M., Backert, S., & Tegtmeyer, N. (2017). Overexpression of serine protease HtrA enhances disruption of adherens junctions, paracellular transmigration and type IV secretion of CagA by Helicobacter pylori. Gut Pathogens, 9(1), 1–12. Hayashi, N., Matsukawa, M., Horinishi, Y., Nakai, K., Shoji, A., Yoneko, Y., & Gotoh, N. (2013). Interplay of flagellar motility and mucin degradation stimulates the association of Pseudomonas aeruginosa with human epithelial colorectal adenocarcinoma (Caco-2) cells. Journal of Infection and Chemotherapy, 19(2), 305–315. Hoy, B., Löwer, M., Weydig, C., Carra, G., Tegtmeyer, N., Geppert, T., & Wessler, S. (2010). Helicobacter pylori HtrA is a new secreted virulence factor that cleaves E‐cadherin to disrupt intercellular adhesion. EMBO Reports, 11(10), 798–804. King, L. B., Pangburn, M. K., & McDaniel, L. S. (2013). Serine protease PKF of Acinetobacter baumannii results in serum resistance and suppression of biofilm formation. The Journal of Infectious Diseases, 207(7), 1128–1134. Mehta, S., Nijhuis, A., Kumagai, T., Lindsay, J., & Silver, A. (2015). Defects in the adherens junction complex (E-cadherin/β-catenin) in inflammatory bowel disease. Cell and Tissue Research, 360(3), 749–760. Mochizuki, Y., Suzuki, T., Oka, N., Zhang, Y., Hayashi, Y., Hayashi, N., & Ohashi, Y. (2014). Pseudomonas aeruginosa MucD protease mediates keratitis by inhibiting neutrophil recruitment and promoting bacterial survival. Investigative Ophthalmology & Visual Science, 55(1), 240–246. Ong, V. A., Marsh, J. W., Lawrence, A., Allan, J. A., Timms, P., & Huston, W. M. (2013). The protease inhibitor JO146 demonstrates a critical role for CtHtrA for Chlamydia trachomatis reversion from penicillin persistence. Frontiers in Cellular and Infection Microbiology, 3, 100. Rai, N., Muthukumaran, R., & Amutha, R. (2018). Identification of inhibitor against H. pylori HtrA protease using structure-based virtual screening and molecular dynamics simulations approaches. Microbial Pathogenesis, 118, 365–377. Ramírez-Larrota, J. S., & Eckhard, U. (2022). An introduction to bacterial biofilms and their proteases, and their roles in host infection and immune evasion. Biomolecules, 12(2), 306. Schmidt, T. P., Perna, A. M., Fugmann, T., Böhm, M., Hiss, J., Haller, S., & Wessler, S. (2016). Identification of E-cadherin signature motifs functioning as cleavage sites for Helicobacter pylori HtrA. Scientific Reports, 6(1), 1–12. Skórko-Glonek, J., Wawrzynów, A., Krzewski, K., Kurpierz, K., & Lipińiska, B. (1995). Site-directed mutagenesis of the HtrA (DegP) serine protease, whose proteolytic activity is indispensable for Escherichia coli survival at elevated temperatures. Gene, 163(1), 47–52. Skorko-Glonek, J., Zurawa–Janicka, D., Koper, T., Jarzab, M., Figaj, D., Glaza, P., & Lipinska, B. (2013). HtrA protease family as therapeutic targets. Current Pharmaceutical Design, 19(6), 977–1009. Strauch, K. L., Johnson, K., & Beckwith, J. (1989). Characterization of degP, a gene required for proteolysis in the cell envelope and essential for growth of Escherichia coli at high temperature. Journal of Bacteriology, 171(5), 2689–2696.

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Wessler, S., Schneider, G., & Backert, S. (2017). Bacterial serine protease HtrA as a promising new target for antimicrobial therapy? Cell Communication and Signaling, 15(1), 1–5. Xue, R. Y., Liu, C., Xiao, Q. T., Sun, S., Zou, Q. M., & Li, H. B. (2021). HtrA family proteases of bacterial pathogens: Pros and cons for their therapeutic use. Clinical Microbiology and Infection, 27(4), 559–564. Yu, H., Schurr, M. J., & Deretic, V. (1995). Functional equivalence of Escherichia coli sigma E and Pseudomonas aeruginosa AlgU: E. coli rpoE restores mucoidy and reduces sensitivity to reactive oxygen intermediates in algU mutants of P. aeruginosa. Journal of Bacteriology, 177(11), 3259–3268. Zhang, S., Cheng, Y., Ma, J., Wang, Y., Chang, Z., & Fu, X. (2019). Degp degrades a wide range of substrate proteins in Escherichia coli under stress conditions. Biochemical Journal, 476(23), 3549–3564.

Biographical sketch Name: Kapil Singh Narayan Affiliation: New Bolton Center, University of Pennsylvania, Kennett Square 19348, PA USA Education: Ph.D.

Research and Professional Experience Postdoc Researcher working in multidiscipline including investigation of the rumen microbiota, methane mitigation strategies, The microbial-gut-brain axis (MGBA), and bacterial resistance acquisition investigation.

Shweta Dhanda✶, Kiran Bala, Priti, Anil K. Sharma, Anil Panwar, and Varruchi Sharma

Chapter 8 Potential targeted therapy for SARS-CoV-2: host serine proteases Abstract: The current pandemic highlights the narrow therapeutic preferences for treating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); therefore, further effective beneficial targets are needed. Angiotensin-converting enzyme 2 (ACE2) is a human receptor for the viral spike S-glycoprotein, which binds to it before host proteases activate it. Evidence suggests that the furin enzyme cleaves the viral S protein in infected host cells in the nonendosomal pathway. S protein is stimulated by the serine protease 2 (TMPRSS2) to aid in the virus’s effective entry, given that the S has already been cut by furin. TMPRSS2 is also essential for the dispersal of the virus inside the host. This chapter will discuss the significant functions of host proteases present in host cells which are infected with SARS-CoV-2. Even though there are currently at least five highly effective vaccinations, the emergence of novel diseasecausing mutations necessitates the creation of novel helpful drugs. Targeted suppression of host proteases as a treatment for infection is possible. Keywords: SARS-CoV-2, ACE2, TMPRSS2, therapeutics

Introduction Since the coronaviruses (CoVs) that caused the SARS in China (2002–2003) and in Saudi Arabia, the Middle East respiratory disease, other outbreaks of CoVs have drawn attention from around the world over the past decades. The CoV is currently causing global

✶ Corresponding author: Shweta Dhanda, National Centre for Veterinary Type Cultures, ICAR-National Research Centre on Equines, Hisar, Haryana, India, e-mail: [email protected] Kiran Bala, Department of Biochemistry, Om Sterling Global University, Hisar, Haryana, India Priti, Department of Biotechnology, K. L. Mehta Dayanand College for Women, Faridabad, Haryana, India Anil K. Sharma, Department of Bioscience and Technology, Maharishi Markandeshwar (Deemed to be University), Mullana, Ambala 133207, Haryana, India Anil Panwar, Department of Bioinformatics and Computational Biology College of Biotechnology, CCS Haryana Agricultural University Hisar, Haryana, 125004 Varruchi Sharma, Department of Biotechnology & Bioinformatics, Sri Guru Gobind Singh College Sector-26, Chandigarh (UT), India

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problems, and it was first discovered in China. Since its discovery, the COVID-19 pandemic caused by SARS-CoV-2 has resulted in millions of confirmed deaths worldwide, with the exact number continually changing as the situation evolves. Many infected individuals with respiratory infections and viral pneumonia have been noted as dangerous and quick person-to-person transfer occurrences (Ashour et al., 2020). The recognition of receptors is crucial for the infection and pathogenesis of the SARS coronavirus, and it also presents a promising target for therapeutic interventions (Wang et al., 2020a; Shang et al., 2020). Similar to SARS-CoV, SARS-CoV-2 utilizes the human angiotensin-converting enzyme 2 (ACE2) as its entry receptor. By exploiting this common receptor, SARS-CoV-2 can target and infect many of the same cell types as SARS-CoV (Rabi et al., 2020). Through in situ analysis, several cell types highly susceptible to SARS-CoV-2 have been found in various organs including AT2 lung cells, cardiomyocytes, thyroid stromal cells, testis cells, and ovary cells. However, certain cell types such as enterocytes, proximal kidney tubules, and cholangiocytes exhibit lower susceptibility to SARS-CoV-2 infection and are not considered primary targets (Zhou et al., 2020). The initial stage of viral invasion involves attachment to host cells, but entry into the cell requires the degradation of S protein by host proteases such as trypsin and cathepsins (Luan et al., 2020; Lambertz et al., 2019) Evidence suggests several respiratory viruses exploit host proteases to accelerate their transmission throughout the host’s body. SARS-CoV-2 can enter and become activated in two ways depending on the availability of proteases: through endocytosis and breaking of the SARS-COV-2-S in endosomes by cathepsin L or on the surface of the target cells by TMPRSS2 if it is coexpressed with ACE2 (Heurich et al., 2014). Virion internalization results from conformational changes in the glycoproteins brought about by the binding process (Shen et al., 2017). The utilization of host proteases as activators by SARS-CoV-2 suggests that inhibitors targeting these proteases could potentially be effective in treating both SARS-CoV and COVID-19 infections (Shrimp et al., 2020). This review will emphasize the significance of host proteases, specifically furin and TMPRSS2, in the infection process, while also exploring potential strategies to suppress the activity of these enzymes.

Virion SARS-COV-2 SARS-CoV-2 is an RNA-positive virus that contains a genome encoding various structural and membrane proteins necessary for its nucleocapsid (N), envelope (E), membrane (M), and spike (S) components (Zumla et al., 2016). Within the virion, the nucleocapsid consists of positive-sense RNA, phosphorylated N-protein, and S and HE spike proteins, all surrounded by phospholipid bilayers (Wu et al., 2020a). SARS-CoV-2 shares approximately 80% similarity with the human SARS-CoV, exhibiting similar pathophysiology and biochemical interactions (Matsuyama et al., 2010). The S protein plays a crucial role in viral entry by facilitating receptor binding and determining the

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host range. It is also a prime target for vaccinations and neutralizing antibodies (Heurich et al., 2014; Hoffmann et al., 2020a). Comprising two active subunits, S1 and S2, the spike protein enables viral attachment to the host ACE2 through a receptorbinding domain (RBD) in S1, while S2 possesses characteristics necessary for virus-cell membrane fusion (Figure 1). The unique composition of SARS-CoV-2’s spike protein contributes to its high susceptibility to cleavage at the S1/S2 catalytic domain, particularly due to the presence of arginine residues.

SARS CoV 2

Attachment

Cleavage and Activation

TMPRSS2 ACE2 Cell membrane of Host Cell

Figure 1: Interaction of SARS CoV-2 with host cell membrane receptor.

In coronaviruses (CoVs), the RBD may exist in either a flat or standing-up position. The flat position allows for receptor binding, while the standing-up position prevents it (Yuan et al., 2017). Unlike SARS-CoV, the SARS-CoV-2-S spike protein typically exists in the lying-down position, which is less accessible to ACE2 despite having a higher affinity. As a result, the entire spike protein of SARS-CoV-2 has a lower or equivalent receptor binding affinity compared to SARS-CoV (Shang et al., 2020). While SARS-CoV RBDs can bind to ACE2 with higher affinity than SARS-CoV, the spike glycoprotein of SARS-CoV-2 cannot connect to human ACE2 with higher affinity (Shang et al., 2020).

SARS-COV-2 entry and priming by the host proteases The heightened virulence of SARS-CoV-2 compared to SARS-CoV can be attributed, in part, to its strong binding affinity for human receptors (Lu et al., 2020; Wu et al., 2020a). Proper processing of the viral S protein by cellular proteases throughout various stages of the viral life cycle plays a critical role in the virus’s ability to infect host cells (Heurich et al., 2014). Similar to SARS-CoV, SARS-CoV-2 engages with human cells (Ragia and Manolopoulos, 2020). The separation of S1 and S2 subunits in SARS-CoV-2

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occurs through proteolysis of the 2-peptide links (Arg685-Ser686), and the activation or priming of these subunits, as well as the dissociation and subsequent activation of the S protein, is facilitated by the proteolytic cleavage of two specific polypeptide bonds (Arg685-Ser686) (Fuentes Prior, 2021). Following proteolytic cleavage at the S1 and S2 site, the C-terminal of the S2 subunit is responsible for the attachment of the viral envelope to the host cell membrane, leading to fusion (Benton et al., 2020). The N-terminal domain of the S1 subunit plays a crucial role in binding to the peptidase domain of ACE2. In SARS-CoV-2 virions, the S proteins are preactivated through furin-mediated proteolytic cleavage, which involves breaking the Arg815-Ser816 bond to initiate the fusion mechanism. The distinctive characteristics of the S protein are vital for the fusion and penetration of SARS-CoV-2 into human cells (Fuentes-Prior, 2021). Furthermore, TMPRSS2 is necessary for the early activation of the S protein and the entry of SARS-CoV-2 (Ragia and Manolopoulos, 2020). The interaction of SARS to ACE2 may facilitate the endocytosis of the virus (Zhao et al., 2021). When SARS-S binds to ACE2, it undergoes structural modifications that can increase its vulnerability to proteolytic enzymes. Cathepsin L, located in the endosome, cleaves the SARS-S, enabling the S protein to become active and fuse with the endosomal membrane. As the endosome acidifies, the HA configuration changes and activates, exposing the viral fusion peptide and allowing it to open the endosomal membrane (Simmons et al., 2013; Shen et al., 2017). Additionally, the transcription and enzyme activity initiated by SARS-CoV-2 cathepsin L can increase viral infection (Zhao et al., 2021).

Host cell proteases The spike protein of SARS-CoV-2 contains cleavage sites targeted by cellular proteases, resulting in the exposure of fusion sequences. Studies have reported that SARS-CoV-2 entry can be facilitated by both lysosomal and cell surface proteases, with furin having a particularly significant impact (Shang et al., 2020). The efficient role of host proteases in SARS-COV-2 infection is summarized in Table 1. Table 1: Role of serine proteases and their inhibitors in SARS-COV-2 infection. S. No. Serine Inhibitors proteases

Function

References

.

Furin

It is a proprotein convertase

Feliciangeli et al., 

.

Cathepsins E-

It breaks peptide bond and helps in the entry of virus in host cell

Sahebnasagh et al., 

Dec-RVKRCMK

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Table 1 (continued) S. No. Serine Inhibitors proteases

Function

References

.

TMPRSS

Camostat mesylate

It acts as an activator of viral infection by Iwatapromoting viral replication and syncytium assembly Yoshikawa et al., b

.

Cellular elastase

Alpha- antitrypsin (AAT)

It releases proinflammatory cytokines

Bai et al., 

.

Plasmin

Aprotinin

It cleaves additional furin sites in the SARS-CoV- S protein, leading to increased viral infectivity

Ji et al., 

Furin The proprotein convertase family includes furin and furin-like proteases. Furin, a type I transmembrane protein present in all eukaryotic cells, acts as a proprotein convertase. Its activation occurs in the acidic pH environment of the trans-Golgi network (Feliciangeli et al., 2006). Furin plays a crucial role in the maturation of various cell surface proteins, including surface receptors and hormones, through proteolytic cleavage. It is also involved in the degradation of viral envelopes in several viruses (Garten, 2018). Recent discoveries indicate that the presence of a furin cleavage site on the S protein of SARS-CoV-2 contributes to its heightened contagiousness compared to other coronaviruses, enabling more efficient spread of the disease.

Cathepsins Human cathepsins are a group of endosomal proteases that exhibit a wide range of proteolytic activity under acidic pH conditions. The activation of cathepsins within lysosomes plays a significant role in the entry of MERS-CoV and SARS-CoV-2 through endocytosis (Sahebnasagh et al., 2020). According to Vargas-Alarcón et al. (2020), cathepsin L is known for its ability to cleave peptide bonds. It has been implicated in the processing of SARS-CoV-2 and Ebola glycoproteins (Pislar et al., 2020; VargasAlarcon et al., 2020). Recent studies have examined the involvement of cathepsins in SARS-CoV-2 entry. While CA-074 did not show significant effects on SARS-CoV-2 entry, the importance of cathepsin L in the entry of the virion has been emphasized (Pislar et al., 2020).

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TMPRSS2 Membrane serine proteases, known as TTSPs, play an essential role in various physiological processes (Hoffmann et al., 2018). TTSPs as the activator of viral infection. TMPRSS2 and HAT cause FLUAV-HA activation and FLUAV dissemination in the host (Hoffmann et al., 2018). According to several previous studies (Matsuyama et al., 2010; Shulla et al., 2011; Simmons et al., 2013; Iwata Yoshikawa et al., 2019a), the presence of TMPRSS2 significantly promotes coronavirus replication and syncytium assembly in vitro and in vivo (Iwata-Yoshikawa et al., 2019b). Furthermore, by activating spike S for cell-cell fusion and virus-cell entry and by neutralizing antibodies that reduce viral detection, TMPRSS2 may enhance viral pathogenicity and transmission (Glowacka et al., 2011).

Cellular elastase Upon exposure to the virus, neutrophils release elastase as a component of the hostdefensive system (Vargas-Alarcón et al., 2020). Induction of proinflammatory cytokines response that increases elastase activity by neutrophil vesicles can lead to enlarged inflammatory responses such as vasodilatation, which promote acute lung impairment (Bai et al., 2020). Under normal circumstances, endogenous protease inhibitors control the activity of NE. However, under pathophysiological conditions, neutrophil oxidants can degrade proteins like collagen-IV and elastin, leading to the loss of the endothelial barrier and intrusion into bronchoalveolar space as well as the hydrolysis of host extracellular matrix molecules (Thierry, 2020., Korkmaz et al., 2020).

Plasmin The cleavage of surface proteins by viruses can facilitate host cell infection or evasion of the immune system, and one mechanism involves the conversion of plasminogen to plasmin (Medcalf et al., 2020). Plasmin, in turn, can cleave additional furin sites in the S protein of SARS-CoV-2, thereby enhancing viral infectivity (Ji et al., 2020). In the early stages of viral infections, plasmin may stimulate the release of cytokines and contribute to the exacerbation of edema, potentially worsening the severity of the disease (Medcalf et al., 2020). Increased levels of plasminogen have been observed in COVID-19 patients (Jin et al., 2020). Moreover, plasmin has the capability to break down hyaline membranes, a characteristic histopathological feature of acute respiratory distress syndrome (ARDS) caused by SARS-CoV-2 (Henry et al., 2020).

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Role of furin in SARS-CoV-2 infection The genome sequence of SARS-CoV-2-S contains a potential polybasic furin cleavage site where amino acid residues (–PRRA–) are inserted between the S1 and S2 subunits. This furin cleavage site has been found to be capable of cleaving and activating the viral spike protein according to genomic markers of SARS-CoV-2 (Mallapaty, 2020). It is worth noting that this furin-like cleavage site is absent in SARSCoV-1, pangolin, and Bat-CoV (Vankadari, 2020; Coutard et al., 2020), highlighting its distinct presence in SARS-CoV-2. Furin also interacts with the SARS-CoV-2-S protein (Vankadari, 2020), and the precleavage of S proteins is required for the subsequent formation of the spike protein by TMPRSS2 (Hoffmann et al., 2020b). To initiate membrane fusion between viral and human cells and allow the viral genome to enter the host cell cytoplasm, the viral spike S glycoprotein requires cleavage at the S1 or S2 and S2” sites. The processing of the S2” site is carried out by TMPRSS2, while the S1/S2 site is cleaved by furin. These enzymes function independently and cannot work together (Hoffmann et al., 2018). The preactivation of furin enables SARSCoV-2 to rely less on host cells, enhancing its ability to enter target cells that have relatively low levels of lysosomal cathepsins and/or TMPRSS2 (Shang et al., 2020). Furin expression has been detected in various tissues and organs such as the intestine, heart, and oral mucosa, suggesting that they may be additional target organs for the coronavirus (Mei et al., 2020). The widespread production of furin in certain tissues and organs may explain the high pathogenicity and mode of transmission of SARS-CoV-2 (Wang et al., 2020a). Consequently, during SARS-CoV-2 infection, the levels of furin may contribute to various symptoms (Dittmann et al., 2015). For instance, the utilization of furin to facilitate SARS-CoV-2 entry into cardiomyocytes may contribute to the cardiac dysfunction observed in COVID-19 patients. Furthermore, the presence of furin protease activity in oral mucosal cells may make them more susceptible to SARS-CoV-2 (Mei et al., 2020). The role of furin and its inhibitors is summarized in Table 1.

Role of TMPRSS2 in viral infection Emerging evidence indicates that the spread of SARS-CoV-2 and related viruses, including MERS-CoV, SARS-CoV, and influenza A (FLUAV), relies on the activity of TMPRSS2 (Hoffmann et al., 2020a; Kim J et al., 2020). Additionally, it has been observed that SARS-CoV-2 infection exhibits high susceptibility to the VeroE6 cell line, which expresses TMPRSS2 (Matsuyama et al., 2020). Both TMPRSS2 and cathepsins play a cumulative role in facilitating the entry of SARS-CoV-2 into specific cells, such as those in the lungs, through the involvement of a calcium-dependent proprotein convertase mechanism (Shang et al., 2020).

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The coexpression of TMPRSS2 and ACE2 in lung cells suggests the potential for SARS-CoV transmission throughout the respiratory tract in humans (Heurich et al., 2014). TMPRSS2 facilitates SARS-CoV infection through two distinct mechanisms. First, it cleaves ACE2, enhancing viral invasion without activating SARS-S for entry and promoting viral uptake through cathepsin L-dependent entry. Second, TMPRSS2 cleaves the spike protein at the host cell surface, facilitating fusion between the viral and host cell membranes (Heurich et al., 2014). The metalloprotease ADAM17 and TMPRSS2 compete to cleave ACE2, with TMPRSS2-mediated cleavage alone inducing SARS-Sdriven entry (Heurich et al., 2014). ADAM17 promotes SARS-CoV entry by facilitating ACE2 shedding into the extracellular environment, leading to increased release of TNF-α and IL-6 receptors. TNF-α signaling enhances ADAM17 activity, while TNF-α itself promotes autocrine and paracrine activity. High levels of ACE2 shedding by ADAM17 result in ACE2 downregulation, leading to elevated angiotensin II levels and further increased ADAM17 activity (Gheblawi et al., 2020). Studies suggest that TMPRSS2 may regulate mitochondrial activity through the estrogen-related receptor (Xu et al., 2018). Viral RNAs can enter the mitochondria during infection to manipulate and utilize host mitochondria. SARS-CoV-2 has the ability to suppress host immunity in COVID-19 cases by affecting ubiquitination and disrupting mitochondrial function through various pathways. One potential mechanism involves the regulation of host USP30 by viral ORFs, which control mitochondrial dynamics and homeostasis, such as fusion and fission. Specific nucleotides in SARS-CoV-2’s open-reading frame 3a (ORF3a) can target a specific locus in mitochondrial USP30 transcripts. When viral ORFs interact with host mitochondria, mitochondrial DNA (mtDNA) is released into the cytoplasm, triggering mtDNA-induced inflammation and suppressing innate and adaptive immunity. Ultimately, the virus induces mitochondrial collapse, leading to cell death (Singh et al., 2020).

The TMPRSS2 gene and its expression in cells The TMPRSS2 gene, located on human chromosome 21, is approximately 44 kb long and consists of 14 exons and 13 introns. Studies have shown that prostate cancer cells exhibit higher expression of TMPRSS2 (Paoloni-Giacobino et al., 1997). Notably, the TMPRSS2 gene possesses multiple 15-bp androgen response elements (AREs) upstream of the transcription start site at position 148 (Lin et al., 1999; Afar et al., 2001). Androgenic hormones activate the TMPRSS2 gene in prostate cancer cells, likely mediated through the androgen receptor (Antalis et al., 2011; Shen et al., 2017; Ashour et al., 2020). Mouse knockout models have demonstrated that these mice are unaffected by the propagation and pathogenicity of various subtypes of FLUAV. As mentioned earlier, androgens and their receptor can regulate TMPRSS2 (Chen et al., 2019), and the presence of AREs on the TMPRSS2 gene promoter may contribute

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to its severity (Zununi Vahed et al., 2020). The promoter region of the human TMPRSS2 gene contains a guanine-rich region that can form G-quadruplex secondary structures, which can inhibit or limit TMPRSS2 transcription in the presence of potassium ions (Shen et al., 2020). Furthermore, investigations into TMPRSS2 gene variants associated with TMPRSS2 overexpression have revealed a correlation with severe flu cases (Hoffmann et al., 2018). Susceptibility to H7N9 influenza has been strongly linked to genetic variations in TMPRSS2 including two identified single nucleotide polymorphisms (SNPs) (rs383510 and rs2070788) (Cheng et al., 2015). Functional SNPs and epigenetic mechanisms of the TMPRSS2 gene play significant roles in differential susceptibility to SARS-CoV-2 among various populations (Paniri et al., 2020). TMPRSS2 gene SNPs are more prevalent in European and American populations compared to Asian populations, suggesting that these groups may be more susceptible to SARS-CoV -2 infection (Irham et al., 2020). It is worth noting that the expression of TMPRSS2 varies significantly among individuals and may be positively correlated with the severity of COVID-19 (Lucas et al., 2008). Clinical indications of COVID-19, such as elevated liver enzymes, acute kidney injury, and cardiac damage, are associated with the disease’s complications. The expression of ACE2 and TMPRSS2 enables SARS-CoV-2 to enter ocular surface cells, while their coexpression in prostate epithelial cells may contribute to the pathogenicity of COVID-19 in men compared to women (Song et al., 2020). TMPRSS2 and TMPRSS4 promote SARSCoV-2 entry into intestinal cells by enhancing spike fusogenic activity (Zang et al., 2020). In various gene expression datasets, it has been observed that TMPRSS2 and ACE2 levels are significantly higher in nasal epithelium compared to saliva and blood. However, their levels decrease in lower airway tissues. Notably, children have lower expression levels of these genes in nasal and bronchial tissues compared to adults. These findings suggest that differences in the expression levels of TMPRSS2 and ACE2 genes in airway tissue may contribute to the severity of SARS-CoV-2 infection in adults and children (Saheb et al., 2020). The analysis of the nasal respiratory transcriptome in offspring has revealed mechanisms that impact SARS-CoV-2 infection. It has been discovered that the interferon response to respiratory viruses increases ACE2 expression, while the gene for the mucus secretory network, TMPRSS2, is upregulated by the action of IL-13α (Sajuthi et al., 2020).

Pharmacological methods to target host proteases for COVID-19 Unfolding the characteristics of the RBD can open up novel possibilities for developing protease inhibitors and blocking spike cleavage sites. Recent research suggests that host proteases are essential for activating SARS-CoV-2 in human epithelial cells, making them potential therapeutic targets for treating COVID-19 (Bestle et al., 2020a).

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Furin and therapeutic TMPRSS2 inhibition could be utilized as a treatment for COVID19. Various compounds and drugs can alter the activity and expression of TMPRSS2, including diminazene, naphthofluorescein, MI-1900, aprotinin, TMPRSS2 protease inhibitors, excavatolide M, geniposide, microcarpin, and others (Saadat et al., 2021).

Furin inhibitors Furin-like enzymes are critical for numerous pathways; as a result, inhibiting these enzymes in the long term may have adverse and harmful effects (Hasan et al., 2020). However, furin inhibition for a short duration may have beneficial impacts (Sarac et al., 2002). It may be necessary to use a combination of protease inhibitors to achieve satisfactory results. Various furin inhibitors targeting separate SARS-CoV-2 proteases might be a promising treatment approach (Wu et al., 2020b). TMPRSS2 and furin inhibitors can be administered together to target these proteases (Bestle et al., 2020a).

Modifying TMPRSS2’s expression and activity In addition to SARS-CoV, TMPRSS2 is vital for the reproduction and pathogenesis of the influenza A virus in mice (Lambertz et al., 2019). The fact that this subtype can also affect people highlights the importance of TMPRSS2 for developing drugs to combat various IAV subtypes (Lambertz et al., 2019). TMPRSS2 plays a role in the persistence and virulence that can stimulate invasion at pre- and postbinding stages (Nickols and Dervan, 2007). TMPRSS2 is involved in coronavirus (Shirato et al., 2013), influenza virus (Shen et al., 2020), and hepatitis C virus (Esumi et al., 2015). Especially for SARS-CoV-2, it would be a desirable alternative against various respiratory infections. The AREs, as noted earlier, are essential for the expression of TMPRSS2 and can be potential therapeutic targets. Nichols and Dervan (2007) reported that polyamide could bind to the AREs. Wang et al. (2020) discovered that estrogen-related substances can downregulate TMPRSS2 by analyzing publicly available gene expression data. These drugs represent potentially effective therapeutic options for managing COVID19 (Wang et al., 2020). In the presence of potassium ions, the human TMPRSS2 gene promoter can adopt G-quadruplex structures that have the capacity to impede or diminish gene transcription (Shen et al., 2020). Notably, benzoselenoxanthene analogues have demonstrated a significant ability to decrease the expression of TMPRSS2 and inhibit the growth and transmission of the influenza A virus by preserving the G-quadruplex structure (Shen et al., 2020). Consequently, modulating the expression of TMPRSS2 mRNA through the use of G-quadruplex structure stabilizers holds great promise as a potential strategy for the development of novel small-molecule drugs to combat SARS-CoV-2.

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Antioxidants play a crucial role in maintaining the balance of proteases, which can effectively hinder viral infection. Antioxidants are chemicals that can prevent or mitigate the damage caused by free radicals to cells. In prostate cancer cell lines, the oxidative transcriptional factor Nrf2 can affect the protease/antiprotease balance by downregulating the expression of TMPRSS2, thus protecting against lung problems (Meyer and Jaspers, 2015). Nrf2 plays a crucial role in eliminating oxidative stress and positively affects the respiratory epithelium’s response to viral infections of the respiratory system. Antioxidants are available in many forms worldwide, and sulforaphane (SFN), a sulfur-containing chemical molecule naturally present in cruciferous vegetables, is a potent antioxidant that belongs to the isothiocyanate class. SFN has antibacterial activity and can reduce oxidative stress and inflammation. It also lowers TMPRSS2 levels, providing immunity from infection (Meyer and Jaspers, 2015). The information available indicates that Nrf2 has a negative effect on TMPRSS2 (Kesic et al., 2011). Bromhexine hydrochloride (BRH), an inhibitor of TMPRSS2, has demonstrated its ability to reduce metastasis in mouse models of cancer. As an FDA-approved mucolytic cough suppressant, BRH is being investigated as a potential treatment for coronavirus infections due to its minimal side effects (Fu et al., 2020; Tolouian et al., 2020). A clinical pilot study conducted by Li et al. on Chinese patients showed positive effects of BRH in mild cases of COVID-19, leading to improved chest computed tomography findings within 20 days (Irham et al., 2020). Nafamostat, an anticoagulant, is another medication known to inhibit TMPRSS2 activity (Yamamoto et al., 2016). It has demonstrated the ability to reduce viral entry and prevent MERS-CoV infection in vitro. Recent evidence suggests potential benefits of nafamostat in COVID-19 patients (Asakura and Ogawa, 2020; Hoffmann et al., 2020). Furthermore, blocking TMPRSS2 may prevent virus fusion and impede viral invasion (Jang and Rhee, 2020). Targeting TMPRSS2 could provide comprehensive therapeutic benefits against respiratory coronavirus infections. Plasminogen activator inhibitor1 (PAI-1), an effective membrane-anchored serine protease inhibitor, has shown the ability to suppress the influenza virus in mice by inhibiting TMPRSS2-mediated hemagglutinin cleavage (Dittmann et al., 2015). Based on their studies, Mc Cord et al. (2020) have also suggested PB125 as a potential treatment option for COVID-19 patients.

Cathepsin inhibitors The study revealed a significant reduction (approximately 92.5%) in the entry of SARS-CoV-2 S pseudovirions into 293/hACE2 cells, emphasizing the importance of calpain or cathepsin in the viral entry process. Specifically, the inhibition of lysosomal cathepsin L resulted in a 76% decrease in the entry of SARS-CoV-2 S pseudovirions, suggesting its involvement in priming the SARS-CoV-2 S protein in 293/hACE2 cells. On

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the other hand, the inhibition of Cathepsin B did not have any effect on virus entry (Korkmaz et al., 2020; Sahebnasagh et al., 2020). Combining E-64D and camostat mesylate completely inhibited SARS-CoV-2 Sprotein-mediated invasion in Caco-2 cells and Vero TMPRSS2 cells, supporting the notion that both enzymes are required for cell priming (Hoffmann et al., 2020a). The use of a cathepsin L inhibitor has been shown to prevent coronavirus infection by blocking virus penetration on the host cell surface and impeding viral material release and replication within host cell endosomes (Liu et al., 2020).

Other protease inhibitors The use of sivelestat, a neutrophil elastase inhibitor has demonstrated its potential as a therapeutic strategy for the treatment of coagulopathy or acute lung injury (ALI)/ ARDS in COVID-19 patients. It has been shown to improve the condition of the alveolar epithelium and reduce damage to the vascular endothelium, providing promising outcomes (Sahebnasagh et al., 2020). By reducing the activity of antiproteases like plasmin, it is possible to hinder the ability of SARS-CoV-2 to infect respiratory cells and potentially enhance clinical outcomes for patients (Ji et al., 2020).

Clinical trials A clinical trial conducted by Ansarin et al. (2020) in Tabriz, Iran, examined the early administration of bromhexine. The treated group showed a significant reduction in ICU hospitalizations, intubations, and mortality compared to the control group after receiving oral bromhexine. However, there were no notable differences in the length of hospital stay between the two groups (Ansarin et al., 2020). In another study an open-label randomized controlled pilot research, oral BRH was found to improve chest computed tomography, oxygen therapy requirement, and discharge rates after 20 days. However, these results did not reach statistical significance (Shang et al., 2020). Hofmann-Winkler et al. (2014) evaluated the effectiveness of camostat mesylate in COVID-19 patients with organ failure. Their study demonstrated that the camostat mesylate-treated group experienced a decrease in the sepsis-related Organ Failure Assessment score, while the hydroxychloroquine group showed persistently high scores. Camostat mesylate treatment also resulted in reduced disease severity, inflammatory markers, and improved symptoms. Within eight days of treatment, patients receiving camostat mesylate exhibited decreased disease severity, inflammatory markers, and improved oxygenation compared to those receiving hydroxychloroquine (HofmannWinkler et al., 2020).

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Furthermore, Doi et al. (2020) demonstrated the efficacy of nafamostat mesylate in combination with favipiravir in critically ill COVID-19 patients at Tokyo Hospital. Nafamostat therapy targeted the virus’s entry into host epithelial cells, preventing intravascular coagulopathy (Doi et al., 2020).

Conclusion and future prospective A low rate of RBD mutations in the SARS-CoV-2 virus has been associated with immune evasion and a strong binding affinity for human ACE2 (hACE2). This unique characteristic of the RBD facilitates efficient cell entry and may contribute to the virus evading immune surveillance. Additionally, preactivation of the spike protein by furin enhances the virus’s ability to enter certain cells, further contributing to its hidden nature and potential for rapid spread. Among the various host factors required for SARS-CoV-2 entry, suppressing the transcription of TMPRSS2 shows promise. The discovery of TMPRSS2 was initially made in the context of cancer research, where its inhibition was found to reduce the severity of prostate cancer in a mouse model. Subsequently, it was found that aprotinin, an HAactivating protease inhibitor, could effectively reduce influenza virus infections, providing additional evidence for the potential inhibition of TMPRSS2. Inhibitors of TMPRSS2 have the potential to mitigate or prevent SARS-CoV-2 infection, offering an economically viable approach to blocking TMPRSS2-mediated virus penetration into host cells. Studies have shown promise in the early administration of bromhexine as a TMPRSS2 inhibitor during the early stages of COVID-19 (Saadat et al., 2021). Importantly, TMPRSS2 appears to have no significant role in normal organ function and its blockade does not interfere with normal development and homeostasis. However, there are contradictory reports regarding the effectiveness of TMPRSS2 inhibition in treating COVID-19.

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Biographical sketch Name: Shweta Dhanda Affiliation: Kurukshetra University, Kurukshetra Education: M.Sc. (Biotechnology) & Ph.D. (Biochemistry)

Research and Professional Experience Young professional fellow position at ICAR-National Research Centre on Equines, Hisar, since 20 September 2022.

Publication from the Last 3 years Research article: In vitro studies on anti-inflammatory, antioxidant and antihyperglycemic activities of potential probiotic Pediococcus acidilactici NCDC 252 in Research Journal of Biotechnology (Vol. 16 (12), 2021). Research article: Identification, purification, characterization and biopreservation potential of antimicrobial peptide of Pediococcus acidilactici NCDC 252 in Journal of Food Processing and Preservation (Communicated). Book chapter: Current scenario of RNA therapeutics application in Systems Biomedicine in RNA therapeutics (Communicated).

Shweta Dhanda✶, Kiran Bala, Anil K. Sharma, Anil Panwar, and Varruchi Sharma

Chapter 9 Role of serine proteases in fertility Abstract: Serine proteases are necessary proteolytic enzymes in cell differentiation, cell death, immunology, reproduction, and embryonic development. Some of the serine proteases genes are present in humans and rats, functionally conserved amongst metazoans and required for male fertility. The testis is an organ that undergoes several dramatic remodeling processes during development and maturation. The plasminogen activation system and a complex network of complementing inhibitors and proteases are essential for these events. The plasminogen activation system plays a crucial role in the development and maintenance of testicular morphology. A PRSS55 is a conserved chymotrypsin-like serine protease in species of mammals. It is a membrane protein that is GPI-anchored, is mainly expressed in adult mouse testes, and is found chiefly on the seminiferous tubules and sperm acrosomes. Studies showed that infertile mice lack PRSS55 despite normal reproduction-related parameters. Serine protease also regulates the motility of sperm in the female reproductive tract. Some studies demonstrated that the pan-serine protease inhibitor prevented semen liquefaction in vivo. Furthermore, this inhibitor significantly reduces the quantity of sperm in the oviduct of female mice. This chapter will confer and emphasize the role of various serine proteases in regulating male fertility in mice. Specifically, we will examine how these proteases affect the in vivo migration of sperm and the in vitro interaction of sperm and egg. Additionally, we will investigate PRSS55’s ability to influence the development of the sperm precursor (ADAM3) and expression of numerous genes in the testis. Keywords: male fertility, fertilization, embryonic development, PRSS55, differentiation

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Corresponding author: Shweta Dhanda, National Centre for Veterinary Type Cultures, ICAR-National Research Centre on Equines, Hisar, Haryana, India, e-mail: [email protected] Kiran Bala, Department of Biochemistry, Om Sterling Global University, Hisar, Haryana, India Anil K. Sharma, Department of Biosciences and Technology, Maharishi Markandeshwar (Deemed to be University), Mullana, Ambala, 133207, Haryana, India Anil Panwar, Department of Bioinformatics and Computational Biology, College of Biotechnology, CCS Haryana Agricultural University, Hisar, Haryana, 125004 Varruchi Sharma, Department of Biotechnology and Bioinformatics, Sri Guru Gobind Singh College Sector-26, Chandigarh (UT), India https://doi.org/10.1515/9783111325040-009

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Introduction Furthermore, reproduction is one of the most essential characteristics of living organisms for survival, and the testis is a particularly potent organ throughout the life cycle, including during synaptogenesis and adulthood. It is divided into two primary portions: the seminiferous tubules and interstitium, which contain steroidogenic Leydig cells. The seminiferous tubules are covered by peritubular cells. Sertoli cells are vital for the production of sperm. They serve as follicle-stimulating hormone (FSH) and testosterone target cells, essential for initiating and maintaining spermatogenesis. Sertoli cells create the tubules and give nutritional and structural support for growing germ cells (Griswold 1998; Figure 1).

Leydig cells

Sertoli cells

Seminiferous cord Germ cells

Testis

Cell proliferation

Fertility Figure 1: Development of testicular function.

The Sry gene is found on the Y chromosome, which regulates whether the gonads develop into a testis or an ovary when they first appear as an outgrowth. Additionally, Sry causes Sertoli cells to differentiate, and with peritubular cells, they generate Mullerianinhibiting substances and consolidate to form cords. As a result, Leydig cells begin to discriminate in the interstitial environment and begin to produce testosterone (Wilhelm et al., 2007). Dynamic changes occur during spermatogenesis development, including the cords’ transition into tubules at puberty. In adulthood, germ cells continue to differentiate as they migrate along the basal to the apical axis of the tubule epithelium. Finally, spermatids are discharged from the seminiferous epithelium’s apex into the tubule lumen, where they develop into spermatozoa (Russell 1980).

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Existing research has revealed that several proteinases and their associated inhibitors participate in this spatial and highly coordinated process, both throughout the testis’ maturation and adolescence (Fritz et al., 1993a; Charron & Wright 2005).

Serine proteases and their inhibitors Proteolytic events are necessary for various significant mechanisms that control the behavior and fate of numerous proteins. As a result, proteases play a crucial role in almost all intricate processes involving tissue upkeep and development. Modifications in the configuration and transcriptional regulation of proteases are also implicated in melanoma, arthritic conditions, osteoporosis, neurological disorders, and cardiometabolic illnesses. Completing the human genome sequence project has demonstrated the significance of proteolytic degradation in human biology, with proteases or protease inhibitors playing important roles (Puente & Lopez-Otin 2004). The proteases activity is monitored at each level in three ways: (i) at production levels, (ii) at stimulation levels (by producing inactive pro-form), and (iii) by synthesizing specific inhibitors. Serine proteases comprise about one-third of the proteolytic enzymes in the genome of rats, mice and humans (Puente & Lopez-Otin 2004). The serine protease family is one of the largest and earliest known multigene proteolytic groups, and it plays several well-known functions, including thrombosis, blood clotting, platelet aggregation, and fibrinolysis. Few serine proteases were listed in Table 1 with their function. Table 1: Serine proteases and their function. S. No. Serine proteases

Function

References

.

HGFA

Convert proHGF into active HGF

Odet et al., 

.

Hepsin

Activate proHGF

Lijnen, H. R., 

.

Kallikrein-

Expressed in Leydig cells and degrades fibronectin

Odet et al., 

.

Matriptase-

Break collagen and fibronectin

Odet et al., 

.

Plasminogen activators (PA) (tPA and uPA)

Convert plasminogen into Lijnen, plasmin H. R., 

.

aPC

Anticoagulant

Odet et al., 

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Plasminogen activators (PAs) in testis The first serine proteases discovered in the testis were PAs, also produced in the testis. The two types of PAs were initially believed to be synthesized by Sertoli cells, and FSH was thought to promote tissue type (tPA) while decreasing urokinase-type (uPA) levels in the gonads of the rat. Cytokines such as interleukin 1alpha (IL1α) and tumor necrosis factor-alpha (TNF) play a significant regulatory role in PAs. Additionally, germ cells have been found to positively regulate PAs both in mixed culture and with the addition of a germ cell-conditioned medium. However, when anti-FGF2 or anti-TNFα antibodies were added simultaneously with the natal fibroblast medium, the effects were not reversed, indicating that neither of these two substances actively promotes PAs (Le Magueresse-Battistoni, 2007). Pachytene and diakinetic spermatocytes, which are interestingly immunoreactive to tPA, suggest that a tPA proteolytic event occurs at these cells’ surface level. Determining whether the immunoreactivity matches a tPA-binding protein/tPA receptor found on the germ cells would be intriguing. Annexin II is a strong contender for this role due to its known function as a tPA receptor and the presence of its mRNA in a testis cDNA library (Charron & Wright 2005). In contrast, the urokinase receptor has been discovered in Leydig and Sertoli cells (Odet et al., 2004), suggesting that plasminogen-related proteolysis may occur near these cells’ membranes. By enhancing uPAs affinity for vitronectin, uPA binding to its receptor encourages cell adhesion (Dellas & Loskutoff 2005, Lijnen 2005). Therefore, the discovery of vitronectin in early spermatids and the finding that proteinase nexin-1 (PAI-1) is a component of peritubular and Sertoli cells (Fritz et al., 1993) are also noteworthy. Sertoli cell PAI-1 may control spermatid adherence by obstructing uPAR’s ability to bind to vitronectin. Among the PAs, TGFβ1 (transforming growth factor-β1), FGF2, TNF, and FSH play significant regulatory roles. Conversely, FSH and testosterone upregulate PAI-3 (SERPINA5) (Anway et al., 2005; Meachem et al., 2005). Notably, androgens also increase the activity of serine proteases such as spin, SERPINs A3n, and A12n (Denolet et al., 2006). Many serine proteases and their inhibitors are found in germ cells (Odet et al., 2006). However, a disintegrin and metalloproteases (ADAMs) have been shown to act within the seminiferous epithelium and are predicted to have a similar function for vitronectin and uPA receptor (Bronson et al., 2000; Rubinstein et al., 2006; Blasi & Carmeliet 2002). The role of Leydig cells in the testicular protease family is still poorly understood. It has been found that Leydig cells exhibit a variety of serine proteases and their associated inhibitors (Puente & Lopez-Otin 2004; Matsui & Takahashi 2001). Studies have also found that many of these serine proteases and inhibitors found in Leydig cells are regulated by luteinizing hormone (LH)-hCG, suggesting that similar transcriptional signals may be responsible for the expression of these molecules (Odet et al., 2006). Additionally, testosterone and estradiol have been shown to regulate the activity of kallikreins (Matsui & Takahashi 2001, Eacker et al., 2007).

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Serine proteases inhibitors (SERPINs) The SERPINs are a superfamily which contains proteins that composed of a conserved tertiary structure domain. Roughly half of the 500 predicted or known coding sequences for SERPINs are inhibitors of proteinases, which is how the superfamily got its name. Clades are created within the SERPIN family based on evolutionary connections. SERPINA5, also known as retinoic acid and the protein C inhibitor or PAI-3, targets both uPA and tPA and activates protein C. PAI-2 slightly inhibits both tPA and uPA. SERPINE1 is PAI-1, and SERPINE2 is a superfamily member (Law et al., 2006). The mechanism of targeting serine proteinases with SERPINs involves the formation of noncovalent complexes by interacting with proteinases, preventing their suicide by dissociating them when boiled in SDS, and making them susceptible to nucleophiles. This process is made possible by the dramatic conformational alteration of SERPINs and the resulting trapped complexity. Furthermore, several SERPINs, including A5, E1, and E2, can interact with highly reactive glycosaminoglycans, leading to activation. The resulting increase in proteinase inhibition levels can increase by up to a thousand times, indicating that rate-limiting glycosaminoglycans are critical factors at SERPIN auction sites. The mechanism involves the attachment of glycosaminoglycans so that both the SERPIN and proteinase can interact properly (Pike et al., 2005).

The function of PA in testis During adult life, regulating tropic hormones is crucial for testis development and male fertility. This regulation influences the organization of proteases and inhibitors in the testis, which directly affects gonad growth. Studies have shown that infertility of male is related with abnormal thickness of membrane around seminiferous tubules in the testis (de Kretser et al., 1975). The basement membrane is essential for the structural organization of the testis cord during gonad growth and for maintaining the specialized activities of Sertoli cells in adulthood (Kuopio & Pelliniemi 1989). Evidence suggests that components of the basement membrane, such as laminin and tPA, as well as growth hormones like HGF and FGF2, play vital roles in prepubertal alterations (Skinner, 2005). Recent research has shown that MMP9 and its inhibitor TIMP-1 also work with ECM components to control tight junction protein expression and lumen formation (Wong & Cheng 2005). Proteases and inhibitors work together to maintain homeostasis and regulate germ cell movement across Sertoli cell membranes. Many cytokines, inhibitors, and proteases are present at appropriate time and location (Xia et al., 2005). For example, there is an increase in PA activity during spermiation at seven and eight stages (Fritz et al., 1993). Before spermiation, a2-macroglobulin

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immunostaining is concentrated at stages I–VI, suggesting that a2-macroglobulin may protect seminiferous epithelium from proteolysis. When Sertoli cells and germ cells cocultured, PA activity and cysteine protease activity were maximized. This activity correlated with assembly configuration of de novo adherent joints among cultured Sertoli cells. Proteases and inhibitors cooperate synergistically, which may regulate the adhesion of germ cells to Sertoli cells by forming intercellular junctions (Wong & Cheng, 2005). Expression of both α2-macroglobulin and cystatin further supports this idea. These findings are consistent with earlier research suggesting that spermatids and Sertoli cells are held together by protease-sensitive substances of unknown type (Russell 1980).

PRSS55 (serine proteases 55) The human PRSS55 protein, also known as T-SP1, was first discovered as a new member of the family of membrane-anchored serine proteases that are similar to chymotrypsin (S1) and are mainly produced in testicles (Neth et al., 2008) and is found on chromosome 8p23.1. A variety of human PRSS55 mRNA variants are generated as a result of alternative splicing. According to reports, PRSS55 plays significant roles in carcinogenesis and spermatogenesis since it is expressed in tissues from some ovarian and prostate cancers (Liu et al., 2013). A 321-aa protein is encoded by the mouse Prss55 gene, which has seven exons and is found on chromosome 14D1. Protein sequence analysis indicates that PRSS55 is highly conserved across a wide range of species, strongly suggesting that it has played a significant evolutionary role, particularly in male reproduction. The biological function of PRSS55 is still not fully understood, but it is a GPI-anchored membrane protein mainly expressed in adult mouse testis. It is primarily found in the sperm acrosome region and luminal border of seminiferous tubules. Functionally, it is necessary for male fertility in mice, playing a role in the recognition and binding of egg cells and sperm motility into the oviduct. These findings reveal a crucial part of PRSS55, suggesting that it may contribute to the pathogenesis of UMI and serve as a viable therapeutic target for UMI patients or male contraceptives (Shang et al., 2018). Serine proteases are prevalent in the male reproductive system, suggesting that they play an important role in spermatogenesis and egg motility. Several serine protease family members have been reported to participate in spermatogenesis or be involved in various sperm functions, all of which are crucial for male reproduction (Kobayashi et al., 2020; Khan et al., 2018). A specific gene expression pattern was observed in testicular spermatids and epididymis sperm, indicating that PRSS55 plays a crucial role in spermatogenesis and motility of sperm (Shang et al., 2018). Prior studies investigating the function of PRSS55 in male reproduction have yielded mixed results. While male mouse sterility was unaffected by the loss of PRSS55, a lack of PRSS55 did affect specific sperm functions, for example, sperm-zona pellucida (ZP) bind-

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ing and sperm uterotubal junction (UTJ) migration, leading to reduced fertility (Shang et al., 2018; Khan et al., 2018). In the testis, Prss55 is expressed by four transcripts, and failure to eliminate all of them could result in insufficient or unstable phenotypes. Sperm’s energy metabolism and differentiated standard tail structure, which includes microtubules and mitochondria, determine its motility. The microtubule complex makes up the axoneme, which connects the sperm neck to the entire flagellum and is crucial for flagellum movement. The cysteine and disulfide bonds in the flagellum’s thick fibers are essential for reliability and elasticity. The mitochondria spiral around the surrounding dense fibers to produce a mitochondrial membrane, and mitochondrial metabolism generates ATP for sperm motility (Holcomb et al., 2020). The sperm tail is a crucial component for maintaining optimal sperm motility. Studies have shown that knockout mice lacking PRSS55 exhibited abnormal sperm tail extension and structural damage. These mice had sperm tails that folded and curled to varying degrees. All movement-related components in sperm tails had anomaly, such as a lack of microtubules, destruction to peripheral heavy fibers, and deficiencies in mitochondrial cristae. The aforementioned abnormal phenotypes were confirmed from various perspectives, that is, morphological observations, structural evaluation, and molecular detection. The irregular metabolism in PRSS55 KO mice was further supported by the fact that their sperm ATP concentration was significantly lower than that of WT mice, as shown by Shang et al., (2018). ATP, produced through aerobic oxidation and anaerobic glycolysis, provides the energy for sperm movement, with aerobic oxidation generating significantly more ATP (Agarwal et al., 2003). Researchers found that the major components of the oxidative respiratory chain, COX-2 and MTATP had significantly lower expression levels in the sperm of Prss55- mice. While decreased sperm motility was observed in Prss55-mice, there were no reported abnormalities in sperm count or testicular structure (Shang et al., 2018 and Kobayashi et al., 2020). However, Zhu et al. (2021) found no reduction in sperm motility or impairments in sperm shape and structure. The scientists reported that sperm exodus through UTJ was disrupted. They attributed it to the lower amount of fully mature ADAM metallopeptidase domain 3 (ADAM3) in sperm, crucial for sperm movement through the female reproductive tract (Yamaguchi et al., 2009). The researchers confirmed that Prss55-mice sperm have decreased ADAM3 expression, and they proposed that decreased sperm motility is a significant factor in the impairment of UTJ migration. Studies have shown that sperm motility is imperative for its movement through the different parts of female reproductive tract (Fujihara et al., 2018). Therefore, the subfertility phenotype of Prss55- mice is further explained by the deficiency in sperm motility. The results of these studies suggest that PRSS55 have important role in spermatid differentiation and can directly impact sperm function, which is persistent with the distinctive expression patterns of PRSS55 observed in epididymal spermatozoa and tubular spermatids. Some researchers propose that PRSS55 is expressed in male germ cells and remains in epididymal sperm, but its function may be more critical in epi-

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didymal sperm than in testicular germ cells (Kobayashi et al., 2020). PRSS55 is active in the testis and epididymis (Shang et al., 2018 and Kobayashi et al., 2020). In these studies, the unique gene knockdown method eliminated all Prss55 transcripts from the testis, whereas previous studies removed only two or more genes.

Conclusion and future prospective The evidence suggests that proteases may play a role in testicular morphology and migration of germ cells during spermatogenic development. Proteases and their inhibitors have a wide range of effects, which may explain their redundancy. For example, male mice lacking in uPA, tPA, both PAs, or PAI-1 can still reproduce, but those lacking in both PAs had decreased weight, reduced lifecycles, and increased fibrin accumulation. However, it is essential to note that systematic investigation of the testes of the mice lacking in these proteases is often only carried out if there are reproductive issues. PRSS55 has significance in sperm motility and structural differentiation. As a protease, it may activate substrate proteins, such as various types of muscle myosin family members, which can serve as PRSS55 precursor protein substrates. These myosins can then help to differentiate the structure of sperm and impact its function, potentially resulting in male infertility. PRSS55 may work by stimulating muscle myosins in the testis as a serine protease. It contributes to various processes such as cell migration, polarization, adherence, regulation of cell shape, and signal transmission that require drive and cytoskeleton translocation. The possible pathogenic role of PRSS55 in male astheno/teratozoospermia still needs to be fully understood. Despite not being essential for in vitro fertilization, this gene is crucial for in vivo sperm movement and the maturation of ADAM3 and spermegg contact in vitro. The loss of PRSS55 in the testis affects many genes related to the cell surface and organelle organization, complex assembly, protein transport, and stimulus-response and signaling. Understanding the functional organization of these genes will help to unravel the maturation process of sperm in the future.

References Agarwal, A., Saleh, R. A., & Bedaiwy, M. A. (2003). Role of reactive oxygen species in the patho-physiology of human reproduction. Fertil and Steril, 79(4), 829–843. Anway, M. D., Show, M. D., & Zirkin, B. R. (2005). Protein C inhibitor expression by adult rat Sertoli cells: Effects of testosterone withdrawal and replacement. Journal of Andrology, 5, 578–585. Blasi, F., & Carmeliet, P. (2002). uPAR: A versatile signalling orchestrator. Nature Reviews. Molecular Cell Biology, 3, 932–943.

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Meachem, S. J., Ruwanpura, S. M., Ziolkowski, J., Ague, J. M., Skinner, M. K., & Loveland, K. L. (2005). Developmentally distinct in vivo effects of FSH on proliferation and apoptosis during testis maturation. Journal of Endocrinology, 186(3), 429–446. Neth, P., Profanter, B., Geissler, C., Nägler, D. K., Nerlich, A., Sommerhoff, C. P., & Jochum, M. (2008). T-SP1: A novel serine protease-like protein predominantly expressed in testis. Biological Chemistry, 389, 1495–1504. Odet, F., Guyot, R., Leduque, P., & Le Magueresse-Battistoni, B. (2004). Evidence for similar expression of protein C inhibitor and the urokinase-type plasminogen activator system during mouse testis development. Endocrinology, 145, 1481–1489. Odet, F., Vérot, A., & Le Magueresse-Battistoni, B. (2006). The mouse testis is the source of various serine proteases and serine protease inhibitors (SERPINs). Serine proteases and SERPINs identified in Leydig cells are under gonadotropin regulation. Endocrinology, 147, 4374–4383. Pike, R. N., Buckle, A. M., Le Bonniec, B. F., & Church, F. C. (2005). Control of the coagulation system by serpins. Getting by with a little help from glycosaminoglycans. FEBS Journal, 272, 4842–4851. Puente, X. S., & Lopez–Otin, C. (2004). A genomic analysis of rat proteases and protease inhibitors. Genome Research, 4, 609–622. Rubinstein, E., Ziyyat, A., Wolf, J. P., Le Naour, F., & Boucheix, C. (2006). The molecular players of spermegg fusion in mammals. Seminars in Cell and Developmental Biology, 17, 254–263. Russell, L. D. (1980). Sertoli-germ cell interrelations: A review. Gamete Research, 3, 179–202. Shang, X., Shen, C., Liu, J., Tang, L., Zhang, H., Wang, Y., Wu, W., Chi, J., Zhuang, H., Fei, J., & Wang, Z. (2018). Serine protease PRSS55 is crucial for male mouse fertility via affecting sperm migration and sperm-egg binding. Cellular and Molecular Life Sciences, 75(23), 4371–4384. Wilhelm, D., Palmer, S., & Koopman, P. (2007). Sex determination and gonadal development in mammals. Physiological Reviews, 87, 1–28. Wong, C. H., & Cheng, C. Y. (2005). The blood-testis barrier: Its biology, regulation, and physiological role in spermatogenesis. Current Topics in Developmental Biology, 71, 263–296. Xia, W., Mruk, D. D., Lee, W. M., & Cheng, C. Y. (2005). Cytokines and junction restructuring during spermatogenesis – A lesson to learn from the testis. Cytokine and Growth Factor Reviews, 16, 469–493. Yamaguchi, R., Muro, Y., Isotani, A., Tokuhiro, K., Takumi, K., Adham, I., Ikawa, M., & Okabe, M. (2009). Disruption of ADAM3 impairs the migration of sperm into the oviduct in mouse. Biology of Reproduction, 81(1), 142–146. Zhu, F., Li, W., Zhou, X., Chen, X., Zheng, M., Cui, Y., Liu, X., Guo, X., & Zhu, H. (2021). PRSS55 plays an important role in the structural differentiation and energy metabolism of sperm and is required for male fertility in mice. Journal of Cellular and Molecular Medicine, 25(4), 2040–2051.

Biographical sketch Name: Shweta Dhanda Affiliation: Kurukshetra University, Kurukshetra Education: M.Sc. (Biotechnology) & Ph.D. (Biochemistry)

Research and Professional Experience Young professional fellow position at ICAR-National Research Centre on Equines, Hisar, since 20 September 2022.

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Publication from the Last 3 years Research article: In vitro studies on anti-inflammatory, antioxidant and antihyperglycemic activities of potential probiotic Pediococcus acidilactici NCDC 252 in Research Journal of Biotechnology (Vol. 16 (12), 2021). Research article: Identification, purification, characterization and biopreservation potential of antimicrobial peptide of Pediococcus acidilactici NCDC 252 in Journal of Food Processing and Preservation (Communicated). Book chapter: Current scenario of RNA therapeutics application in Systems Biomedicine in RNA therapeutics (Communicated).

Sonali Sangwan✶, Shikha Yashveer, Mahiti Gupta, Priti, Himani Punia, Jayanti Tokas, and Bhupesh Gupta

Chapter 10 Serine protease inhibitors and its therapeutics Abstract: Serine protease account for about 40% of all protease in human and forms the largest group of proteases. They are crucial in health; however, unlimited protease activity can lead to disease conditions when checkpoints fail during defined physiological conditions. As a counter, serine protease inhibitors are present that can control the unbalanced proteolytic activity of serine proteases. They allow for achieving a state of equilibrium between proteolytic activity and its inhibition that further prevent comorbidities. Regarding this, several sources were initially evaluated for different types of serine protease inhibitors, followed by a study of their structures and active sites involved in their action. The recent focus is on designing new compounds acting as serine protease inhibitors with better selectivity, inhibition, and efficiency. This paves the way for more potential as a therapeutic agent in the management of diseases. Keywords: serine proteases, serine protease inhibitors, therapeutics, designing

Introduction All living organisms have proteases with a proteolytic activity vital to survival. Serine protease is one such protease in its numerous isoforms and homologues. This enzyme occurs as elastase, collagenase, trypsin, chymotrypsin, subtilisin, and thrombin, playing

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Corresponding Author: Sonali Sangwan, Department of Biosciences and Technology, Maharishi Markandeshwar (Deemed to be University), Mullana, Ambala 133207, Haryana, India, e-mail: [email protected] Mahiti Gupta, Department of Biosciences and Technology, Maharishi Markandeshwar (Deemed to be University), Mullana, Ambala, 133207 Haryana, India Shikha Yashveer, Department of Molecular Biology and Biotechnology, and Bioinformatics, College of Biotechnology, Chaudhary Charan Singh Haryana Agricultural University, Hisar 125004, Haryana, India Priti, Department of Biotechnology K. L. Mehta Dayanand College for Women, Faridabad, Haryana, India Himani Punia, Department of Sciences, Chandigarh School of Business, Chandigarh Group of Colleges, Jhanjeri 140307, Mohali, India Jayanti Tokas, Department of Biochemistry, COBS&H Chaudhary Charan Singh Haryana Agricultural University Hisar 125004, Haryana, India Bhupesh Gupta, Department of Computer Science, Maharishi Markandeshwar (Deemed to be University), Mullana, Ambala 125004, Haryana, India

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a wide array of functions, including pathological roles as coagulatory to hemorrhagic, blood pressure regulation, inflammation, and corticosteroid-binding (Di Cera, 2009). At times, noninhibitory functions like hormone transporters (Pemberton et al., 1988), molecular chaperones (Nagata, 1996), and tumor suppressors (Zou et al., 1994) are also significant. Consequently, they are also well-studied for their structural characteristics, various functions, and selective inhibition. Inhibition of serine proteases can be achieved using multiple compounds such as dithiothreitol, lima bean trypsin inhibitor, ovomucoid, phenylmethylsulfonyl fluoride, pepstatin, soybean trypsin inhibitor, and aprotinin that inhibit but are not effective universally (Zheng et al., 2011). Serine proteases can be inhibited by serine protease inhibitors, one such class of molecules comprising peptides or polysaccharides that irreversibly inhibit them, ultimately protecting proteins from degradation and maintaining homeostasis (Huntington et al., 2000; Farady and Craik, 2010). The presence and availability of protease inhibitors were first reported by Fermi and Pernossi in 1894 (Fermi and Pernossi, 1894). Since then, serine protease inhibitors have been found in sources like the venom of spiders (Wan et al., 2013), the saliva of ticks (Chmelar et al., 2012), scorpion (Scorpiops jendeki) venom (Chen et al., 2013), termite derived (Negulescu et al., 2015), helminths (Molehin et al., 2012), jellyfish (Cnidaria phylum) (Jouiaei et al., 2015), echinoderm (Holothuria glaberrima) (Jouiaei et al., 2015), toad species (Rhinella schnideri) (Shibao et al., 2015), mushrooms (Sabotic et al., 2012), in plants like Solanum nigrum (Hartl et al., 2011), barley (Carrillo et al., 2011), fungi (Phytophthorainfestans) (Tian et al., 2007), chicken egg white (Ou et al., 2001), and the human body (Murakami et al., 2012). Protease inhibitors from different species also resemble each other, which indicate sharing of folds in the peptide structure (Chen et al., 2013). The presence of inhibitors is responsible for their various functions, including the survival of parasites by interfering host’s immune response and taking part in immune regulation. In plants, the expression of inhibitors is induced when herbivory takes place, eventually leading to the inactivation of the digestive proteases of insects. Inhibitors in fungi can deter the host plant proteases (Tian et al., 2007). They can selectively inhibit any form of serine protease, that is, chymotrypsin, trypsin, and elastase, and their specificity differs. Kazal-type and Kunitz-type are wellstudied among many families to which protease inhibitors belong (Ohmuraya and Yamamura, 2011; Liu et al., 2015). They inactivate their target with a lock-and-key mechanism (Farady and Craik, 2010). Standard features, that is, knottins, barrel fold, and Bowman-Birk family are present in peptide inhibitors. Inhibitors assist their function at nM and µM levels. Serine protease and its inhibitors are studied extensively because delicate tune equilibrium must be current between protease and its inhibitor to enable normal body functions accentuating their pivotal role in biological processes. Any upregulation or downregulation in these molecules is deleterious. It causes skin disease, coagulation abnormalities, inflammation, immunological disorders, cancer, pulmonary and neuronal disorders, atherosclerosis, and other pathologies. Serine protease inhibitors advancement as therapeutics has been depicted in Figure 1

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Serine protease inhibitor advancement as therapeutics

Different sources of serine protease inhibitor

Study of serine protease inhibitor structures and active sites

Designing of new compounds

Figure 1: Advancement of serine protease as therapeutics.

Various serine proteases and their inhibitors SERPINS SERPINs (serine protease inhibitors) represent a superfamily of proteins segregated into 38 clans based on tertiary structure (Rawlings, 2010; Heit et al., 2013). Serpins are classified as clade and group based on clade-based classification and group-based classification, respectively. In a clade-based classification system, the sequence similarity and specific phylogenetic relationships are grouped and determined as clades, whereas those which cannot be grouped are known as orphans. They are divided into 16 clades, A-P, where A-I includes human serpins (Irving et al., 2000). Clades comprise six subgroups based on the amino acid sites and gene structure (Ragg et al., 2001). Also, they are classified based on function and activity into inhibitory and noninhibitory groups (Law et al., 2006). In the group-based classification system of vertebrates, serpin genes are categorized into six groups, namely V1-V6, based on gene structure (Kumar et al., 2008). Serpins are specific in nature and target serine proteases mainly. It may even target papain-like cysteine proteases (Irving et al., 2000), caspases known as cathepsins (Ray et al., 1992) and some proteases involved in blood pressure regulation and hormone transport (Silverman et al., 2001)

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Thrombin inhibitors Thrombin is pivotal in activating the coagulation cascade, establishing itself as an excellent antithrombotic therapy target. This trypsin-like serine protease cleaves fibrinogen to release fibrin and work as the final enzyme of a coupled system. It also activates factors like Factor V, VIII, and XIII to amplify itself through a positive feedback mechanism (Goldsack et al., 1998). The use of thrombin inhibitors can directly decrease thrombin levels in the blood. Heparin and warfarin are the most widely used agents to inhibit precursor coagulation proteins (Goldsack et al., 1998; Coburn, 2001). Their use was accompanied by several other significant side effects such as thrombocytopenia, bleeding, osteoporosis, interference with several commonly used drugs, and hemorrhage (Coburn, 2001; Goldsack et al., 1998; Steinmetzer et al., 2001). Eventually, molecular modeling and X-ray crystallographic analysis of enzyme-inhibitor complexes based on structural considerations led to the development of two main types of inhibitors, that is, tripeptide and nonpeptide (Sanderson, 1999; Coburn, 2001). Leupeptin, a peptide aldehyde natural product that could inhibit serine proteases reversibly (Sanderson, 1999), formed the basis for the development of first thrombin inhibitor in the category of peptide aldehydes (Aoyagi et al., 1969; Umezawa, 1977). Later in the early 80s, the first synthetic lowmolecular-weight thrombin inhibitors, namely NAPAP and argatroban, were independently developed by two groups in a class of nonpeptide inhibitors (Sturzebecher, 1983; Okamoto et al., 1980; Kikumoto et al., 1980a; b).

Factor Xa inhibitors Factor Xa-based inhibitors provide another possibility for the interference of the coagulation cascade as fXa is present at the convergence point of intrinsic and extrinsic pathways, making them ideal for inhibiting either of these processes. A smaller dose of fXa inhibitor works well compared to the dose requirement for thrombin inhibitors to obtain therapeutic effects (Quan and Wexler, 2001). This owes to the fact that the concentration of fXa is considerably lower than that of thrombin, and thrombin activation is a highly amplified process. Recent trials based on direct fXa inhibitor DX9065a for stable coronary disease in patients showed that larger safety margins exist when compared with other antithrombotic therapies that include the use of thrombin inhibitors and low molecular weight heparins (Hauptmann and Sturzebecher, 1999; Post et al., 2002; Leadley, 2001; Dyke et al., 2002)

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Human neutrophil elastase inhibitors Human neutrophil elastase (HNE) is a highly destructive protease that can degrade a broad range of proteins with implications in several inflammatory diseases, namely, cystic fibrosis, acute respiratory distress syndrome (ARDS), emphysema, chronic bronchitis and ischemia–reperfusion injury. It is a glycoprotein with 218 amino acids constituting a weight of 33 kDa and the catalytic triad of trypsin-like serine proteases (AnZhi et al., 1988; Edwards, 2002). They can degrade various structural proteins in blood vessels, skin, and lungs. HNE participate in multiple conditions. They assist neutrophils in migration toward the inflammation site. They are involved in wound healing and tissue remodeling, thus making them a target for novel therapies. All main serine protease inhibitors can inhibit them, including the halomethyl dihydro coumarins, the lactones, the boronic acids, the alkylating agents, the benzoisothiazolinones, and some oligopeptidyl aldehydes/ketones (Edwards, 2002). These compounds are of little interest from a pharmaceutical point of development as they inhibit many nontarget serine proteases and have a high toxicity. Another hurdle in the implication of HNE inhibitors is prolonged clinical trials. Thus, the development of inhibitors is now accompanied by changed orientations of strategies adopted by pharmaceutical companies. They now focus on acute diseases requiring shorter clinical trials, and ONO-5046 (silvistat) is one such most advanced HNE inhibitor for treating septic shock and ARDS registered in Japan (Tomizawa et al., 1999).

Therapeutic effect of serine protease inhibitors Proteolytic enzymes expand the injured area, cause cellular injury, and induce inflammation. The inhibition of serine proteases by implementing selective serine protease inhibitors is a logical therapeutic strategy when an excess of the proteolytic activity of serine proteases causes complications in health conditions like chronic inflammatory disorders and cardiovascular diseases (Korkmaz et al., 2020). The discovery of novel serine protease inhibitors took speed when the “structure-based” approach to drug discovery existed. In addition, to attain a prolonged half-life and good oral bioavailability, a strong emphasis is being laid on ADME (parameters like drug absorption, distribution, metabolism, and excretion). This revolution results from technological advances that include molecular biology, genomics, protein X-ray crystallography, and computational methods (high-throughput synthesis and high-throughput in vitro screening). Many valuable targets have been validated, and attractive, promising compounds have emerged (Table 1) in recent years, which are in advanced clinical trials (Ilies et al., 2002).

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Table 1: Serine protease and inhibitors designed. Serine protease

Inhibitor

Side chain replaced

Functional group inserted

References

Thrombin

Boc-D-trimethylsilylalanine-prolineboro-X pinanediol derivatives

D-phenyl

D-trimethylsilyl

Von Matt et al., 

Trypsin

Tripeptide boronate

No side chain replaced

Butyl formamide Zhu et al., (added) 

Thrombin

N-arylsulfonylarginine amides

Guanidine/ arginine

Dimethyl tetrahydro quinoline

Okayama et al., 

Factor Xa

(R)--[(-Chloro--naphthalenyl) Amidine sulfonyl]-oxo--[[-(-pyridinyl)--piperidinyl] methyl]--piperazinecarboxylic acid

Pyridine

Nishida et al., 

Factor Xa

Melagatran

Benzamide

Naphthylamidine Furugohri et al., 

Human neutrophil elastase

Sivelestat

N-CO

-(Sulfamoyl) phenyl pivalate

Crocetti et al., 

Urokinase-type plasminogen activator

-(-Hydroxybiphenyl--yl)- H-indole- Hydrogen -carboxamidine

Chlorine

Mackman et al., 

Tryptase

(S,S)--(-Carboxy)phenoxy-[diphenylmethyl aminocarbonyl]-(-ethoxybenzyl) azetidine--one

Ethyl

Aoyama et al., 

Cytomegalovirus protease

α-Methylpyrrolidine-,-trans-lactam

Dansyl-(S)proline (added)

Borthwick et al., 

Longer carbon chain

Wu et al., 

HIV serine protease β-Hydroxy γ-lactam inhibitor

alanine

Methyl

Shorter carbon chain

Conclusion and future prospective Serine proteases are a widespread class of proteases, and recent developments have made designing inhibitors and their further therapeutic application possible. Diverse members of these proteolytic enzymes offer a chance for a real revolution in this field to provide more specific and potent compounds that can serve the purpose of serine protease inhibitors with more selectivity, inhibition activity and efficiency. This book chapter discussed popular serine protease inhibitors and therapeutics offered by

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them. However, the path from bench to marketplace for serine protease inhibitorsbased drugs is difficult; still, many potent inhibitors were detected and improved further to obtain good oral bioavailability. Further efforts are needed to optimize various pharmacodynamics and pharmacokinetics properties of these compounds to enhance the output of drug design. In addition, steps are also required in vivo evaluation of these designed inhibitors for the desired goal. Bacteria also possess serine proteases that are involved in critical processes such as the acquisition of nutrients, colonization, tissue damage during infection, and host immune response evasion. This field is developing and paving the way for novel antibiotics, as only a few potent bacterial inhibitors have been reported till now.

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Biographical sketch Name: Sonali Sangwan Affiliation:Maharishi Markandeshwar University, Mullana-Ambala Education: M.Sc. & Ph.D. Molecular Biology and Biotechnology

Research and Professional Experience – –

Experienced in supervision and perform Molecular Biology laboratory operations (Research Methodologies/Molecular work/Data analysis/Review Data) Worked on synthesis of Salicylic acid nanoformulations to mitigate heat stress in wheat (Triticum aestivum L. em Thell)

Publications 1. Sangwan S, Shameem N, Yashveer S, Tanwar H, Parray JA, Jatav HS, Sharma S, Punia H, Sayyed RZ, Almalki WH, Poczai P (2022) Role of Salicylic Acid in Combating Heat Stress in Plants: Insights into Modulation of Vital Processes. Frontiers in Bioscience-Landmark 27(11): 310 https://doi.org/10.31083/j.fbl2711310

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2. Hembade VL, Yashveer S, Taunk J, Sangwan S, Tokas J, Singh V, Redhu NS, Grewal S, Malhotra S, Kumar M (2022) Chitosan-Salicylic acid and Zinc sulphate nano-formulations defend against yellow rust in wheat by activating pathogenesis-related genes and enzymes. Plant Physiology and Biochemistry 1–12. https://doi. org/10.1016/j.plaphy.2022.10.002 3. Sharma S, Singh V, Tanwar H, Mor VS, Kumar M, Punia RC, Dalal MS, Khan M, Sangwan S, Bhuker A, Dagar CS, Yashveer S and Singh J (2022) Impact of High Temperature on Germination, Seedling Growth and Enzymatic Activity of Wheat. Agriculture, 12, 1500. https://doi.org/10.3390/agriculture12091500

Index 6-gingerol 72 aberrant expression 54 accentuating 142 ACE2 109, 116 Acinetobacter baumannii 103 acquisition 147 acute respiratory distress syndrome 145 acylation 4 adaptive immunity 24, 116 adenocarcinoma 44 adenosine deaminase 67 adipokine 66 aggrecanase 90 albumin-to-creatinine ratio 69 albuminuria 66 alkaline-fibrinolytic 25 amino-terminus 5, 53 amplification 18 anaerobic glycolysis 135 androgen response elements 116 angiogenesis 37 Annexin II 132 antibiotics 104 antioxidants 119 antiprotease imbalance 51 antiproteases 17–18 antithrombotic therapy 144 anti-TNFα 132 Arthrobacter 9 aspartate 18 Aspergillus oryzae 25 asthmatics 24 autosomal recessive 22 azurophilic granules 23 Bacillus 8 bacteriostatic 102 benzoselenoxanthene analogues 118 bio-active molecules 34 biofilm 102 biofilm formation 104 biomarkers 37 blood coagulation 4 blood pressure 143 Borrelia burgdorferi 104 Bowman-Birk 142 https://doi.org/10.1515/9783111325040-011

bromhexine 120 bromhexine hydrochloride 119 bronchiolar 21 calcium-dependent 115 camostat mesylate 120 cancer 51 carboxy-terminus 5 carcinogenesis 40 cardiometabolic illnesses 131 cardiomyocytes 110 cardiovascular diseases 145 cartilage 84 cartilage destruction 89 cascades 37 catabolism 24 catalyst 1 catalytic domain 111 catalytic triad 4 cathepsin G 19 cathepsin Inhibitors 119 cathepsin L 112 cathepsin L-dependent entry 116 cathepsins 113 cell adhesion molecules 6 cell migration 136 cell signaling 6 cellular elastase 114 centrilobular 56 chemotaxis process 55 cholangiocytes 110 chondrocytes 88 chronic bronchitis 57 chronic inflammatory 19 chymases 57 chymotrypsin 5 Clades 143 classification 1 coagulation processes 18 collagenases 25 collagenolytic activity 25 colonization 106 complement system 34 coronaviruses 113 cotransporter-2 70 COVID-19 110 cruciferous 119

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Index

cysteine 34 cystic fibrosis 22 cytokines-mediated signaling 55 cytokine-stimulated 86 cytoplasmic extension 36 deacylation 4 dedifferentiated 40 degradative 11 degranulation 57 diabetic nephropathy 66 diagnosis 58 differentiation 3 dipeptidyl peptidase 56 downregulation 44 DPP-IV inhibitors 66 dyad 4 dysregulation 7 E-cadherin 41, 101 E-cadherin protein 103 effectiveness 10, 121 elastase 145 elastase-like 52 elevated 39 embryonic development 129 embryonic growth 38 emphysema 56, 145 endocytosis 112–113 endopeptidases 2 endothelial lining 88 envelope 110 enzyme activity 112 Enzyme Commission classification 52 epididymal 135 epithelial cells 55 erythroblastosis 41 Escherichia coli 102 esophageal adenocarcinoma 45 estrogen-related substances 118 excavatolide M 118 exopeptidases 2 extracellular matrix 33, 36 factor Xa inhibitors 144 fertilization 129 fibrinolysis 131 fibroblasts 44, 56 fibronectin 105

flagellum 135 Flavobacterium 9 flavonoids 76 FLUAV-HA activation 114 follicle-stimulating hormone 130 Food and Drug Administration 10 furin 113 furin inhibitors 118 fusion sequences 112 gemigliptin 70 generally regarded as safe 10 glucose-dependent insulinotropic polypeptide 65 glycoprotein 111 glycosaminoglycans 133 glycosyl phosphatidylinositol 5, 53 glycyl-prolyl-β-naphthylamide 66 GPI-anchored 39 G-quadruplex 117–118 Gram-negative bacteria 103 granzyme A 85 granzymes 33 H7N9 influenza 117 HA-activating protease 121 haemagglutinin 21 haemophilia 11 Helicobacter HtrA inhibitor 105 hemagglutinin 40 hemorrhage 144 hemorrhagic 141 heparins 144 hepatocellular 45 hepsin 36 higher stability 7 high-temperature requirement 101 high-throughput 145 histidine 18, 35 histopathological 114 histopathological profile 69 HNE inhibitor 145 homeostasis 18–19 homologous 35 hormone transport 143 host 35 host proteases 111 HtrA family 103 human genome 52 human orthologue 40

Index

hydrolysates 11 hydrolytic enzymes 10 immune complexes 85 immunofluorescence 69 immunomodulation 4 immunomodulatory 90 immunosuppression 33, 44 in silico 72 in vitro 72 in vitro screening 145 in vivo 72 inactive 53 infertility of male 133 inflammation 17, 83, 87 inflammatory cells 86 influenza 20, 57 inhibitors 101 interleukin 132 interstitium 57, 130 intracellular protein 34 intravascular 121 kallikreins 37 keratolytic reactivity 25 kringle domain 42 lactic acid bacteria 9 Lactobacillus 10 Leydig cells 132 lung cancers 45 lung disorders 17 lung fibrosis 19, 21 lung infections 51 lung inflammation 18, 59 lysosomal 115 macophage stimulating protein 39 macroglobulin 134 macrophages 23 male fertility 129 male reproductive system 134 malignant pleural mesothelioma 68 manipulation 11 matriptase 88 mechanism 1 melanoma 44 membrane attack complex 87 membrane-anchored 5

meniscectomy 87 metabolic reactions 3 metagenomics 10 metalloprotease 116 metalloproteases 38, 132 metalloproteinase 83–84 metalloproteinases 88 metastasis 7, 33, 37 metastatic 42 microbes 8 micro-environment 58 microtubules 135 motif 36 mucociliary 55 mucus plugging 55 multifaceted 102 musculoskeletal 90 Mycobacterium tuberculosis 23 myosins 136 nafamostat 119, 121 naphthofluorescein 118 naringenin 66 nasal epithelium 117 nasopharyngitis 70 neuraminic acid 67 neutrophil 145 neutrophil serine protease 22 nexin-1 133 NOTCH signaling 41 nucleocapsid 110 nucleophilic serine 3 obstructive pulmonary 54 oligopeptidyl aldehydes 145 open-reading frame 3a 116 oral bioavailability 147 oral mucosa 115 osteoarthritis 84 osteophytes 90 ovarian cancers 39 oviduct 134 PA clan superfamily 52 pandemic 110 pangolin 115 parasites 142 pathogen-associated receptors 21 pathogenicity 117

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pathogen-selective HtrA 106 pathological 90 pathological processes 35 pathophysiological 114 pathophysiological processes 55 pathophysiology 43 penultimate region 68 peptidase 2 pericellular 6 perivascular 56 phenolic compounds 76 phenotype 104 phenylmethylsulfonyl fluoride 142 phosphatidylinositol 36 phosphorylation 104 photogenicity 17 phylogenetic relationships 143 physiological functioning 3 phytocompounds 70 plantarum 10 plasmin 86, 114 plasminogen 114 plasminogen activator 33 plasminogen activator inhibitor-1 119 plasminogen activators 86 polybasic furin 115 polymorphonuclear leukocytes 85 postbinding stages 118 postsynaptic density protein 102 post-transcriptional 3 post-translational 3 potent 106 potent inhibitors 147 prepubertal 133 prognostic factor 58 progression 59 proinflammation 57 proinflammatory 69, 85, 87 proinflammatory cells 24 proprotein convertases 84 prostate malignancies 43 protease-activated receptors 83 proteinaceous 10 proteinase-3 84 protein-engineering 10 protein-protein interactions 52 proteoglycans 86 proteolysis 5 PRSS55 129

Pseudomonas aeruginosa 103 pseudovirions 119 randomized 120 receptor-binding domain 111 regeneration 54 remodeling 24 reno-protection 69 replication 120 resemblance 58 respiratory tract 21 resveratrol 66 retinoic acid 133 rheumatoid arthritis 84 Salmonella enterica 103 SARS-CoV 58 SARS-CoV-2 109 sax gliptin 76 secretory serine protease 40 serine alkaline proteases 8 serine carboxypeptidases 7 serine proteases 1 SERPINS 143 Sertoli cells 132 S-glycoprotein 58 shelf life 10 sialic acid 57 signaling 85 sitagliptin 65 site directed mutagenesis 11 sivelestat 120 sodium-chloride ions 55 Solanum nigrum 142 sperm motility 134, 136 sperm shape 135 sperm tail 135 spermatids 130, 134 spermatozoa 130 spermiation 133 sperm-zona pellucida 134 spike protein 111, 121 splicing 134 sputum 22 Streptomyces 9 streptozotocin 69 structurally 70 subfertility 135 substrate specificity 23

Index

subtilase 8 subtilisin 89 sulphonylurea 70 superfamily 4 survivability 102 synaptogenesis 130 syncytium 114 synovial cells 88 synoviocytes 90 tenascin C 56 teratozoospermia 136 testicular 134 testis 133 tetrahedral intermediate 4 therapeutic approaches 105 therapeutic effects 144 therapeutics 141 threonine 34 thrombin inhibitors 144 thrombolysis 11 tight junction 133 TMPRSS2 41, 109, 114 tooth enamel formation 43 transcriptome 117 transforming growth factor β 83 trans-golgi network 89 transmembrane domain 52 trypsin 5

trypsin-like protease 21 tubulointerstitial lesions 65 tumor cell invasion 42 tumor development 33 tumor necrosis factor 86 tumorigenesis 37 tumorigenicity 59 tumour necrosis factor 132 twentieth century 20 tyrosine-kinase-signaling pathways 39 ubiquitous 2, 51 urokinase 58, 86 urokinase-type plasminogen activator 42 urothelial carcinoma 45 uterotubal junction 135 vasoconstriction 68 vasodilatation 114 viral genome 115 virion 110 virulence 111 vitronectin 132 widespread 11 X-ray crystallographic 144 zymogens 3

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