Misplaced Pages

#356643

100-1304: 1by7 A:1-415 1ova A:1-385 1uhg A:1-385 1jti B:1-385 1att B:77-433 1nq9 L:76-461 1oyh I:76-461 1e03 L:76-461 1e05 I:76-461 1br8 L:76-461 1r1l L:76-461 1lk6 L:76-461 1ant L:76-461 2beh L:76-461 1dzh L:76-461 1ath A:78-461 1tb6 I:76-461 2ant I:76-461p 1dzg I:76-461 1azx L:76-461 1jvq I:76-461 1sr5 A:76-461 1e04 I:76-461 1xqg A:1-375 1xu8 B:1-375 1wz9 B:1-375 1xqj A:1-375 1c8o A:1-300 1m93 A:1-55 1f0c A:1-305 1k9o I:18-392 1sek :18-369 1atu :45-415 1ezx B:383-415 8api A:43-382 1qmb A:49-376 1iz2 A:43-415 1oo8 A:43-415 1d5s B:378-415 7api A:44-382 1qlp A:43-415 1oph A:43-415 1kct :44-415 2d26 A:43-382 9api B:383-415 1psi :47-415 1hp7 A:43-415 3caa A:50-383 1qmn A:43-420 4caa B:390-420 2ach A:47-383 1as4 A:48-383 1yxa B:42-417 1lq8 F:376-406 2pai B:374-406 1pai B:374-406 1jmo A:119-496 1jmj A:119-496 1oc0 A:25-402 1dvn A:25-402 1b3k D:25-402 1dvm D:25-402 1a7c A:25-402 1c5g A:25-402 1db2 B:26-402 9pai A:25-402 1lj5 A:25-402 1m6q A:138-498 1jjo D:101-361 Serpins are

200-455: A covalent bond with the serine protease, inhibiting its function. The best-studied serpins are antithrombin and alpha 1-antitrypsin , studied for their role in coagulation / thrombosis and emphysema / A1AT , respectively. Artificial irreversible small molecule inhibitors include AEBSF and PMSF . A family of arthropod serine peptidase inhibitors, called pacifastin , has been identified in locusts and crayfish , and may function in

300-452: A nucleophilic serine residue in a catalytic triad in their active site . Examples include thrombin , trypsin , and human neutrophil elastase . Serpins act as irreversible , suicide inhibitors by trapping an intermediate of the protease's catalytic mechanism. Some serpins inhibit other protease classes, typically cysteine proteases , and are termed "cross-class inhibitors". These enzymes differ from serine proteases in that they use

400-457: A superfamily of proteins with similar structures that were first identified for their protease inhibition activity and are found in all kingdoms of life . The acronym serpin was originally coined because the first serpins to be identified act on chymotrypsin-like serine proteases ( ser ine p rotease in hibitors). They are notable for their unusual mechanism of action, in which they irreversibly inhibit their target protease by undergoing

500-487: A conformation wherein the first two amino acids of the RCL are inserted into the top of the A β-sheet . The partially inserted conformation is important because co-factors are able to conformationally switch certain partially inserted serpins into a fully expelled form. This conformational rearrangement makes the serpin a more effective inhibitor. The archetypal example of this situation is antithrombin, which circulates in plasma in

600-418: A distinctive structure, consisting of two beta-barrel domains that converge at the catalytic active site. These enzymes can be further categorised based on their substrate specificity as either trypsin-like, chymotrypsin-like or elastase-like. Trypsin-like proteases cleave peptide bonds following a positively charged amino acid ( lysine or arginine ). This specificity is driven by the residue which lies at

700-513: A general and controllable role in determining cell fate. Plant serpins were amongst the first members of the superfamily that were identified. The serpin barley protein Z is highly abundant in barley grain, and one of the major protein components in beer. The genome of the model plant, Arabidopsis thaliana contain 18 serpin-like genes, although only 8 of these are full-length serpin sequences. Plant serpins are potent inhibitors of mammalian chymotrypsin-like serine proteases in vitro ,

800-674: A human serpin counterpart. Examples include numerous rodent serpins (particularly some of the murine intracellular serpins) as well as the uterine serpins . The term uterine serpin refers to members of the serpin A clade that are encoded by the SERPINA14 gene. Uterine serpins are produced by the endometrium of a restricted group of mammals in the Laurasiatheria clade under the influence of progesterone or estrogen . They are probably not functional proteinase inhibitors and may function during pregnancy to inhibit maternal immune responses against

900-469: A large conformational change to disrupt the target's active site . This contrasts with the more common competitive mechanism for protease inhibitors that bind to and block access to the protease active site. Protease inhibition by serpins controls an array of biological processes, including coagulation and inflammation , and consequently these proteins are the target of medical research . Their unique conformational change also makes them of interest to

1000-410: A methionine in alpha1-antitrypsin as an inhibitor of tissue elastase and on arginine in antithrombin as an inhibitor of thrombin. The critical role of the active centre residue in determining the specificity of inhibition of serpins was unequivocally confirmed by the finding that a natural mutation of the active centre methionine  in alpha1-antitrypsin to an arginine, as in antithrombin, resulted in

1100-415: A more effective inhibitor of thrombin and factor Xa . Furthermore, both of these coagulation proteases also contain binding sites (called exosites ) for heparin. Heparin, therefore, also acts as a template for binding of both protease and serpin, further dramatically accelerating the interaction between the two parties. After the initial interaction, the final serpin complex is formed and the heparin moiety

SECTION 10

#1732855356357

1200-549: A new protein family became apparent on their further alignment with the non-inhibitory egg-white protein ovalbumin , to give what was initially called the alpha1-antitrypsin-antithrombin III-ovalbumin superfamily of serine proteinase inhibitors, but was subsequently succinctly renamed as the Serpins. The initial characterisation of the new family centred on alpha1-antitrypsin , a serpin present in high concentration in blood plasma,

1300-551: A nomenclature system has been adopted that is based on the position of serpin genes on the Drosophila chromosomes . Thirteen of the Drosophila serpins occur as isolated genes in the genome (including Serpin-27A, see below), with the remaining 16 organised into five gene clusters that occur at chromosome positions 28D (2 serpins), 42D (5 serpins), 43A (4 serpins), 77B (3 serpins) and 88E (2 serpins). Studies on Drosophila serpins reveal that Serpin-27A inhibits

1400-622: A nucleophilic cysteine residue, rather than a serine , in their active site. Nonetheless, the enzymatic chemistry is similar, and the mechanism of inhibition by serpins is the same for both classes of protease. Examples of cross-class inhibitory serpins include serpin B4 a squamous cell carcinoma antigen 1 (SCCA-1) and the avian serpin myeloid and erythroid nuclear termination stage-specific protein (MENT), which both inhibit papain-like cysteine proteases . Approximately two-thirds of human serpins perform extracellular roles, inhibiting proteases in

1500-471: A partially inserted relatively inactive state. The primary specificity determining residue (the P1 arginine) points toward the body of the serpin and is unavailable to the protease. Upon binding a high-affinity pentasaccharide sequence within long-chain heparin , antithrombin undergoes a conformational change, RCL expulsion, and exposure of the P1 arginine. The heparin pentasaccharide-bound form of antithrombin is, thus,

1600-406: A protective function and guards against stress-induced calpain -associated lysosomal disruption. Further, SRP-6 inhibits lysosomal cysteine proteases released after lysosomal rupture. Accordingly, worms lacking SRP-6 are sensitive to stress. Most notably, SRP-6 knockout worms die when placed in water (the hypo-osmotic stress lethal phenotype or Osl). It has therefore been suggested that lysosomes play

1700-539: A protein often correspond to functionally important regions like catalytic sites and binding sites, since these regions are less tolerant to sequence changes. Using sequence similarity to infer homology has several limitations. There is no minimum level of sequence similarity guaranteed to produce identical structures. Over long periods of evolution, related proteins may show no detectable sequence similarity to one another. Sequences with many insertions and deletions can also sometimes be difficult to align and so identify

1800-461: A rare type of dementia caused by neuroserpin polymerisation). Each monomer of the serpin aggregate exists in the inactive, relaxed conformation (with the RCL inserted into the A-sheet). The polymers are therefore hyperstable to temperature and unable to inhibit proteases. Serpinopathies therefore cause pathologies similarly to other proteopathies (e.g. prion diseases) via two main mechanisms. First,

1900-643: A result of one of the α-helices (the F-helix) partially switching to a β-strand conformation, completing the β-sheet hydrogen bonding. It is unclear whether other serpins can adopt this conformer, and whether this conformation has a functional role, but it is speculated that the δ-conformation may be adopted by Thyroxine-binding globulin during thyroxine release. The non-inhibitory proteins related to serpins can also cause diseases when mutated. For example, mutations in SERPINF1 cause osteogenesis imperfecta type VI in humans. In

2000-909: A serpin's RCL alters its specificity to target the wrong protease. For example, the Antitrypsin-Pittsburgh mutation (M358R) causes the α1-antitrypsin serpin to inhibit thrombin, causing a bleeding disorder. The majority of serpin diseases are due to protein aggregation and are termed "serpinopathies". Serpins are vulnerable to disease-causing mutations that promote formation of misfolded polymers due to their inherently unstable structures. Well-characterised serpinopathies include α1-antitrypsin deficiency (alpha-1), which may cause familial emphysema , and sometimes liver cirrhosis , certain familial forms of thrombosis related to antithrombin deficiency , types 1 and 2 hereditary angioedema (HAE) related to deficiency of C1-inhibitor , and familial encephalopathy with neuroserpin inclusion bodies (FENIB;

2100-419: A serpin, that of post-cleavage alpha1-antitrypsin. This together with the subsequent solving of the structure of native (uncleaved) ovalbumin indicated that the inhibitory mechanism of the  serpins involved a remarkable conformational shift,  with the movement of the exposed peptide loop containing the reactive site and its incorporation as a middle strand in the main beta-pleated sheet that characterises

SECTION 20

#1732855356357

2200-424: A severe bleeding disorder. This active-centre specificity of inhibition was also evident in the many other families of protease inhibitors but the serpins differed from them in being much larger proteins and also in possessing what was soon apparent as an inherent ability to undergo a change in shape. The nature of this conformational change was revealed with the determination in 1984 of the first crystal structure of

2300-460: A signalling cascade. When a serpin inhibits a target protease, it forms a permanent complex, which needs to be disposed of. For extracellular serpins, the final serpin-enzyme complexes are rapidly cleared from circulation. One mechanism by which this occurs in mammals is via the low-density lipoprotein receptor-related protein ( LRP ), which binds to inhibitory complexes made by antithrombin, PA1-1, and neuroserpin, causing cellular uptake . Similarly,

2400-530: A superfamily is commonly conserved, although substrate specificity may be significantly different. Catalytic residues also tend to occur in the same order in the protein sequence. For the families within the PA clan of proteases, although there has been divergent evolution of the catalytic triad residues used to perform catalysis, all members use a similar mechanism to perform covalent, nucleophilic catalysis on proteins, peptides or amino acids. However, mechanism alone

2500-477: A wide array of important roles. Thyroxine-binding globulin and transcortin transport the hormones thyroxine and cortisol , respectively. The non-inhibitory serpin ovalbumin is the most abundant protein in egg white . Its exact function is unknown, but it is thought to be a storage protein for the developing foetus . Heat shock serpin 47 is a chaperone , essential for proper folding of collagen . It acts by stabilising collagen's triple helix whilst it

2600-590: Is a chaperone essential for proper folding of collagen , and cycles between the cis-Golgi and the endoplasmic reticulum . Protease-inhibition is thought to be the ancestral function, with non-inhibitory members the results of evolutionary neofunctionalisation of the structure. The S to R conformational change has also been adapted by some binding serpins to regulate affinity for their targets. The human genome encodes 16 serpin clades, termed serpinA through serpinP, including 29 inhibitory and 7 non-inhibitory serpin proteins. The human serpin naming system

2700-401: Is a serine protease in prokaryotes . Subtilisin is evolutionarily unrelated to the chymotrypsin-clan, but shares the same catalytic mechanism utilising a catalytic triad , to create a nucleophilic serine . This is the classic example used to illustrate convergent evolution , since the same mechanism evolved twice independently during evolution . The main player in the catalytic mechanism in

2800-547: Is a tightly conserved framework, which allows the precise alignment of their key structural and functional components based on the template structure of alpha1-antitrypsin. In the 2000s, a systematic nomenclature was introduced in order to categorise members of the serpin superfamily based on their evolutionary relationships. Serpins are therefore the largest and most diverse superfamily of protease inhibitors. Most serpins are protease inhibitors, targeting extracellular, chymotrypsin -like serine proteases . These proteases possess

2900-437: Is based upon a phylogenetic analysis of approximately 500 serpins from 2001, with proteins named serpinXY, where X is the clade of the protein and Y the number of the protein within that clade. The functions of human serpins have been determined by a combination of biochemical studies, human genetic disorders , and knockout mouse models . Many mammalian serpins have been identified that share no obvious orthology with

3000-511: Is being processed in the endoplasmic reticulum . Some serpins are both protease inhibitors and perform additional roles. For example, the nuclear cysteine protease inhibitor MENT , in birds also acts as a chromatin remodelling molecule in a bird's red blood cells . All serpins share a common structure (or fold), despite their varied functions. All typically have three β-sheets (named A, B and C) and eight or nine α-helices (named hA–hI). The most significant regions to serpin function are

3100-557: Is considered a good predictor of relatedness, since similar sequences are more likely the result of gene duplication and divergent evolution , rather than the result of convergent evolution . Amino acid sequence is typically more conserved than DNA sequence (due to the degenerate genetic code ), so it is a more sensitive detection method. Since some of the amino acids have similar properties (e.g., charge, hydrophobicity, size), conservative mutations that interchange them are often neutral to function. The most conserved sequence regions of

Serpin - Misplaced Pages Continue

3200-424: Is currently possible. They are therefore amongst the most ancient evolutionary events currently studied. Some superfamilies have members present in all kingdoms of life , indicating that the last common ancestor of that superfamily was in the last universal common ancestor of all life (LUCA). Superfamily members may be in different species, with the ancestral protein being the form of the protein that existed in

3300-400: Is essential, because it activates its own reaction, as well as the reaction of both chymotrypsin and elastase . Therefore, it is essential that this activation does not occur prematurely. There are several protective measures taken by the organism to prevent self-digestion: There are certain inhibitors that resemble the tetrahedral intermediate, and thus fill up the active site, preventing

3400-445: Is not sufficient to infer relatedness. Some catalytic mechanisms have been convergently evolved multiple times independently, and so form separate superfamilies, and in some superfamilies display a range of different (though often chemically similar) mechanisms. Protein superfamilies represent the current limits of our ability to identify common ancestry. They are the largest evolutionary grouping based on direct evidence that

3500-494: Is released (the N-terminus "half" of the peptide with carboxyl group visible). Each amino acid in the triad performs a specific task in this process: The whole reaction can be summarized as follows: It was discovered that additional amino acids of the protease, Gly 193 and Ser 195 , are involved in creating what is called an oxyanion hole . Both Gly 193 and Ser 195 can donate backbone hydrogens for hydrogen bonding. When

3600-412: Is released. This interaction is physiologically important. For example, after injury to the blood vessel wall, heparin is exposed, and antithrombin is activated to control the clotting response. Understanding of the molecular basis of this interaction enabled the development of Fondaparinux , a synthetic form of Heparin pentasaccharide used as an anti-clotting drug . Certain serpins spontaneously undergo

3700-427: Is such a condition, in which there is premature activation of the digestive enzymes in the pancreas, resulting in self-digestion (autolysis). It also complicates postmortem investigations , as the pancreas often digests itself before it can be assessed visually. Zymogens are large, inactive structures, which have the ability to break apart or change into the smaller activated enzymes. The difference between zymogens and

3800-403: Is used in order to avoid inflammatory and apoptotic responses of infected host cells. CrmA increases infectivity by suppressing its host's inflammatory response through inhibition of IL-1 and IL-18 processing by the cysteine protease caspase -1. In eukaryotes , a plant serpin inhibits both metacaspases and a papain-like cysteine protease. Non-inhibitory extracellular serpins also perform

3900-403: Is used to describe these members as well, despite their non-inhibitory function, since they are evolutionarily related. Protease inhibitory activity in blood plasma was first reported in the late 1800s, but it was not until the 1950s that the serpins antithrombin and alpha 1-antitrypsin were isolated, with the subsequent recognition of their close family homology in 1979. That they belonged to

4000-530: Is very rare to find “consistently isolated superfamilies”. When domains do combine, the N- to C-terminal domain order (the "domain architecture") is typically well conserved. Additionally, the number of domain combinations seen in nature is small compared to the number of possibilities, suggesting that selection acts on all combinations. Several biological databases document protein superfamilies and protein folds, for example: Similarly there are algorithms that search

4100-595: The Drosophila necrotic serpin is degraded in the lysosome after being trafficked into the cell by the Lipophorin Receptor-1 (homologous to the mammalian LDL receptor family). Serpins are involved in a wide array of physiological functions, and so mutations in genes encoding them can cause a range of diseases. Mutations that change the activity, specificity or aggregation properties of serpins all affect how they function. The majority of serpin-related diseases are

Serpin - Misplaced Pages Continue

4200-687: The PDB for proteins with structural homology to a target structure, for example: Serine protease Serine proteases (or serine endopeptidases ) are enzymes that cleave peptide bonds in proteins . Serine serves as the nucleophilic amino acid at the (enzyme's) active site . They are found ubiquitously in both eukaryotes and prokaryotes . Serine proteases fall into two broad categories based on their structure: chymotrypsin -like (trypsin-like) or subtilisin -like. The MEROPS protease classification system counts 16 superfamilies (as of 2013) each containing many families . Each superfamily uses

4300-580: The catalytic triad or dyad in a different protein fold and so represent convergent evolution of the catalytic mechanism . The majority belong to the S1 family of the PA clan (superfamily) of proteases. For superfamilies , P: superfamily, containing a mixture of nucleophile class families, S: purely serine proteases. superfamily. Within each superfamily, families are designated by their catalytic nucleophile, (S: serine proteases). Serine proteases are characterised by

4400-503: The cellulosome . It is suggested that the role of cellulosome-associated serpins may be to prevent unwanted protease activity against the cellulosome. Serpins are also expressed by viruses as a way to evade the host's immune defense. In particular, serpins expressed by pox viruses , including cow pox (vaccinia) and rabbit pox (myxoma), are of interest because of their potential use as novel therapeutics for immune and inflammatory disorders as well as transplant therapy. Serp1 suppresses

4500-421: The conceptus or to participate in transplacental transport. The Drosophila melanogaster genome contains 29 serpin encoding genes. Amino acid sequence analysis has placed 14 of these serpins in serpin clade Q and three in serpin clade K with the remaining twelve classified as orphan serpins not belonging to any clade. The clade classification system is difficult to use for Drosophila serpins and instead

4600-420: The structural biology and protein folding research communities. The conformational-change mechanism confers certain advantages, but it also has drawbacks: serpins are vulnerable to mutations that can result in serpinopathies such as protein misfolding and the formation of inactive long-chain polymers . Serpin polymerisation not only reduces the amount of active inhibitor, but also leads to accumulation of

4700-407: The tetrahedral intermediate of step 1 and step 3 are generated, the negative oxygen ion, having accepted the electrons from the carbonyl double bond, fits perfectly into the oxyanion hole. In effect, serine proteases preferentially bind the transition state and the overall structure is favored, lowering the activation energy of the reaction. This "preferential binding" is responsible for much of

4800-463: The 'Responsive to Desiccation-21' (RD21) papain-like cysteine protease. AtSerpin1 also inhibits metacaspase -like proteases in vitro . Two other Arabidopsis serpins, AtSRP2 (At2g14540) and AtSRP3 (At1g64030) appear to be involved in responses to DNA damage. A single fungal serpin has been characterized to date: celpin from Piromyces spp. strain E2. Piromyces is a genus of anaerobic fungi found in

4900-582: The A-sheet and the reactive centre loop (RCL). The A-sheet includes two β-strands that are in a parallel orientation with a region between them called the 'shutter', and upper region called the 'breach'. The RCL forms the initial interaction with the target protease in inhibitory molecules. Structures have been solved showing the RCL either fully exposed or partially inserted into the A-sheet, and serpins are thought to be in dynamic equilibrium between these two states. The RCL also only makes temporary interactions with

5000-518: The B-sheet (with each molecule's RCL inserted into its own A-sheet). It has also been proposed that serpins may form domain-swaps by inserting the RCL of one protein into the A-sheet of another (A-sheet polymerisation). These domain-swapped dimer and trimer structures are thought to be the building blocks of the disease-causing polymer aggregates, but the exact mechanism is still unclear. Several therapeutic approaches are in use or under investigation to treat

5100-536: The D-helix as well as significant portions of the A- and E-helices. Protein superfamily Superfamilies of proteins are identified using a number of methods. Closely related members can be identified by different methods to those needed to group the most evolutionarily divergent members. Historically, the similarity of different amino acid sequences has been the most common method of inferring homology . Sequence similarity

SECTION 50

#1732855356357

5200-544: The Easter protease (the final protease in the Nudel, Gastrulation Defective, Snake and Easter proteolytic cascade) and thus controls dorsoventral patterning . Easter functions to cleave Spätzle (a chemokine-type ligand), which results in toll-mediated signaling. As well as its central role in embryonic patterning, toll signaling is also important for the innate immune response in insects. Accordingly, serpin-27A also functions to control

5300-456: The N-terminus of tengpin, a serpin from Thermoanaerobacter tengcongensis , is required to lock the molecule in the native inhibitory state. Disruption of interactions made by the N-terminal region results in spontaneous conformational change of this serpin to the latent conformation. Certain non-inhibitory serpins also use the serpin conformational change as part of their function. For example,

5400-450: The S to R transition can activate cell signalling events. In these cases, a serpin that has formed a complex with its target protease, is then recognised by a receptor. The binding event then leads to downstream signalling by the receptor. The S to R transition is therefore used to alert cells to the presence of protease activity. This differs from the usual mechanism whereby serpins affect signalling simply by inhibiting proteases involved in

5500-488: The S to R transition without having been cleaved by a protease, to form a conformation termed the latent state. Latent serpins are unable to interact with proteases and so are no longer protease inhibitors. The conformational change to latency is not exactly the same as the S to R transition of a cleaved serpin. Since the RCL is still intact, the first strand of the C-sheet has to peel off to allow full RCL insertion. Regulation of

5600-464: The TLR-mediated innate immune response and allows indefinite cardiac allograft survival in rats. Crma and Serp2 are both cross-class inhibitors and target both serine (granzyme B; albeit weakly) and cysteine proteases (caspase 1 and caspase 8). In comparison to their mammalian counterparts, viral serpins contain significant deletions of elements of secondary structure. Specifically, crmA lacks

5700-437: The ability of the mammalian liver to secrete active antitrypsin. Small molecules have also been developed that block antitrypsin polymerisation in vitro . Serpins are the most widely distributed and largest superfamily of protease inhibitors. They were initially believed to be restricted to eukaryote organisms, but have since been found in bacteria , archaea and some viruses . It remains unclear whether prokaryote genes are

5800-405: The absence of a required serpin, the protease that it normally would regulate is over-active, leading to pathologies. Consequently, simple deficiency of a serpin (e.g. a null mutation ) can result in disease. Gene knockouts , particularly in mice , are used experimentally to determine the normal functions of serpins by the effect of their absence. In some rare cases, a single amino acid change in

5900-399: The activated enzymes lies in the fact that the active site for catalysis of the zymogens is distorted. As a result, the substrate polypeptide cannot bind effectively, and proteolysis does not occur. Only after activation, during which the conformation and structure of the zymogen change and the active site is opened, can proteolysis occur. As can be seen, trypsinogen activation to trypsin

6000-409: The amount of active inhibitory serpin. For example, the disease-linked antithrombin variants wibble and wobble , both promote formation of the latent state . The structure of the disease-linked mutant of antichymotrypsin (L55P) revealed another, inactive "δ-conformation". In the δ-conformation, four residues of the RCL are inserted into the top of β-sheet A. The bottom half of the sheet is filled as

6100-438: The ancestral species ( orthology ). Conversely, the proteins may be in the same species, but evolved from a single protein whose gene was duplicated in the genome ( paralogy ). A majority of proteins contain multiple domains. Between 66-80% of eukaryotic proteins have multiple domains while about 40-60% of prokaryotic proteins have multiple domains. Over time, many of the superfamilies of domains have mixed together. In fact, it

SECTION 60

#1732855356357

6200-418: The arthropod immune system . Mutations may lead to decreased or increased activity of enzymes. This may have different consequences, depending on the normal function of the serine protease. For example, mutations in protein C can lead to protein C deficiency and predisposing to thrombosis . Also, some proteases play a vital role in host cell-virus fusion activation by priming virus's Spike protein to show

6300-483: The base of the enzyme's S1 pocket (generally a negatively charged aspartic acid or glutamic acid ). The S1 pocket of chymotrypsin-like enzymes is more hydrophobic than in trypsin-like proteases. This results in a specificity for medium to large sized hydrophobic residues, such as tyrosine , phenylalanine and tryptophan . These include thrombin , tissue activating plasminogen and plasmin . They have been found to have roles in coagulation and digestion as well as in

6400-683: The best-studied example being barley serpin Zx (BSZx), which is able to inhibit trypsin and chymotrypsin as well as several blood coagulation factors. However, close relatives of chymotrypsin-like serine proteases are absent in plants. The RCL of several serpins from wheat grain and rye contain poly-Q repeat sequences similar to those present in the prolamin storage proteins of the endosperm. It has therefore been suggested that plant serpins may function to inhibit proteases from insects or microbes that would otherwise digest grain storage proteins. In support of this hypothesis, specific plant serpins have been identified in

6500-435: The bloodstream in order to modulate their activities. For example, extracellular serpins regulate the proteolytic cascades central to blood clotting (antithrombin), the inflammatory and immune responses (antitrypsin, antichymotrypsin , and C1-inhibitor ) and tissue remodelling (PAI-1) . By inhibiting signalling cascade proteases, they can also affect development . The table of human serpins (below) provides examples of

6600-446: The catalytic efficiency of the enzyme. Host organisms must ensure that the activity of serine proteases is adequately regulated. This is achieved by a requirement for initial protease activation, and the secretion of inhibitors. Zymogens are the usually inactive precursors of an enzyme. If the digestive enzymes were active when synthesized, they would immediately start chewing up the synthesizing organs and tissues. Acute pancreatitis

6700-399: The cell, serpin polymers are slowly removed via degradation in the endoplasmic reticulum. However, the details of how serpin polymers cause cell death remains to be fully understood. Physiological serpin polymers are thought to form via domain swapping events, where a segment of one serpin protein inserts into another. Domain-swaps occur when mutations or environmental factors interfere with

6800-545: The cellulosome. Predicted serpin genes are sporadically distributed in prokaryotes . In vitro studies on some of these molecules have revealed that they are able to inhibit proteases, and it is suggested that they function as inhibitors in vivo . Several prokaryote serpins are found in extremophiles . Accordingly, and in contrast to mammalian serpins, these molecules possess elevated resistance to heat denaturation. The precise role of most bacterial serpins remains obscure, although Clostridium thermocellum serpin localises to

6900-401: The common genetic disorder of which was shown to cause a predisposition to the lung disease emphysema and to liver cirrhosis . The identification of the S and Z mutations responsible for the genetic deficiency and the subsequent sequence alignments of alpha1-antitrypsin and antithrombin in 1982 led to the recognition of the close homologies of the active sites of the two proteins, centred on

7000-447: The descendants of an ancestral prokaryotic serpin or the product of horizontal gene transfer from eukaryotes. Most intracellular serpins belong to a single phylogenetic clade, whether they come from plants or animals, indicating that the intracellular and extracellular serpins may have diverged before the plants and animals. Exceptions include the intracellular heat shock serpin HSP47, which

7100-585: The enzyme from working properly. Trypsin, a powerful digestive enzyme, is generated in the pancreas. Inhibitors prevent self-digestion of the pancreas itself. Serine proteases are paired with serine protease inhibitors , which turn off their activity when they are no longer needed. Serine proteases are inhibited by a diverse group of inhibitors , including synthetic chemical inhibitors for research or therapeutic purposes, and also natural proteinaceous inhibitors. One family of natural inhibitors called "serpins" (abbreviated from serine protease inhibitors ) can form

7200-458: The event of catalysis, an ordered mechanism occurs in which several intermediates are generated. The catalysis of the peptide cleavage can be seen as a ping-pong catalysis, in which a substrate binds (in this case, the polypeptide being cleaved), a product is released (the C-terminus "half" of the peptide with amino group visible), another substrate binds (in this case, water), and another product

7300-406: The final stages of serpin folding to the native state, causing high-energy intermediates to misfold. Both dimer and trimer domain-swap structures have been solved. In the dimer (of antithrombin), the RCL and part of the A-sheet incorporates into the A-sheet of another serpin molecule. The domain-swapped trimer (of antitrypsin) forms via the exchange of an entirely different region of the structure,

7400-479: The gut of ruminants and is important for digesting plant material. Celpin is predicted to be inhibitory and contains two N-terminal dockerin domains in addition to its serpin domain. Dockerins are commonly found in proteins that localise to the fungal cellulosome , a large extracellular multiprotein complex that breaks down cellulose. It is therefore suggested that celpin may protect the cellulosome against plant proteases. Certain bacterial serpins similarly localize to

7500-504: The homologous sequence regions. In the PA clan of proteases , for example, not a single residue is conserved through the superfamily, not even those in the catalytic triad . Conversely, the individual families that make up a superfamily are defined on the basis of their sequence alignment, for example the C04 protease family within the PA clan. Nevertheless, sequence similarity is the most commonly used form of evidence to infer relatedness, since

7600-576: The inappropriate activity of proteases inside the cell. For example, one of the best-characterised human intracellular serpins is Serpin B9 , which inhibits the cytotoxic granule protease granzyme B . In doing so, Serpin B9 may protect against inadvertent release of granzyme B and premature or unwanted activation of cell death pathways. Some viruses use serpins to disrupt protease functions in their host. The cowpox viral serpin CrmA (cytokine response modifier A)

7700-431: The insect immune response. In Tenebrio molitor (a large beetle), a protein (SPN93) comprising two discrete tandem serpin domains functions to regulate the toll proteolytic cascade. The genome of the nematode worm C. elegans contains 9 serpins, all of which lack signal sequences and so are likely intracellular. However, only 5 of these serpins appear to function as protease inhibitors. One, SRP-6, performs

7800-413: The lack of active serpin results in uncontrolled protease activity and tissue destruction. Second, the hyperstable polymers themselves clog up the endoplasmic reticulum of cells that synthesize serpins, eventually resulting in cell death and tissue damage. In the case of antitrypsin deficiency, antitrypsin polymers cause the death of liver cells , sometimes resulting in liver damage and cirrhosis . Within

7900-416: The latency transition can act as a control mechanism in some serpins, such as PAI-1 . Although PAI-1 is produced in the inhibitory S conformation, it "auto-inactivates" by changing to the latent state unless it is bound to the cofactor vitronectin . Similarly, antithrombin can also spontaneously convert to the latent state, as an additional modulation mechanism to its allosteric activation by heparin. Finally,

8000-558: The most common serpinopathy: antitrypsin deficiency. Antitrypsin augmentation therapy is approved for severe antitrypsin deficiency-related emphysema. In this therapy, antitrypsin is purified from the plasma of blood donors and administered intravenously (first marketed as Prolastin ). To treat severe antitrypsin deficiency-related disease, lung and liver transplantation has proven effective. In animal models, gene targeting in induced pluripotent stem cells has been successfully used to correct an antitrypsin polymerisation defect and to restore

8100-413: The native (S) form of thyroxine-binding globulin has high affinity for thyroxine, whereas the cleaved (R) form has low affinity. Similarly, transcortin has higher affinity for cortisol when in its native (S) state, than its cleaved (R) state. Thus, in these serpins, RCL cleavage and the S to R transition has been commandeered to allow for ligand release, rather than protease inhibition. In some serpins,

8200-645: The number of known sequences vastly outnumbers the number of known tertiary structures . In the absence of structural information, sequence similarity constrains the limits of which proteins can be assigned to a superfamily. Structure is much more evolutionarily conserved than sequence, such that proteins with highly similar structures can have entirely different sequences. Over very long evolutionary timescales, very few residues show detectable amino acid sequence conservation, however secondary structural elements and tertiary structural motifs are highly conserved. Some protein dynamics and conformational changes of

8300-398: The pathophysiology of neurodegenerative disorders such as Alzheimer's and Parkinson's induced dementia. Many highly-toxic thrombin-like serine protease isoforms are found in snake venoms. Elastase-like proteases have a much smaller S1 cleft than either trypsin- or chymotrypsin-like proteases. Consequently, residues such as alanine , glycine and valine tend to be preferred. Subtilisin

8400-481: The phloem sap of pumpkin (CmPS-1) and cucumber plants. Although an inverse correlation between up-regulation of CmPS-1 expression and aphid survival was observed, in vitro feeding experiments revealed that recombinant CmPS-1 did not appear to affect insect survival. Alternative roles and protease targets for plant serpins have been proposed. The Arabidopsis serpin, AtSerpin1 (At1g47710; 3LE2 ​), mediates set-point control over programmed cell death by targeting

8500-427: The polymers, causing cell death and organ failure . Although most serpins control proteolytic cascades, some proteins with a serpin structure are not enzyme inhibitors , but instead perform diverse functions such as storage (as in egg white — ovalbumin ), transport as in hormone carriage proteins ( thyroxine-binding globulin , cortisol-binding globulin ) and molecular chaperoning ( HSP47 ). The term serpin

8600-642: The post-inhibitory complex of alpha1-antitrypsin with trypsin, showing how the displacement results in the deformation and inactivation of the attached protease. Subsequent structural studies have revealed an additional advantage of the conformational mechanism in allowing the subtle modulation of inhibitory activity, as notably seen at tissue level with the functionally diverse serpins in human plasma. Over 1000 serpins have now been identified, including 36 human proteins, as well as molecules in all kingdoms of life— animals , plants , fungi , bacteria , and archaea —and some viruses . The central feature of all

8700-614: The protease remains covalently attached for days to weeks. Serpins are classed as irreversible inhibitors and as suicide inhibitors since each serpin protein permanently inactivates a single protease, and can only function once. The conformational mobility of serpins provides a key advantage over static lock-and-key protease inhibitors. In particular, the function of inhibitory serpins can be regulated by allosteric interactions with specific cofactors . The X-ray crystal structures of antithrombin , heparin cofactor II , MENT and murine antichymotrypsin reveal that these serpins adopt

8800-425: The proteases. While the amino acid members of the triad are located far from one another on the sequence of the protein, due to folding, they will be very close to one another in the heart of the enzyme. The particular geometry of the triad members are highly characteristic to their specific function: it was shown that the position of just four points of the triad characterize the function of the containing enzyme. In

8900-462: The protein named "fusion protein" ( TMPRSS2 activate SARS-CoV-2 fusion). Exogenous snake venom serine proteases cause a vast array of coagulopathies when injected in a host due to the lack of regulation of their activity. Determination of serine protease levels may be useful in the context of particular diseases. Due to their catalytic activity, some serine proteases possess potent antimicrobial properties. Several in vitro studies have demonstrated

9000-563: The protein structure may also be conserved, as is seen in the serpin superfamily . Consequently, protein tertiary structure can be used to detect homology between proteins even when no evidence of relatedness remains in their sequences. Structural alignment programs, such as DALI , use the 3D structure of a protein of interest to find proteins with similar folds. However, on rare occasions, related proteins may evolve to be structurally dissimilar and relatedness can only be inferred by other methods. The catalytic mechanism of enzymes within

9100-465: The range of functions performed by human serpin, as well as some of the diseases that result from serpin deficiency. The protease targets of intracellular inhibitory serpins have been difficult to identify, since many of these molecules appear to perform overlapping roles. Further, many human serpins lack precise functional equivalents in model organisms such as the mouse. Nevertheless, an important function of intracellular serpins may be to protect against

9200-426: The rate or the extent of RCL insertion into the A-sheet can cause the serpin to undergo its S to R conformational change before having engaged a protease. Since a serpin can only make this conformational change once, the resulting misfired serpin is inactive and unable to properly control its target protease. Similarly, mutations that promote inappropriate transition to the monomeric latent state cause disease by reducing

9300-415: The relative kinetic rate of the conformational change is several orders of magnitude faster than hydrolysis by the protease. Since the RCL is still covalently attached to the protease via the ester bond, the S to R transition pulls protease from the top to the bottom of the serpin and distorts the catalytic triad. The distorted protease can only hydrolyse the acyl enzyme intermediate extremely slowly and so

9400-439: The rest of the structure, and is therefore highly flexible and exposed to the solvent. The serpin structures that have been determined cover several different conformations, which has been necessary for the understanding of their multiple-step mechanism of action. Structural biology has therefore played a central role in the understanding of serpin function and biology. Inhibitory serpins do not inhibit their target proteases by

9500-457: The result of serpin polymerisation into aggregates, though several other types of disease-linked mutations also occur. The disorder alpha-1 antitrypsin deficiency is one of the most common hereditary diseases . Since the stressed serpin fold is high-energy, mutations can cause them to incorrectly change into their lower-energy conformations (e.g. relaxed or latent) before they have correctly performed their inhibitory role. Mutations that affect

9600-416: The serine proteases is the catalytic triad. The triad is located in the active site of the enzyme, where catalysis occurs, and is preserved in all superfamilies of serine protease enzymes. The triad is a coordinated structure consisting of three amino acids : His 57, Ser 195 (hence the name "serine protease") and Asp 102. These three key amino acids each play an essential role in the cleaving ability of

9700-399: The serpin from a stressed state, to a lower-energy relaxed state (S to R transition). Serine and cysteine proteases catalyse peptide bond cleavage by a two-step process. Initially, the catalytic residue of the active site triad performs a nucleophilic attack on the peptide bond of the substrate. This releases the new N-terminus and forms a covalent ester -bond between the enzyme and

9800-409: The serpin molecule. Early evidence of the essential role of this loop movement in the inhibitory mechanism came from the finding that even minor aberrations in the amino acid residues that form the hinge of the movement in antithrombin resulted in thrombotic disease. Ultimate confirmation of the linked displacement of the target protease by this loop movement was provided  in 2000 by the structure of

9900-417: The substrate. This covalent complex between enzyme and substrate is called an acyl-enzyme intermediate . For standard substrates , the ester bond is hydrolysed and the new C-terminus is released to complete catalysis. However, when a serpin is cleaved by a protease, it rapidly undergoes the S to R transition before the acyl-enzyme intermediate is hydrolysed. The efficiency of inhibition depends on fact that

10000-442: The typical competitive ( lock-and-key ) mechanism used by most small protease inhibitors (e.g. Kunitz-type inhibitors ). Instead, serpins use an unusual conformational change , which disrupts the structure of the protease and prevents it from completing catalysis. The conformational change involves the RCL moving to the opposite end of the protein and inserting into β-sheet A, forming an extra antiparallel β-strand. This converts

#356643