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Serine Protease and Serine Protease Inhibitors: Advanced Article
Serine Protease and Serine Protease Inhibitors: Advanced Article
Advanced Article
Article Contents
Serine Proteases and their Inhibitors Serine Protease Inhibitors Characterization of Protease-Inhibitor Interactions Serine Proteases and their Inhibitors in Disease
Serine proteases are among the largest group of proteolytic enzymes in the human genome that play vital roles in health and disease. Regulation of their activity in vivo is mediated by a diverse group of serine protease inhibitors. An overview of the interplay between serine proteases and their inhibitors is provided. In addition, approaches to characterize this relationship are discussed with subsequent emphasis on how such measures apply to pathologies that result from defect of serine proteases or their inhibitors.
Cellular life is brokered on the activity of proteins, and, in turn, control over their concentration and state is vital. Enzymes that hydrolyze peptide bonds, the proteases, are therefore critical ingredients of the genome. Proteases may acts as nonspecic agents of digestion or high-selectivity mediators of posttranslational modication. Proteases are a diverse group of enzymes. Over 180 phylogenetically distinct families of proteases have been identied by the MEROPS protease classication system on the basis of protein fold and additionally separated through phylogenetic relationship (1). Of these families, the largest contingent comprises serine proteases, which are named based on their application of the hydroxyl group of a serine side chain as catalytic nucleophile. Regulation of protease function in vivo is mediated by a minimum of 90 families of protease inhibitors. Together, these two groups control biological functions both inside and outside of the cell. Genetic defects in either protease or inhibitor, therefore, can result in signicant pathology with potentially multiple biological pathways impacted.
subtilisin, prolyl oligopeptidase, and ClpP protease. Many other enzyme families use the same catalytic triad, such as asparaginases, esterases, acylases, and lactamases (5). Mutagenesis of the aspartic acid to alanine impacts peptide bond hydrolysis to a greater extent than ester hydrolysis, which indicates that a complete catalytic triad is required for the hydrolysis of the stronger peptide bond. It should be noted that several serine protease families use a dyad mechanism in which lysine or histidine is paired with the catalytic serine. Yet, other serine proteases present novel catalytic triads, such as a pair of histidines combined with the nucleophilic serine. In nearly all reported cases, the active site serine can be rendered inactive by generic inhibitors such as diisopropyluorophosphate and phenylmethanesulfonyl uoride. We will focus on the most abundant serine protease and serine protease inhibitor families in the human genome. The reader is also referred throughout the text to other recent and more expansive reviews and to the MEROPS database for a more detailed description of the impressive diversity of serine protease and inhibitor structure, function, and activity. A typical genome contains 24% of genes that encode for proteolytic enzymes. The entire complement of peptidases within a genome is referred to as the degradome (6). Of these proteases, a select subset of peptidases underwent considerable gene duplication and divergence. The trypsin-like serine peptidases (Clan PA Family S1 Subfamily A in MEROPS nomenclature) are the largest group of homologous peptidases in the human genome responsible for various critical biological processes. Similar degradome composition is observed in all vertebrates, which indicates that expansion of the S1A peptidase family occurred before emergence of the lineage roughly 450 million years ago. Of 699 peptidases in humans, 178 are serine peptidases, and 138 of them belong to the S1 peptidase family. The chymotrypsin-like fold of the S1 peptidase family presents an ideal catalytic platform that enables high turnover, substrate 1
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selectivity, and various modes of regulation in a package readily combined with additional protein domains (7).
(a)
(b)
Figure 1 (a) Two -barrel architecture of the S1A peptidase family (PDB 1OS8). Following activation, the zymogen arm stabilizes the active site cleft that lies in between the two barrels. An additional -helix is at the C-terminus. (b) The canonical catalytic triad is generated by the spatial arrangement of Asp-102, His-57, and Ser-195 positioned to facilitate hydrogen bond formation and abstraction of the proton of hydroxyl moiety.
protein. Cleavage of the proprotein precursor occurs at an identical position in all known members of the family: between residues 15 and 16. The nascent N-terminus induces conformational change in the enzyme through formation of an intramolecular electrostatic interaction with Asp-194 that stabilizes both oxyanion hole and substrate-binding site (10). Zymogen activation provides a powerful mechanism of regulation that endows temporal control over protease activity, an ability to escape premature enzyme inhibition, and places these enzymes within the context of chains of proteolytic events. Many proteases of the coagulation and immune pathways are regulated more through allosteric mechanisms that involve monovalent cations (Na+ ), divalent cations (Ca2+ ), glycosaminoglycans, and protein cofactors (11). These properties derive from the structure of the chymotrypsin fold and combine to produce proteolytic networks responsible for key biological processes responsible for human health. Several vital processes rely on clan PA peptidases. Chief among them are blood coagulation and the immune response, which involve cascades of sequential zymogen activation. In both systems, the chymotrypsin-fold peptidase domain is combined with one more associated protein domains, including apple, CUB, EGF, bronectin, kringle, sushi, and von Willebrand factor domains. These protein domains are on the N-terminus as an extension of the propeptide segment of the peptidase. Such a trend of N-terminal-associated domains in the S1A peptidase family is common across all forms of life. The domain architecture pairs well with the zymogen activation mechanism, which liberates the proper N-terminus to enable catalytic activity. Often, the associated protein domains remain attached to
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the peptidase domain through a covalent disulphide bond on the opposing surface of the protease active site. Many associated domains are entirely encoded by a single exon in their peptidase gene and suggest an important role for exon shufing during molecular evolution of clan PA. S1 peptidases in the human genome are, for the most part, phylogenetically grouped into six functional categories: digestion, coagulation and immunity, tryptase, matriptase, kallikrein, and granzymes. Various enzymes are involved in the breakdown of proteins in the digestive system. The trypsins, chymotrypsins, and elastases are endopeptidases that breakdown polypeptides into shorter chains. More digestion is mediated by various exopeptidases (12). In particular, carboxypeptidases A and B from the M14 family of zinc-dependent metalloproteases shorten the nascent peptides through complementary selectivity toward basic or aromatic residues. Inappropriate release of trypsin from the digestive system signals proinammatory responses typically mediated by tryptase-like S1 peptidases. Tryptases are major components in secretory granules of mast cells that are unique among clan PA peptidases because of their homotetrameric quaternary structure (13). Like trypsin, tryptases mediate proinammatory signaling through protease-activated receptors 2, yet denition of other substrates in health and disease states remain elusive. Matriptases are membrane-bound S1 peptidases that bear primary substrate selectivity similar to trypsin (14). Again, physiological substrates of this subfamily of peptidases are largely unknown, yet high gene expression levels for matriptases are associated with various cancer types. Similar association with cancer has led to great interest in the large family of kallikreins (15), a family commonly known for its role in regulation of blood pressure through the kinin system (16). Granzymes are mediators of directed apoptosis by natural killer cells and cytotoxic T cells that play key roles in the defense against viral infection (17). Notably, unique among clan PA is the primary selectivity of granzymes toward acidic residues in the P1 position of the substrates. Of the wide diversity of proteases in clan PA family S1, the mediators of immunity and blood coagulation have been particularly well studied.
catalytic activity in acidic environments. Nearly all serine peptidases have activity restricted within the range of neutral to alkaline pH. Many clan SC peptidases hydrolyze substrates on the C-terminal side of a proline residue with several exceptions. Both endoproteolytic and exoproteolytic activities occur in clan SC, which contrasts the trend in other serine peptidase families in which members are predominantly one or the other. For examples of differing selectivity in clan SC, prolyl oligopeptidase from family S9 cleaves peptide bonds within peptides, and prolyl aminopeptidase from family S33 removes N-terminal proline and hydroxyproline residues from peptides (20). Substrate selectivity for peptides shorter than 30 amino acids in length is derived from the two-domain architecture. An N-terminal seven-bladed propeller restricts access to the C-terminal / hydrolase domain and, in turn, the site of peptide bond hydrolysis (21). On the basis of their selectivity toward smaller peptides and not full-length proteins, clan SC peptidases are thought to be particularly important in cell signaling mechanisms. In humans, clan SC peptidases are often associated with proline-specic N-terminal processing of peptides and proteins, yet many present a nonproteolytic function. S9 is the largest family of clan SC peptidases with 41 homologs in the human genome. Of these homologs, prolyl oligopeptidase (POP) and dipeptidyl peptidase IV (DPP-IV) are the best characterized. The crystal structure of POP revealed the two-domain architecture and basis for substrate selectivity. Notably, no naturally occurring inhibitor of this family of proteases has been found. A putative role for POP has been suggested in the metabolism of various neuropeptides (22). DPP-IV presents a similar two-domain architecture (23). DPP-IV is a transmembrane protein responsible for processing hormones and chemokines. Only three S10 family peptidases have been identied in the human genome, and their biological roles remain to be uncovered. Of three S28 family peptidases in humans, only dipeptidyl-peptidase II (DPP-II) is characterized. DPP-II catalyzes release of two N-terminal amino acids when proline or alanine is in the P1 position. Eighteen S33 family peptidases are in the human genome; however, many of them do not display peptidase activity. For example, several of these enzymes catalyze hydrolysis of epoxide bonds into diols and play a role in detoxication or cellular signaling (24).
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Figure 2 Subtilisin (PDB 1SCN) presents an identical catalytic triad to that observed in other serine proteases and enzymes yet within an entirely different protein fold.
rather than through partial positive charges. Subtilisins have proven extremely useful for protein engineering studies. Successful examples of engineering the subtilisin scaffold include substrate selectivity, thermal stability, cold adaptation, stability in nonaqueous solvents, uoride activation, and ability to act as a peptide ligase (26). Many engineering studies on subtilisin have led to greatly improved cleaning agents for use in laundry detergent. Physiological function of clan SB peptidases tends to be nutrition-oriented with select roles in protein processing. Most clan SB peptidases prefer to hydrolyze substrates on the C-terminal side of large hydrophobic residues. However, proprotein processing peptidases such as kexin and furin cleave following a pair of dibasic residues (27). Most clan SB peptidases are secreted outside the cell or localized to the cell membrane. A notable exception is the tripeptidyl-peptidases responsible for intracellular protein turnover. Within the human genome, 10 clan SB peptidases have been identied; nine belong to the S8 family, and only one is from the S53 family. Although well known for their role in processing proteins along the secretion pathway, new roles for proprotein convertases are emerging, including regulation of plasma protein levels. Tripeptidyl-peptidase I (TPP-I) is the sole representative from family S53 in the human genome and one of many lysosomal peptidases responsible for protein turnover (28). TPP-I removes three amino acids from the N-terminus of small peptides. Mutations in TPP-I are associated with infantile neuronal ceroid lipofuscinosis (Batten disease), the most common neurodegenerative disorder in children, which is characterized by intracellular accumulation of autouorescent lipopigments. 4
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domains in which one domain prefers trypsin, and the other prefers chymotrypsin.
Figure 3 Complex formation between trypsin and soybean trypsin inhibitor exemplies the mechanism of Kunitz-type inhibitors (PDB 1AVW). Greatly reduced peptide bond hydrolysis rates lead to inhibition.
complex of soybean trypsin inhibitor with trypsin by Sweet and colleagues (31). The structure presents a -barrel architecture capped by a pair of -strands stabilized by two disulphide bonds (Fig. 3). The physiologic function of the Kunitz-type proteases remains unknown for many family members other than those in man. Prevention against digestion or invasion from pathogens has been suggested based on a common abundance in seeds. In man, tissue factor pathway inhibitor is a key Kunitz-type inhibitor responsible for regulating blood clot formation. On the basis of their potency, Kunitz-type inhibitors were among the rst examined for therapeutic application. Aprotinin was approved for clinical application in coronary-artery bypass graft surgery in 1993. Fifteen years later, considerable controversy has developed over its use given an associated risk of mortality and the availability of less expensive lysine analogs that achieve the same goals (32). Kazal-type inhibitors are classied as family I1 by the MEROPS database. The name is derived from pancreatic secretory inhibitor, which is now termed SPINK1, originally isolated by Kazal and coworkers in 1948. The SPINK family (serine protease inhibitor, Kazal) plays important roles in the digestive system, lungs, skin, and likely many other tissues in the body. Six SPINKs can be identied in the human genome, and each contains multiple repeats of the Kazal-type fold. Mutations in SPINK1 are associated with hereditary pancreatitis (33). Netherton syndrome is a rare disorder that affects the skin of patients and results in ichthyosiform dermatosis and hair shaft abnormalities. Patients with Netherton syndrome are found to have a mutation on chromosome 5 in the SPINK5 gene, which encodes an array of 15 Kazal-type inhibitor domains (34). Considerable biochemical characterization has been carried out on the ovomucoid inhibitors of the Kazal family (35). Notably, as multiple Kazal-type domains are often found within a single polypeptide chain, they need not inhibit the same type of protease or protease specicity. For example, dog bikazin contains two Kazal-type
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Figure 4 A large conformational change denes the mechanism of serpin inhibition. Conversion of the Michaelis complex (PDB 1OPH) into cleaved trapped conformation (PDB 1EZX) traps the RCL of serpin into the -strand core of inhibitor. A signicant gain in stability, therefore, is imparted to the entire serpin structure.
converted from a metastable free state into a more energetically favored relaxed bound state. Serpins have a considerably lower melting temperature (TM 60 C) in isolation than when cleaved (TM >100 C). Unrelated in sequence or structure, the macroglobulin family of protease inhibitors similarly applies scissile bond cleavage, yet the subsequent step involves entrapment of the protease in a cage-like architecture (40). Although most serpins apply this mechanism to inhibit serine proteases irreversibly, a select group has been shown to act reversibly. For example, protein C inhibitor, also known as PAI-1, acts as a reversible inhibitor to the single-chain urokinase-type plasminogen activator. Moreover, several serpins are known to integrate their cleaved RCL into another serpin molecule in trans (41). In turn, serpins can undergo polymerization, which becomes relevant in several pathological conditions. Serpins play key regulatory functions in man. 1 -antitrypsin serves a major role in protecting the connective tissue of the lungs from leukocyte-released elastase. The C1 inhibitor restricts proteases of the immune system from unwanted proteolysis and inammation. Two plasminogen activator inhibitors control brinolysis. Viral serpins have also been described that broker their survival and propagation through restricting these same proteolytic pathways. Control of blood clot formation is through antithrombin. However, unlike other serpin family members, the interaction between clotting factor protease and antithrombin is greatly facilitated by heparin or heparin sulfate glycosaminoglycans, which bind to the inhibitor to mediate 6
this effect (42). In turn, antithrombin is directed toward regulation of protease activity at cell surfaces such as the vascular endothelium, which display heparin in various forms.
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short half-life, and development of antihirudin antibodies. Additional modication of the hirudin platform and other medicinal chemistry strategies aimed at thrombin inhibition is well described in Reference 43.
such imbalances are within the blood coagulation cascade. For example, hemophilia B results from deciency in coagulation factor IX. In contrast, excessive activation of immune system serine proteases produces inammatory states. Errors in serine protease inhibitors can have consequences on multiple biological systems. However, overlapping inhibition by multiple families of inhibitor can, in certain instances, lessen the severity of the pathology. Genetic abnormalities in the serpins have also been associated with polymerization and therefore belong to the category of conformational disease. Emphysema, cirrhosis, and thrombosis may result from such aberrant conformational transitions. Neuroserpin may also play a key role in familial encephalopathy because of the formation of inclusion body-like material (45). Understanding the molecular mechanisms of limited proteolysis and their regulation in vivo remains a challenging and insightful venue to improve human health.
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Further Reading
The MEROPS database of peptidases and inhibitors is an invaluable resource that can be found at http://merops.sanger.ac.uk/
See Also
Enzyme Kinetics Protease Inhibitors, Mechanisms of Approaches to Enzyme Inhibition
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