Search

Human plasminogen activator inhibitor-1 (PAI-1) is an important component of the coagulation system and has been recognized as a potential therapeutic target of diverse cardiovascular disorders. Previously, it was found that the extracts from the Chinese medicine Dracaena dragon blood have potent inhibitory activity against PAI-1, but it is unclear which constituents directly participate in the inhibition and how do they regulate PAI-1 at molecular level. Here, we describe an integrated strategy to identify the dragon blood's chemical constituents that can directly target PAI-1. With the strategy, five compounds 1-5 are hit as promising PAI-1 inhibitor candidates, from which three are measured to have high or moderate activity against PAI-1. In particular, the compound 3 is determined to exhibit the highest potency; this value is roughly comparable with the widely used PAI-1 inhibitor Tiplaxtinin. We further examine the molecular effect of compound 3 on PAI-1 conformation at structural level. It is supposed that small-molecule inhibitor regulates the reactive center loop (RCL) of PAI-1 through an allosterism, that is, binding of compound 3 to PAI-1 can allosterically stabilize RCL in latent form, thus promoting PAI-1 conformational conversion from metastable active form to the inactive latent form. Long-term atomistic simulations also demonstrate that removal of compound 3 can destabilize the structured _-stranded conformation of RCL in latent form, although the current simulations are still not sufficient to characterize the full conversion dynamics trajectory. [ABSTRACT FROM AUTHOR]

Abstract Serine protease inhibitors (serpins), a superfamily of protease inhibitors, are known to be involved in several physiological processes, such as development, metamorphosis, and innate immunity. In our study, a full-length serpin cDNA, designated Haserpin1, was isolated from the cotton bollworm Helicoverpa armigera. The cDNA sequence of Haserpin1 is 1176 nt long, with an open reading frame encoding 391 amino acids; there is one exon and no intron. The predicted molecular weight of Haserpin1 is 43.53 kDa, with an isoelectric point of 4.98. InterProScan was employed for Haserpin1 functional characterization, which revealed that Haserpin1 contains highly conserved signature motifs, including a reactive center loop (RCL) with a hinge region (E341–N350), the serpin signature, (F367–F375) and a predicted P1–P1′ cleavage site (L357–S358), which are useful for identifying serpins. Transcripts of Haserpin1 were constitutively expressed in the fat body, suggesting that it is the major site for serpin synthesis. During the developmental stages, a fluctuation in the expression level of Haserpin1 was observed, with low expression detected at the 5th-instar larval stage. In contrast, relatively high expression was detected at the prepupal stage, suggesting that Haserpin1 might play a critical role at the H. armigera wandering stage. Although the detailed function of this serpin (Haserpin1) needs to be elucidated, our study provides a perspective for the functional investigation of serine protease inhibitor genes.

Neuroserpin is a serine protease inhibitor expressed mainly in the brain and at low levels in other tissues like the kidney, testis, heart, and spinal cord. It is involved in the inhibition of tissue plasminogen activator (tPA), plasmin, and to a lesser extent, urokinase‐type plasminogen (uPA). Neuroserpin has also been shown to plays noninhibitory roles in the regulation of N‐cadherin‐mediated cell adhesion. It is involved in neuroprotection from seizure and stroke through tPA‐mediated inhibition and also through its other protease targets. Mutations in critical domains of neuroserpin lead to its polymerization and neuronal death. In this study, a novel truncated isoform of human neuroserpin was identified in the brain and liver, which was confirmed by reverse transcriptase‐PCR and DNA sequencing using exon‐specific primers. Structural characterization of novel isoform using MD simulations studies indicated that it lacks the reactive center loop (RCL) but largely maintains its secondary structure fold. The novel truncated variant was cloned, expressed, and purified. A comparative intrinsic fluorescence and 4,4′‐bis‐1‐anilino naphthalene 8‐sulfonate studies revealed a decrease in fluorescence emission intensity and a more exposed hydrophobic surface as compared to the reported isoform. However, the novel isoform has lost its ability for tPA inhibition and complex formation. The absence of RCL indicates a noninhibitory role for the truncated isoform, prompting a detailed search and identification of two smaller isoforms in the human brain. With indications of the noninhibitory role of neuroserpin, identifying novel isoforms that appear to be without the tPA recognition domain is significant. [ABSTRACT FROM AUTHOR]

Copyright of Brazilian Journal of Biology is the property of Instituto Internacional de Ecologia and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Serine proteases drive important physiological processes such as coagulation, fibrinolysis, inflammation and angiogenesis. These proteases are controlled by serine protease inhibitors (SERPINs) that neutralize their activity. Currently, over 1,500 SERPINs are known in nature, but only 37 SERPINs are found in humans. Thirty of these are functional protease inhibitors. The inhibitory potential of SERPINs is in perfect balance with the proteolytic activities of its targets to enable physiological protease activity. Hence, SERPIN deficiency (either qualitative or quantitative) can lead to disease. Several SERPIN resupplementation strategies have been developed to treat SERPIN deficiencies, including concentrates derived from plasma and recombinant SERPINs. SERPINs usually inhibit multiple proteases, but only in their active state. Over the past decades, considerable insights have been acquired in the identification of SERPIN biological functions, their inhibitory mechanisms and specificity determinants. This paves the way for the development of therapeutic SERPINs. Through rational design, the inhibitory properties (selectivity and inhibitory potential) of SERPINs can be reformed and optimized. This review explores the current state of SERPIN engineering with a focus on reactive center loop modifications and backbone stabilization. We will discuss the lessons learned from these recombinant SERPINs and explore novel techniques and strategies that will be essential for the creation and application of the future generation of therapeutic SERPINs.

Serine protease inhibitors (serpins) are a unique family of protease inhibitor containing mobile reactive center loop. The availability of genome sequences of pigeonpea, soybean, commanbean, cowpea, mungbean and adzuki bean provided an opportunity to search for the serpin genes. A total of seven serpin genes were identified in the above legume genomes. Phylogenetic analysis of legume serpins, devided them into two major clades (Clade-I:VanSerpin, VraSerpin, VunSerpin, PvuSerpin and clade-II: GmaSerpin1, GmaSerpin2 and CcaSerpin) based on conserved reactive center (P2-P1’), domains and exon-intron boundary in the gene structure. The common reactive center of all the seven serpins indicated their ability to target cognate proteases and play an important role in response to insects, fungal pathogens and also the drought.

Corticosteroid-binding globulin (CBG) delivers anti-inflammatory cortisol to inflamed tissues through proteolysis of an exposed reactive center loop (RCL) by neutrophil elastase (NE). We previously demonstrated that RCL-localized Asn347-linked N-glycans impact NE proteolysis, but a comprehensive structure-function characterization of the RCL glycosylation is still required to better understand CBG glycobiology. Herein, we first performed RCL-centric glycoprofiling of serum-derived CBG to elucidate the Asn347-glycans and then used molecular dynamics simulations to study their impact on NE proteolysis. Importantly, we also identified O-glycosylation (di/sialyl T) across four RCL sites (Thr338/Thr342/Thr345/Ser350) of serum CBG close to the NE-targeted Val344-Thr345 cleavage site. A restricted N- and O-glycan co-occurrence pattern on the RCL involving exclusively Asn347 and Thr338 glycosylation was experimentally observed and supported in silico by modeling of a CBG-GalNAc-transferase (GalNAc-T) complex with various RCL glycans. GalNAc-T2 and GalNAc-T3 abundantly expressed by liver and gall bladder, respectively, showed in vitro a capacity to transfer GalNAc (Tn) to multiple RCL sites suggesting their involvement in RCL O-glycosylation. Recombinant CBG was then used to determine roles of RCL O-glycosylation through longitudinal NE-centric proteolysis experiments, which demonstrated that both sialoglycans (disialyl T) and asialoglycans (T) decorating Thr345 inhibit NE proteolysis. Synthetic RCL O-glycopeptides expanded on these findings by showing that Thr345-Tn and Thr342-Tn confer strong and moderate protection against NE cleavage, respectively. Molecular dynamics substantiated that short Thr345-linked O-glycans abrogate NE interactions. In conclusion, we report on biologically relevant CBG RCL glycosylation events, which improve our understanding of mechanisms governing cortisol delivery to inflamed tissues., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Abstract Insect-derived serine protease inhibitors (serpins) exhibit multiple inhibitory activities. Todate some functional roles for serpins in Hyphantria cunea Drury have been identified. Here, new functional features of the H. cunea serine protease inhibitor (dHC-serpin) were characterized. In this study, the cDNA encoding serpin was amplified from H. cunea (dHC) pupa fat body total RNA using RT-PCR. The full-length dHC-serpin cDNA encoded a protein of 440 amino acids with a predicted 19-amino acid signal peptide and a 421-amino acid functional domain. The functional domain was cloned into a pSUMO vector and transformed into Escherichia coli , resulting in the production of a pSUMO-dHC-serpin fusion protein. The soluble form of this protein was then purified by Ni-IDA chromatography. The SUMO-dHC-serpin fusion protein was then cleaved using a SUMO protease and purified again by Ni-IDA chromatography. dHC-serpin did not inhibit trypsin, elastase, proteinase K or cathepsin B, but strongly inhibited papain. The inhibitor retained its inhibitory activity over a broad range of pH (pH 2–12), temperature (20–50 °C), and DTT concentration (up to 100 mM). A complete loss of inhibitory activity was observed at pH 13 and 70 °C. Serpins generally serve as inhibitors that use a mobile reactive center loop (RCL) as bait to trap protease targets. dHC-serpin, like others serpins, binds papain using the RCL structure. Highlights • The full length dHC-serpin gene cloned from Hyphantria cunea Drury. • The SUMO-dHC-serpin fusion protein is highly soluble in Escherichia coli. • dHC-serpin exhibits activity against papain. • dHC-serpin remained active and stable between 20 °C and 50 °C and pH 2 and 12. • The reactive center loop region is important for dHC-serpin binding with papain. [ABSTRACT FROM AUTHOR]

IntroductionHuman α1-antitrypsin (hAAT) is a 394-amino acid long anti-inflammatory, neutrophil elastase inhibitor, which binds elastase via a sequence-specific molecular protrusion (reactive center loop, RCL; positions 357–366). hAAT formulations that lack protease inhibition were shown to maintain their anti-inflammatory activities, suggesting that some attributes of the molecule may reside in extra-RCL segments. Here, we compare the protease-inhibitory and anti-inflammatory profiles of an extra-RCL mutation (cys232pro) and two intra-RCL mutations (pro357cys, pro357ala), to naïve [wild-type (WT)] recombinant hAAT, in vitro, and in vivo.MethodsHis-tag recombinant point-mutated hAAT constructs were expressed in HEK-293F cells. Purified proteins were evaluated for elastase inhibition, and their anti-inflammatory activities were assessed using several cell-types: RAW264.7 cells, mouse bone marrow-derived macrophages, and primary peritoneal macrophages. The pharmacokinetics of the recombinant variants and their effect on LPS-induced peritonitis were determined in vivo.ResultsCompared to WT and to RCL-mutated hAAT variants, cys232pro exhibited superior anti-inflammatory activities, as well as a longer circulating half-life, despite all three mutated forms of hAAT lacking anti-elastase activity. TNFα expression and its proteolytic membranal shedding were differently affected by the variants; specifically, cys232pro and pro357cys altered supernatant and serum TNFα dynamics without suppressing transcription or shedding.ConclusionOur data suggest that the anti-inflammatory profile of hAAT extends beyond direct RCL regions. Such regions might be relevant for the elaboration of hAAT formulations, as well as hAAT-based drugs, with enhanced anti-inflammatory attributes.

Introduction: Human α1-antitrypsin (hAAT) is a 394-amino acid long anti-inflammatory, neutrophil elastase inhibitor, which binds elastase via a sequence-specific molecular protrusion (reactive center loop, RCL; positions 357-366). hAAT formulations that lack protease inhibition were shown to maintain their anti-inflammatory activities, suggesting that some attributes of the molecule may reside in extra-RCL segments. Here, we compare the protease-inhibitory and anti-inflammatory profiles of an extra-RCL mutation (cys232pro) and two intra-RCL mutations (pro357cys, pro357ala), to naïve [wild-type (WT)] recombinant hAAT, in vitro, and in vivo. Methods: His-tag recombinant point-mutated hAAT constructs were expressed in HEK-293F cells. Purified proteins were evaluated for elastase inhibition, and their anti-inflammatory activities were assessed using several cell-types: RAW264.7 cells, mouse bone marrow-derived macrophages, and primary peritoneal macrophages. The pharmacokinetics of the recombinant variants and their effect on LPS-induced peritonitis were determined in vivo. Results: Compared to WT and to RCL-mutated hAAT variants, cys232pro exhibited superior anti-inflammatory activities, as well as a longer circulating half-life, despite all three mutated forms of hAAT lacking anti-elastase activity. TNFα expression and its proteolytic membranal shedding were differently affected by the variants; specifically, cys232pro and pro357cys altered supernatant and serum TNFα dynamics without suppressing transcription or shedding. Conclusion: Our data suggest that the anti-inflammatory profile of hAAT extends beyond direct RCL regions. Such regions might be relevant for the elaboration of hAAT formulations, as well as hAAT-based drugs, with enhanced anti-inflammatory attributes. [ABSTRACT FROM AUTHOR]

For successful protease inhibition, the reactive center loop (RCL) of the two-domain serine protease inhibitor, α1-antitrypsin (α1-AT), needs to remain exposed in a metastable active conformation. The α1-AT RCL is sequestered in a β-sheet in the stable latent conformation. Thus, to be functional, α1-AT must always fold to a metastable conformation while avoiding folding to a stable conformation. We explore the structural basis of this choice using folding simulations of coarse-grained structure-based models of the two α1-AT conformations. Our simulations capture the key features of folding experiments performed on both conformations. The simulations also show that the free energy barrier to fold to the latent conformation is much larger than the barrier to fold to the active conformation. An entropically stabilized on-pathway intermediate lowers the barrier for folding to the active conformation. In this intermediate, the RCL is in an exposed configuration, and only one of the two α1-AT domains is folded. In contrast, early conversion of the RCL into a β-strand increases the coupling between the two α1-AT domains in the transition state and creates a larger barrier for folding to the latent conformation. Thus, unlike what happens in several proteins, where separate regions promote folding and function, the structure of the RCL, formed early during folding, determines both the conformational and the functional fate of α1-AT. Further, the short 12-residue RCLmodulates the free energy barrier and the folding cooperativity of the large 370-residue α1-AT. Finally, we suggest experiments to test the predicted folding mechanism for the latent state. [ABSTRACT FROM AUTHOR]

Serpins are a superfamily of proteins, most of which inhibit cognate serine proteases by forming inactive acyl-enzyme complexes. In the tobacco hornworm Manduca sexta , serpin-1, -3 through -7 negatively regulate a hemolymph serine protease system that activates precursors of the serine protease homologs (SPHs), phenoloxidases (POs), Spätzles, and other cytokines. Here we report the cloning and characterization of M. sexta serpin-9 and -13. Serpin-9, a 402-residue protein most similar to Drosophila Spn77Ba, has R 366 at the P1 position right before the cleavage site; Serpin-13, a 444-residue ortholog of Drosophila Spn28Dc, is longer than the other seven serpins and has R 410 as the P1 residue. Both serpins are mainly produced in fat body and secreted into plasma to function. While their mRNA and protein levels were not up-regulated upon immune challenge, they blocked protease activities and affected proPO activation in hemolymph. Serpin-9 inhibited human neutrophil elastase, cathepsin G, trypsin, and chymotrypsin to different extents; serpin-13 reduced trypsin activity to approximately 10% at a molar ratio of 4:1 (serpin: enzyme). Serpin-9 was cleaved at Arg 366 by the enzymes with different specificity, but serpin-13 had four P1 sites (Arg 410 for trypsin-like proteases, Gly 406 and Ala 409 for the elastase and Thr 404 for cathepsin G). Supplementation of induced cell-free hemolymph (IP, P for plasma) with recombinant serpin-9 did not noticeably affect proPO activation, but slightly reduced the PO activity increase after 0–50% ammonium sulfate fraction of the IP had been elicited by bacteria. In comparison, addition of recombinant serpin-13 significantly inhibited proPO activation in IP and the suppression was stronger in the fraction of IP. Serpin-9- and -13-containing protein complexes were isolated from IP using their antibodies. Hemolymph protease-1 precursor (proHP1), HP6 and HP8 were found to be associated with serpin-9, whereas proHP1, HP2 and HP6 were pulled downed with serpin-13. These results indicate that both serpins regulate immune proteases in hemolymph of M. sexta larvae. [ABSTRACT FROM AUTHOR]

The adipokine vaspin (serpinA12) is mainly expressed in white adipose tissue and exhibits various beneficial effects on obesity-related processes. Kallikrein 7 is the only known target protease of vaspin and is inhibited by the classical serpin inhibitory mechanism involving a cleavage of the reactive center loop between P1 (M378) and P1' (E379). Here, we present the X-ray structure of vaspin, cleaved between M378 and E379. We provide a comprehensive analysis of differences between the uncleaved and cleaved forms in the shutter, breach, and hinge regions with relation to common molecular features underlying the serpin inhibitory mode. Furthermore, we point out differences towards other serpins and provide novel data underlining the remarkable stability of vaspin. We speculate that the previously reported FKGx1Wx2x3 motif in the breach region may play a decisive role in determining the reactive center loop configuration in the native vaspin state and might contribute to the high thermostability of vaspin. Thus, this structure may provide a basis for future mutational studies. [ABSTRACT FROM AUTHOR]

Corticosteroid-binding globulin (CBG) is the principal transport protein of glucocorticoids. Approximately 80–90% of serum cortisol binds to CBG with high affinity and only about 5% of cortisol remain unbound and is considered biologically active. CBG seems to modulate and influence the bioavailability of cortisol to local tissues. In this review, we will discuss physicochemical properties of CBG and structure of CBG in the mechanisms of binding and release of cortisol. This review describes several factors affecting CBG functions, such as genetic factors or temperature. Furthermore, clinical implications of CBG abnormalities and the measurement of CBG and its use for assessment of free cortisol levels are described in this review. [ABSTRACT FROM AUTHOR]

Serpin constitute about 10% of blood protein and are associated with mutations that results in aberrant intermolecular linkages which leads to polymer formation. Studies with short peptides have shown promise in depolymerization of serpins however a reactive center loop based peptide also makes the serpin inactive. A chemical chaperone based approach is a better option in terms of maintaining activity and retarding polymerization but not much is known about its binding and mechanism. Specific target for chemical chaperones and its effectiveness across many serpin is not known. We did an analysis of serpin cavity using CASTp and show that cavities are distributed throughout the molecule where the largest cavities are generally present in areas of major conformational change like shutter region, helix D and helix F. An analysis of different conformational states of serpins showed that this large cavity undergoes increase in size in latent and cleaved states as compared to native state. We targeted serpins with a variety of carbohydrate, methylamine and amino acid based chemical chaperones and selected those that have highest binding energy across different serpins to assess their ability to bind large cavities. The results show that carbohydrate based chemical chaperone like sorbitol, sucrose, arabitol and trehalose and amino acid based chaperones like dopamine, phenylalanine, arginine and glutamic acid are the most effective in binding serpins. Most of these chemical chaperone interacted with residues in the shutter region and the helix D arm at the C-terminal which are part of the largest cavities. We selected the carbohydrate based chemical chaperone with best binding energies and did experimental study under the condition that induce polymerization and show that indeed they were able to retard polymer formation with moderate effect on inhibition rates. However a fluorometric study with native antithrombin showed that chemical chaperone may effect the conformation of the proteins. Our study shows that chemical chaperones have the best binding affinities for the cavities around shutter region and helix D and that a cavity targeting based approach seems to be a better option for retarding polymerization in serpins, but a thorough analysis of its effect on folding, inhibition and cofactor binding is required. [ABSTRACT FROM AUTHOR]

Background: The reactive center loop (RCL) of native antithrombin is partially inserted in the main serpin body. It must be fully exposed for optimal inhibitory function. Objective: To test the hypothesis that P14-s2B interaction affects loop insertion in antithrombin. By mutating Phe274 to Tyr274, the objective was to introduce P14-s2B interaction in antithrombin. Methods: Site-directed mutagenesis and affinity chromatography were used to obtain purified recombinant protein. Antithrombin’s ability to form sodium dodecyl sulfate (SDS)-stable complex with thrombin, stoichiometry of thrombin inhibition, second-order rate constant for thrombin and factor Xa (fXa) inhibition (M-1 s-1), and heparin dissociation constant (KD; tryptophan fluorescence emission spectra) were determined. Results and Conclusion: A marginal, but inconclusive, difference between the wild type and the mutant was observed. The result highlights the variable effect of P14-s2B interaction in different serpins. Alternate hypothesis for achieving loop expulsion is proposed. [ABSTRACT FROM PUBLISHER]

Abstract: Corticosteroid-binding globulin (CBG, transcortin) belongs to the serpin family of serine protease inhibitors (SERPINA6) and is mainly secreted by the liver. The negative acute phase protein CBG regulates free cortisol levels in the blood and distributes cortisol to its target tissues. So far no CBG serpin partner protease has been identified. However, its cleavage by human neutrophil elastase destroys ligand binding capacity and supposedly liberates cortisol at sites of inflammation. Here we report on the recombinant expression and secretion of human wild-type CBG and several novel mutants by human 293-EBNA cells. Functional characterization of wild-type and mutant CBG revealed distinct differences in ligand binding sensitivity to heat or elastase. Certain mutants are almost devoid of cortisol binding activity (Q232R and CBG Lyon), some display higher sensitivity for heat inactivation (G335V, Q232R and CBG Lyon) or for elastase cleavage (G335V). CBG mutant T342A is more resistant to elastase cleavage. Our data support the validity of the serpin structural concept. The expression system used provides functionally active human recombinant transcortin for further functional characterization of wild-type and human CBG mutant variants, which have been associated with altered serum free cortisol levels or pathophysiological constellations such as increased body weight, fatigue or hypotension. [Copyright &y& Elsevier]

Abstract: Corticosteroid-binding globulin (CBG), a non-inhibitory member of the serine proteinase inhibitor (serpin) super-family, is the high-affinity transport protein for glucocorticoids in vertebrate blood. Plasma CBG is a glycoprotein with 30% of its mass represented by N-linked oligosaccharide chains. Its well-characterized steroid-binding properties represent a “bench-mark data set” used extensively for in silico studies of protein–ligand interactions and drug design. Recent crystal structure analyses of intact rat CBG and cleaved human CBG have revealed the precise topography of the steroid-binding site, and shown that cortisol-bound CBG displays a typical stressed (S) serpin conformation with the reactive center loop (RCL) fully exposed from the central β-sheet A, while proteolytic cleavage of the RCL results in CBG adopting a relaxed (R) conformation with the cleaved RCL fully inserted within the protein core. These crystal structures have set the stage for mechanistic studies of CBG function which have so far shown that helix D plays a key role in coupling RCL movement and steroid-binding site integrity, and provided evidence for an allosteric mechanism that modulates steroid binding and release from CBG. These studies have also revealed how the irreversible release of steroids occurs after proteolysis and re-orientation of the RCL within the R conformation. This recent insight into the structure and function of CBG reveals how naturally occurring genetic CBG mutations affect steroid binding, and helps understand how proteolysis of CBG enhances the targeted delivery of biologically active steroids to their sites of action. [Copyright &y& Elsevier]

Abstract: Squamous cell carcinoma antigen 1 (SCCA1), which belongs to serine proteinase inhibitor (serpin) superfamily, inhibits papain-like cysteine proteinase. Recently, it has been reported that SCCA1 acts not only as a proteinase inhibitor but also as an inhibitor of UV-induced apoptosis via suppression of the activity of c-Jun NH2-terminal kinase (JNK1). The present study determined the crystal structure of SCCA1, suggesting that the reactive center loop (RCL) of SCCA1, a recognition site of proteinase, is very flexible and located away form the main-body of SCCA1. We show that the inhibitory effect of SCCA1 on the kinase activity of JNK1 is lost when the RCL was truncated. Furthermore, we found that a mutant protein created by replacing one amino acid in RCL maintain the suppressive activity to JNK1, whereas the inhibitory effect to proteinase is obviously decreased. [Copyright &y& Elsevier]

Abstract: A serpin of the intracellular type from the tapeworm Echinococcus multilocularis was expressed in Escherichia coli, purified by ion exchange chromatography and tested for inhibitory activity against several proteinases. The recombinant protein, which after transcriptional induction, represents about 20 % of total cellular protein, is biochemically active and inhibits trypsin and the trypsin-like plasmin as well as pig pancreatic and human neutrophil elastase. Implications regarding its biochemistry and biolological function are discussed. [Copyright &y& Elsevier]

Abstract: Ovalbumin is a serpin without inhibitory activity against proteases. During embryonic development, ovalbumin in the native (N) form undergoes changes and takes a heat-stable form, which was previously named HS-ovalbumin. It has been known that N-ovalbumin is artificially converted to another thermostable form called S-ovalbumin by heating at an alkaline pH. Here, we characterized further the three ovalbumin forms, N, HS, and S. The epitope of the monoclonal antibody 2B3/2H11, which recognizes N- and HS-ovalbumin but not S-ovalbumin, was found to reside in the region Glu–Val–Val–Gly–Ala–Ser–Glu–Ala–Gly–Val–Asp–Ala–Ala–Ser–Val–Ser–Glu–Glu–Phe–Arg, which corresponds to 340–359 of amino acid residues and is contained in the reactive center loop (RCL). Removal of RCL by elastase or subtilisin mitigated binding of the antibody. Dephosphorylation experiments indicated that the phosphorylated Ser-344 residue located on RCL is crucial for the epitope recognition. We suggest that the shift to the heat-stable form of ovalbumin accompanies a movement of RCL. [Copyright &y& Elsevier]

Abstract: Pigment epithelium derived factor (PEDF) is non-inhibitory serpin with neurotrophic and antiangiogenic functions. In this study, we have assembled PEDF sequences for 9 additional species by data base mining and performed cross-species alignment for 14 PEDF sequences to identify conserved structural domains. We found evolutionary conservation of a leader sequence, a single C-terminal glycosylation site, collagen-binding residues, and four specific conserved PEDF peptides. The C-terminus, 384–415 and an N-terminal region 78–95, show close homology with many other serpins, and there is strong conservation of 39 of 51 consensus key residues involved in serpin structure and function. Two peptide regions, 40–67 and 277–301, are unique to PEDF but conserved in all species. Conserved residues at the N-terminus, helix d (hD), and helix A (hA) of PEDF form a structure similar to the heparin-binding groove of other serpins. We identified a motif in PEDF that is homologous to the nuclear localization signals of other proteins. A bitopographical localization of PEDF was confirmed by immunocytochemistry and Western blots. Our results suggest that secretion is required for PEDF’s activity, that PEDF can migrate to the nucleus, and that PEDF has structural and functional features more common with inhibitory serpins. [Copyright &y& Elsevier]

Human plasminogen activator inhibitor-1 (PAI-1) is an important component of the coagulation system and has been recognized as a potential therapeutic target of diverse cardiovascular disorders. Previously, it was found that the extracts from the Chinese medicine Dracaena dragon blood have potent inhibitory activity against PAI-1, but it is unclear which constituents directly participate in the inhibition and how do they regulate PAI-1 at molecular level. Here, we describe an integrated strategy to identify the dragon blood's chemical constituents that can directly target PAI-1. With the strategy, five compounds 1-5 are hit as promising PAI-1 inhibitor candidates, from which three are measured to have high or moderate activity against PAI-1. In particular, the compound 3 is determined to exhibit the highest potency; this value is roughly comparable with the widely used PAI-1 inhibitor Tiplaxtinin. We further examine the molecular effect of compound 3 on PAI-1 conformation at structural level. It is supposed that small-molecule inhibitor regulates the reactive center loop (RCL) of PAI-1 through an allosterism, that is, binding of compound 3 to PAI-1 can allosterically stabilize RCL in latent form, thus promoting PAI-1 conformational conversion from metastable active form to the inactive latent form. Long-term atomistic simulations also demonstrate that removal of compound 3 can destabilize the structured β-stranded conformation of RCL in latent form, although the current simulations are still not sufficient to characterize the full conversion dynamics trajectory., (© 2021 International Union of Biochemistry and Molecular Biology, Inc.)

Serine proteases drive important physiological processes such as coagulation, fibrinolysis, inflammation and angiogenesis. These proteases are controlled by serine protease inhibitors (SERPINs) that neutralize their activity. Currently, over 1,500 SERPINs are known in nature, but only 37 SERPINs are found in humans. Thirty of these are functional protease inhibitors. The inhibitory potential of SERPINs is in perfect balance with the proteolytic activities of its targets to enable physiological protease activity. Hence, SERPIN deficiency (either qualitative or quantitative) can lead to disease. Several SERPIN resupplementation strategies have been developed to treat SERPIN deficiencies, including concentrates derived from plasma and recombinant SERPINs. SERPINs usually inhibit multiple proteases, but only in their active state. Over the past decades, considerable insights have been acquired in the identification of SERPIN biological functions, their inhibitory mechanisms and specificity determinants. This paves the way for the development of therapeutic SERPINs. Through rational design, the inhibitory properties (selectivity and inhibitory potential) of SERPINs can be reformed and optimized. This review explores the current state of SERPIN engineering with a focus on reactive center loop modifications and backbone stabilization. We will discuss the lessons learned from these recombinant SERPINs and explore novel techniques and strategies that will be essential for the creation and application of the future generation of therapeutic SERPINs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Maas and de Maat.)

Viruses have devised highly effective approaches that modulate the host immune response, blocking immune responses that are designed to eradicate viral infections. Over millions of years of evolution, virus-derived immune-modulating proteins have become extraordinarily potent, in some cases working at picomolar concentrations when expressed into surrounding tissues and effectively blocking host defenses against viral invasion and replication. The marked efficiency of these immune-modulating proteins is postulated to be due to viral engineering of host immune modulators as well as design and development of new strategies (i.e., some derived from host proteins and some entirely unique). Two key characteristics of viral immune modulators confer both adaptive advantages and desirable functions for therapeutic translation. First, many virus-derived immune modulators have evolved structures that are not readily recognized or regulated by mammalian immune pathways, ensuring little to no neutralizing antibody responses or proteasome-mediated degradation. Second, these immune modulators tend to target early steps in central immune responses, producing a powerful downstream inhibitory "domino effect" which may alter cell activation and gene expression.We have proposed that peptide metabolites of these immune-modulating proteins can enhance and extend protein function. Active immunomodulating peptides have been derived from both mammalian and viral proteins. We previously demonstrated that peptides derived from computationally predicted cleavage sites in the reactive center loop (RCL) of a viral serine proteinase inhibitor (serpin ) from myxoma virus, Serp-1 , can modify immune response activation. We have also demonstrated modulation of host gut microbiota produced by Serp-1 and RCL-derived peptide , S7, in a vascular inflammation model. Of interest, generation of derived peptides that maintain therapeutic function from a serpin can act by a different mechanism. Whereas Serp-1 has canonical serpin-like function to inhibit serine proteases, S7 instead targets mammalian serpins. Here we describe the derivation of active Serp- RCL peptides and their testing in inflammatory vasculitis models.

Here, we assessed the effect of a systematic change in reactive center loop (RCL) length, N-terminal to the reactive center, on the inhibitory activity of the recombinant Apserpin-6. The domain prediction results indicated that the RCL is located between the amino acid numbered 359-379 at the C-terminal of Apserpin-6. The N-terminal variable region for amino acid positions P7-P1 of the RCL of Apserpin-6 was truncated or extended by residue deletion or insertion using site-directed mutagenesis. The recombinant Apserpin-6 with one or two residues insertion in RCL had no effect on prophenoloxidase (proPO) activity, whereas deletion of one or two residues in RCL lowered the efficiency of inhibition of Apserpin-6. The results of this study will facilitate the understanding of inhibition mechanism of RCL on proPO activity., (© 2020 Wiley Periodicals LLC.)

Serpins function as a trap for serine proteases, presenting the reactive center loop (RCL) as a target for individual proteases. When the protease cleaves the RCL, the serpin and protease become covalently linked leading to a loss of function of both the protease and the serpin; this suicide inhibition is often referred to as a "mouse trap." When the RCL P1-P1' scissile bond is cut by the protease, the resulting bond between the protease and the RCL leads to insertion of the cleaved RCL into the β-sheet A and relocation of the protease to the opposite pole of the serpin, forming a suicide complex. Only a relatively small part of the serpin molecule can be removed in deletion mutations before the serpin RCL inhibitory function is lost. Serpin RCL peptides have been developed to block formation of serpin aggregates in serpinopathies, genetic serpin mutations wherein the abnormal serpins insert their RCL into adjacent serpins forming aggregates of inactive serpins.We have further posited that this natural cleavage site in the serpin RCL may form active serpin metabolites with potential to add to the serpin's inhibitory functions. We have developed RCL peptides based upon predicted serpin RCL cleavage (or metabolism) sites and tested these serpins for inhibitory function. In this chapter we describe the development of RCL-derived peptides, peptides derived based upon the RCL sequences of two myxomaviral serpins. Methods used to develop peptides are described for RCL-derived peptides from Serp-1, a thrombotic and thrombolytic serine protease inhibitor, and Serp-2, a cross class serine and cysteine protease inhibitor (Subheadings 2.1 and 3.1). Approaches to testing RCL peptide functions, in vitro by molecular assays and in vivo in models of cell migration, MHV-68 infection, and aortic allograft transplant are described (Subheadings 2.2 and 3.2).