Serpins are asuperfamily ofproteins with similar structures that were first identified for theirprotease inhibition activity and are found in allkingdoms of life.[1][2] The acronym serpin was originally coined because the first serpins to be identified act on chymotrypsin-likeserine proteases (serineproteaseinhibitors).[3][4][5] They are notable for their unusual mechanism of action, in which theyirreversibly inhibit their targetprotease by undergoing a largeconformational change to disrupt the target'sactive site.[6][7] This contrasts with the more commoncompetitive mechanism for protease inhibitors that bind to and block access to the protease active site.[8][9]
Protease inhibition by serpins controls an array of biological processes, includingcoagulation andinflammation, and consequently these proteins are the target ofmedical research.[10] Their unique conformational change also makes them of interest to thestructural biology andprotein folding research communities.[7][8] The conformational-change mechanism confers certain advantages, but it also has drawbacks: serpins are vulnerable tomutations that can result in serpinopathies such asprotein misfolding and the formation of inactive long-chainpolymers.[11][12] Serpinpolymerisation not only reduces the amount of active inhibitor, but also leads to accumulation of the polymers, causingcell death andorgan failure.[10]
Protease inhibitory activity in blood plasma was first reported in the late 1800s,[13] but it was not until the 1950s that the serpinsantithrombin andalpha 1-antitrypsin were isolated,[14] with the subsequent recognition of their close family homology in 1979.[15][16] That they belonged to a new protein family became apparent on their further alignment with the non-inhibitory egg-white proteinovalbumin, to give what was initially called thealpha1-antitrypsin-antithrombin III-ovalbuminsuperfamily of serine proteinase inhibitors,[17] but was subsequently succinctly renamed as the Serpins.[18] The initial characterisation of the new family centred onalpha1-antitrypsin, a serpin present in high concentration in blood plasma, the commongenetic disorder of which was shown to cause a predisposition to the lung diseaseemphysema[19] and to livercirrhosis.[20] The identification of the S and Z mutations[21][22] 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,[23][24] centred on a methionine[25] in alpha1-antitrypsin as an inhibitor of tissue elastase and on arginine in antithrombin[26] as an inhibitor of thrombin.[27]
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 a severe bleeding disorder.[28] This active-centre specificity of inhibition was also evident in the many other families of protease inhibitors[7] 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 crystalstructure of a serpin, that of post-cleavage alpha1-antitrypsin.[29] This together with the subsequent solving of the structure of native (uncleaved) ovalbumin[30] 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 the serpin molecule.[31][32] 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.[31][33] Ultimate confirmation of the linked displacement of the target protease by this loop movement was provided in 2000 by the structure of the post-inhibitory complex of alpha1-antitrypsin with trypsin,[6] 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[34] in allowing the subtle modulation of inhibitory activity, as notably seen at tissue level[35] with the functionally diverse serpins in human plasma.
Over 1000 serpins have now been identified, including 36 human proteins, as well as molecules in allkingdoms of life—animals,plants,fungi,bacteria, andarchaea—and someviruses.[36][37][38] The central feature of all 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.[39] In the 2000s, a systematic nomenclature was introduced in order to categorise members of the serpin superfamily based on their evolutionary relationships.[1] Serpins are therefore the largest and most diverse superfamily of protease inhibitors.[40]
Approximately two-thirds of human serpins perform extracellular roles, inhibiting proteases in the bloodstream in order to modulate their activities. For example, extracellular serpins regulate the proteolytic cascades central toblood clotting (antithrombin), theinflammatory andimmune responses (antitrypsin,antichymotrypsin, andC1-inhibitor) and tissue remodelling(PAI-1).[9] By inhibitingsignalling cascade proteases, they can also affectdevelopment.[47][48] The table of human serpins (below) provides examples of 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 the inappropriate activity of proteases inside the cell.[49] For example, one of the best-characterised human intracellular serpins isSerpin B9, which inhibits thecytotoxicgranule proteasegranzyme B. In doing so, Serpin B9 may protect against inadvertent release of granzyme B and premature or unwanted activation ofcell death pathways.[50]
Someviruses use serpins to disrupt protease functions in their host. Thecowpoxviral serpin CrmA (cytokine response modifier A) is used in order to avoid inflammatory andapoptotic responses of infected host cells. CrmA increases infectivity by suppressing its host's inflammatory response through inhibition ofIL-1 andIL-18 processing by the cysteine proteasecaspase-1.[51] Ineukaryotes, a plant serpin inhibits bothmetacaspases[52] and a papain-like cysteine protease.[53]
Some serpins are both protease inhibitors and perform additional roles. For example, the nuclear cysteine protease inhibitorMENT, inbirds also acts as achromatin remodelling molecule in a bird'sred blood cells.[45][58]
The native state of serpins is an equilibrium between a fully stressed state (left) and a partially relaxed state (right). The five-stranded A-sheet (light blue) contains two functionally important regions for the serpin's mechanism, the breach and the shutter. The reactive centre loop (RCL, blue) exists in a dynamic equilibrium between the fully exposed conformation (left) and a conformation where it is partially inserted into the breach of the A-sheet (right).(PDB:1QLP,1YXA)[59][60]
All serpins share a commonstructure (or fold), despite their varied functions. All typically have threeβ-sheets (named A, B and C) and eight or nineα-helices (named hA–hI).[29][30] The most significant regions to serpin function are the A-sheet and the reactive centre loop (RCL). The A-sheet includes twoβ-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 indynamic equilibrium between these two states.[8] The RCL also only makes temporary interactions with the rest of the structure, and is therefore highly flexible and exposed to the solvent.[8]
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.[8]
Inhibitory serpins do not inhibit their target proteases by the typicalcompetitive (lock-and-key) mechanism used by most smallprotease inhibitors (e.g.Kunitz-type inhibitors). Instead, serpins use an unusualconformational 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 extraantiparallel β-strand. This converts the serpin from a stressed state, to a lower-energy relaxed state (S to R transition).[7][8][61]
Serine andcysteine proteases catalyse peptide bond cleavage by a two-step process. Initially, thecatalytic residue of the active sitetriad performs anucleophilic attack on the peptide bond of the substrate. This releases the newN-terminus and forms a covalentester-bond between the enzyme and the substrate.[7] This covalent complex between enzyme and substrate is called anacyl-enzyme intermediate. For standardsubstrates, the ester bond ishydrolysed and the newC-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.[7] The efficiency of inhibition depends on fact that the relativekinetic 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 the protease remains covalently attached for days to weeks.[6] Serpins are classed asirreversible inhibitors and assuicide inhibitors since each serpin protein permanently inactivates a single protease, and can only function once.[7]
The inhibitory mechanism of serpins involves a largeconformational change (S to R transition). The serpin (white) first binds a protease (grey) with the exposed reactive centre loop (blue). When this loop is cleaved by the protease, it rapidly inserts into the A-sheet (light blue), deforming and inhibiting the protease. (PDB:1K9O,1EZX)
Serine andcysteine proteases operate by a two-step catalytic mechanism. First, thesubstrate (blue) isattacked by the cysteine or serine of thecatalytic triad (red) to form anacyl-enzyme intermediate. For typical substrates, the intermediate is resolved byhydrolysis by water. However, when the reactive centre loop (RCL) of a serpin is attacked, theconformational change (blue arrow) pulls the catalytic triad out of position, preventing it from completing catalysis. (Based onPDB:1K9O,1EZX)
Some serpins are activated by cofactors. The serpinantithrombin has an RCL (blue) where the P1 arginine (blue sticks) points inwards, preventing protease binding. Binding ofheparin (green sticks) causes the P1 arginine residue to flip to an exposed position. The target protease (grey) then binds to both the exposed P1 arginine as well as the heparin. The serpin then activates and heparin is released. (PDB:1TB6,2ANT,1TB6,1EZX)
Theconformational mobility of serpins provides a key advantage over static lock-and-key protease inhibitors.[34] In particular, the function of inhibitory serpins can beregulated byallosteric interactions with specificcofactors. TheX-ray crystal structures ofantithrombin,heparin cofactor II,MENT and murineantichymotrypsin reveal that these serpins adopt a conformation wherein the first two amino acids of the RCL are inserted into the top of the Aβ-sheet. The partially inserted conformation is important because co-factors are able to conformationally switch certain partially inserted serpins into a fully expelled form.[62][63] This conformational rearrangement makes the serpin a more effective inhibitor.
The archetypal example of this situation is antithrombin, which circulates in plasma in 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-chainheparin, antithrombin undergoes a conformational change, RCL expulsion, and exposure of the P1 arginine. The heparin pentasaccharide-bound form of antithrombin is, thus, a more effective inhibitor ofthrombin andfactor Xa.[64][65] Furthermore, both of these coagulation proteases also contain binding sites (calledexosites) 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 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 ofFondaparinux, a synthetic form of Heparin pentasaccharide used as ananti-clotting drug.[66][67]
Some serpins can spontaneously convert to an inactive latent state. The serpinPAI-1 remains in the active conformation when bound tovitronectin (green). However, in the absence of vitronectin, PAI-1 can change to the inactive latent state. The uncleaved RCL (blue; disordered regions as dashed lines) inserts into the A-sheet, pulling a β-strand off the C-sheet (yellow). (PDB:1OC0,1DVM,1LJ5)
Certain serpins spontaneously undergo 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.[68]
Regulation of the latency transition can act as a control mechanism in some serpins, such asPAI-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 cofactorvitronectin.[68] Similarly, antithrombin can also spontaneously convert to the latent state, as an additional modulation mechanism to its allosteric activation by heparin.[69] Finally, the N-terminus of tengpin, a serpin fromThermoanaerobacter 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.[70][71]
Certain non-inhibitory serpins also use the serpin conformational change as part of their function. For example, the native (S) form ofthyroxine-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.[54][55][72]
In some serpins, the S to R transition can activatecell 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.[73] The S to R transition is therefore used to alert cells to the presence of protease activity.[73] This differs from the usual mechanism whereby serpins affect signalling simply by inhibiting proteases involved in a signalling cascade.[47][48]
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, causingcellular uptake.[73][74] Similarly, theDrosophila necrotic serpin is degraded in thelysosome after being trafficked into the cell by the Lipophorin Receptor-1 (homologous to the mammalianLDL receptor family).[75]
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 the result of serpin polymerisation into aggregates, though several other types of disease-linked mutations also occur.[8][76] The disorderalpha-1 antitrypsin deficiency is one of the most commonhereditary diseases.[11][77]
The inactive δ-conformation of the disease-linked antichymotrypsin mutant (L55P). Four residues of the RCL (blue; disordered region as dashed line) are inserted into the top of the A-sheet. Part of the F α-helix (yellow) has unwound and fills the bottom half of the A-sheet. (PDB:1QMN)
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.[10]
Mutations that affect 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.[10][78] Similarly, mutations that promote inappropriate transition to the monomeric latent state cause disease by reducing the amount of active inhibitory serpin. For example, the disease-linked antithrombin variantswibble andwobble,[79] both promote formation of thelatent 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 a result of one of the α-helices (the F-helix) partially switching to a β-strand conformation, completing the β-sheet hydrogen bonding.[80] 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.[55] The non-inhibitory proteins related to serpins can also cause diseases when mutated. For example, mutations in SERPINF1 causeosteogenesis imperfecta type VI in humans.[81]
In the absence of a required serpin, the protease that it normally would regulate is over-active, leading to pathologies.[10] Consequently, simple deficiency of a serpin (e.g. anull mutation) can result in disease.[82]Gene knockouts, particularly inmice, are used experimentally to determine the normal functions of serpins by the effect of their absence.[83]
In some rare cases, a single amino acid change in a serpin's RCL alters its specificity to target the wrong protease. For example, the Antitrypsin-Pittsburgh mutation (M358R) causes theα1-antitrypsin serpin to inhibit thrombin, causing ableeding disorder.[28]
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 otherproteopathies (e.g.prion diseases) via two main mechanisms.[11][12] First, the lack of active serpin results in uncontrolled protease activity and tissue destruction. Second, the hyperstable polymers themselves clog up theendoplasmic 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 ofliver cells, sometimes resulting in liver damage andcirrhosis. Within the cell, serpin polymers are slowly removed via degradation in the endoplasmic reticulum.[85] However, the details of how serpin polymers cause cell death remains to be fully understood.[11]
Physiological serpin polymers are thought to form viadomain swapping events, where a segment of one serpin protein inserts into another.[86] Domain-swaps occur when mutations or environmental factors interfere with the final stages of serpin folding to the native state, causing high-energy intermediates to misfold.[87] Bothdimer andtrimer 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.[86] The domain-swapped trimer (of antitrypsin) forms via the exchange of an entirely different region of the structure, the B-sheet (with each molecule's RCL inserted into its own A-sheet).[88] 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).[84][89] 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.[86][87][88][90]
Several therapeutic approaches are in use or under investigation to treat the most common serpinopathy: antitrypsin deficiency.[11] Antitrypsin augmentation therapy is approved for severe antitrypsin deficiency-related emphysema.[91] In this therapy, antitrypsin is purified from the plasma of blood donors and administered intravenously (first marketed asProlastin).[11][92] To treat severe antitrypsin deficiency-related disease, lung and livertransplantation has proven effective.[11][93] In animal models, gene targeting ininduced pluripotent stem cells has been successfully used to correct an antitrypsin polymerisation defect and to restore the ability of the mammalian liver to secrete active antitrypsin.[94] Small molecules have also been developed that block antitrypsin polymerisationin vitro.[95][96]
Serpins are the most widely distributed and largest superfamily of protease inhibitors.[1][40] They were initially believed to be restricted toeukaryote organisms, but have since been found inbacteria,archaea and someviruses.[36][37][97] It remains unclear whether prokaryote genes are the descendants of an ancestral prokaryotic serpin or the product ofhorizontal gene transfer from eukaryotes. Most intracellular serpins belong to a singlephylogenetic clade, whether they come from plants or animals, indicating that the intracellular and extracellular serpins may have diverged before the plants and animals.[98] Exceptions include the intracellular heat shock serpin HSP47, which is a chaperone essential for proper folding ofcollagen, and cycles between thecis-Golgi and theendoplasmic reticulum.[57]
Protease-inhibition is thought to be the ancestral function, with non-inhibitory members the results of evolutionaryneofunctionalisation of the structure. The S to R conformational change has also been adapted by some binding serpins to regulate affinity for their targets.[55]
The human genome encodes 16 serpin clades, termed serpinA through serpinP, including 29 inhibitory and 7 non-inhibitory serpin proteins.[9][83] The human serpin naming system is based upon aphylogenetic 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.[1][36][83] The functions of human serpins have been determined by a combination ofbiochemical studies, humangenetic disorders, andknockout mouse models.[83]
Knockout in mice originally reported as lethal,[140] but subsequently shown to have no obvious phenotype.[139] Expression may be a prognostic indicator that reflects expression of a neighbouring tumour suppressor gene (the phosphatasePHLPP1).[139]
Murine Serpinb11 is an active inhibitor whereas the human orthalogue is inactive.[153] Deficiency in ponies is associated with hoof wall separation disease.[154]
Manymammalian serpins have been identified that share no obvious orthology with a human serpin counterpart. Examples include numerousrodent serpins (particularly some of themurine intracellular serpins) as well as theuterine 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 theendometrium of a restricted group of mammals in theLaurasiatheria clade under the influence ofprogesterone orestrogen.[184] They are probably not functional proteinase inhibitors and may function during pregnancy to inhibit maternal immune responses against theconceptus or to participate in transplacental transport.[185]
TheDrosophila 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.[186] The clade classification system is difficult to use forDrosophila serpins and instead a nomenclature system has been adopted that is based on the position of serpin genes on theDrosophilachromosomes. Thirteen of theDrosophila 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).[186][187][188]
Studies onDrosophila serpins reveal that Serpin-27A inhibits the Easter protease (the final protease in the Nudel, Gastrulation Defective, Snake and Easter proteolytic cascade) and thus controlsdorsoventral patterning. Easter functions to cleave Spätzle (a chemokine-type ligand), which results intoll-mediated signaling. As well as its central role in embryonic patterning, toll signaling is also important for theinnate immune response in insects. Accordingly, serpin-27A also functions to control the insect immune response.[48][189][190] InTenebrio molitor (a large beetle), a protein (SPN93) comprising two discrete tandem serpin domains functions to regulate the toll proteolytic cascade.[191]
The genome of thenematode wormC. elegans contains 9 serpins, all of which lack signal sequences and so are likely intracellular.[193] However, only 5 of these serpins appear to function as protease inhibitors.[193] One, SRP-6, performs a protective function and guards against stress-inducedcalpain-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 a general and controllable role in determining cell fate.[194]
Plant serpins were amongst the first members of the superfamily that were identified.[195] 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 proteasesin vitro, the best-studied example being barley serpin Zx (BSZx), which is able to inhibit trypsin and chymotrypsin as well as several blood coagulation factors.[196] 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 theprolamin storage proteins of the endosperm.[197][198] 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 the phloem sap of pumpkin (CmPS-1)[199] and cucumber plants.[200][201] 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.[199]
Alternative roles and protease targets for plant serpins have been proposed. TheArabidopsis serpin, AtSerpin1 (At1g47710;3LE2), mediates set-point control over programmed cell death by targeting the 'Responsive to Desiccation-21' (RD21) papain-like cysteine protease.[53][202] AtSerpin1 also inhibitsmetacaspase-like proteasesin vitro.[52] Two otherArabidopsis serpins, AtSRP2 (At2g14540) and AtSRP3 (At1g64030) appear to be involved in responses to DNA damage.[203]
A singlefungal serpin has been characterized to date: celpin fromPiromycesspp. strain E2.Piromyces is agenus of anaerobic fungi found in the gut of ruminants and is important for digesting plant material. Celpin is predicted to be inhibitory and contains two N-terminaldockerin domains in addition to its serpin domain. Dockerins are commonly found in proteins that localise to the fungalcellulosome, a large extracellular multiprotein complex that breaks down cellulose.[38] It is therefore suggested that celpin may protect the cellulosome against plant proteases. Certain bacterial serpins similarly localize to the cellulosome.[204]
Predicted serpin genes are sporadically distributed inprokaryotes.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 inhibitorsin vivo. Several prokaryote serpins are found inextremophiles. Accordingly, and in contrast to mammalian serpins, these molecules possess elevated resistance to heat denaturation.[205][206] The precise role of most bacterial serpins remains obscure, althoughClostridium thermocellum serpin localises to thecellulosome. It is suggested that the role of cellulosome-associated serpins may be to prevent unwanted protease activity against the cellulosome.[204]
Serpins are also expressed byviruses as a way to evade the host's immune defense.[207] In particular, serpins expressed bypox viruses, includingcow pox (vaccinia) andrabbit pox (myxoma), are of interest because of their potential use as novel therapeutics for immune and inflammatory disorders as well as transplant therapy.[208][209] Serp1 suppresses the TLR-mediated innate immune response and allows indefinite cardiacallograft survival in rats.[208][210] Crma and Serp2 are both cross-class inhibitors and target both serine (granzyme B; albeit weakly) and cysteine proteases (caspase 1 and caspase 8).[211][212] In comparison to their mammalian counterparts, viral serpins contain significant deletions of elements of secondary structure. Specifically, crmA lacks the D-helix as well as significant portions of the A- and E-helices.[213]
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