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1
Pippel, Jan, E. Bartholomeus Kuettner, David Ulbricht, Jan Daberger, Stephan Schultz, John T. Heiker et Norbert Sträter. « Crystal structure of cleaved vaspin (serpinA12) ». Biological Chemistry 397, no 2 (1 janvier 2016) : 111–23. http://dx.doi.org/10.1515/hsz-2015-0229.
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Abstract 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.
2
Patston, PA, RL Medcalf, Y. Kourteva et M. Schapira. « C1-inhibitor-serine proteinase complexes and the biosynthesis of C1- inhibitor by Hep G2 and U 937 cells ». Blood 82, no 11 (1 décembre 1993) : 3371–79. http://dx.doi.org/10.1182/blood.v82.11.3371.3371.
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Abstract The biosynthesis of the serpin alpha 1-proteinase inhibitor is regulated by a feedback mechanism whereby complexes between alpha 1- proteinase inhibitor and serine proteinases bind to liver cells and monocytes, a reaction that activates alpha 1-proteinase-inhibitor gene transcription. Such a mechanism may form the basis for the development of new therapeutic strategies for serpin deficiency states with reduced levels of otherwise normally functioning serpins. This issue was addressed for C1-inhibitor, the missing serpin in hereditary angioedema. C1-inhibitor biosynthesis by Hep G2 hepatoma cells was assessed by enzyme-linked immunosorbant assay, by metabolic labeling followed by immunoprecipitation, and by Northern blotting. C1-inhibitor biosynthesis was stimulated by gamma-interferon (100 U/mL) but not by cell exposure to C1-inhibitor-kallikrein (1 mumol/L), C1-inhibitor-C1s (1 mumol/L), and C1-inhibitor-plasmin complexes (1 mumol/L) or to reactive site-cleaved C1-inhibitor (1 mumol/L). Moreover, radioiodinated C1s-C1-inhibitor complex did not bind to Hep G2 cells. C1-inhibitor-kallikrein complex was also without effect on C1-inhibitor mRNA in U 937 cells. Therefore, the proposed mechanism, by which serpin- enzyme complex or reactive site-cleaved serpin binding to a specific receptor provides a signal for the stimulation of the biosynthesis of that serpin, is not operative for the biosynthesis of C1-inhibitor by Hep G2 or U 937 cells.
3
Patston, PA, RL Medcalf, Y. Kourteva et M. Schapira. « C1-inhibitor-serine proteinase complexes and the biosynthesis of C1- inhibitor by Hep G2 and U 937 cells ». Blood 82, no 11 (1 décembre 1993) : 3371–79. http://dx.doi.org/10.1182/blood.v82.11.3371.bloodjournal82113371.
Texte intégralRésumé :
The biosynthesis of the serpin alpha 1-proteinase inhibitor is regulated by a feedback mechanism whereby complexes between alpha 1- proteinase inhibitor and serine proteinases bind to liver cells and monocytes, a reaction that activates alpha 1-proteinase-inhibitor gene transcription. Such a mechanism may form the basis for the development of new therapeutic strategies for serpin deficiency states with reduced levels of otherwise normally functioning serpins. This issue was addressed for C1-inhibitor, the missing serpin in hereditary angioedema. C1-inhibitor biosynthesis by Hep G2 hepatoma cells was assessed by enzyme-linked immunosorbant assay, by metabolic labeling followed by immunoprecipitation, and by Northern blotting. C1-inhibitor biosynthesis was stimulated by gamma-interferon (100 U/mL) but not by cell exposure to C1-inhibitor-kallikrein (1 mumol/L), C1-inhibitor-C1s (1 mumol/L), and C1-inhibitor-plasmin complexes (1 mumol/L) or to reactive site-cleaved C1-inhibitor (1 mumol/L). Moreover, radioiodinated C1s-C1-inhibitor complex did not bind to Hep G2 cells. C1-inhibitor-kallikrein complex was also without effect on C1-inhibitor mRNA in U 937 cells. Therefore, the proposed mechanism, by which serpin- enzyme complex or reactive site-cleaved serpin binding to a specific receptor provides a signal for the stimulation of the biosynthesis of that serpin, is not operative for the biosynthesis of C1-inhibitor by Hep G2 or U 937 cells.
4
Kousted, Tina, Karsten Skjoedt, Steen Petersen, Claus Koch, Lars Vitved, Maja Sochalska, Céline Lacroix et al. « Three monoclonal antibodies against the serpin protease nexin-1 prevent protease translocation ». Thrombosis and Haemostasis 111, no 01 (2014) : 29–40. http://dx.doi.org/10.1160/th13-04-0340.
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SummaryProtease nexin-1 (PN-1) belongs to the serpin family and is an inhibitor of thrombin, plasmin, urokinase-type plasminogen activator, and matriptase. Recent studies have suggested PN-1 to play important roles in vascular-, neuro-, and tumour-biology. The serpin inhibitory mechanism consists of the serpin presenting its so-called reactive centre loop as a substrate to its target protease, resulting in a covalent complex with the inactivated enzyme. Previously, three mechanisms have been proposed for the inactivation of serpins by monoclonal antibodies: steric blockage of protease recognition, conversion to an inactive conformation or induction of serpin substrate behaviour. Until now, no inhibitory antibodies against PN-1 have been thoroughly characterised. Here we report the development of three monoclonal antibodies binding specifically and with high affinity to human PN-1. The antibodies all abolish the protease inhibitory activity of PN-1. In the presence of the antibodies, PN-1 does not form a complex with its target proteases, but is recovered in a reactive centre cleaved form. Using site-directed mutagenesis, we mapped the three overlapping epitopes to an area spanning the gap between the loop connecting α-helix F with β-strand 3A and the loop connecting α-helix A with β-strand 1B. We conclude that antibody binding causes a direct blockage of the final critical step of protease translocation, resulting in abortive inhibition and premature release of reactive centre cleaved PN-1. These new antibodies will provide a powerful tool to study the in vivo role of PN-1’s protease inhibitory activity.
5
Zhang, Weiqing, Richard Swanson, Gonzalo Izaguirre, Yan Xiong, Lester F. Lau et Steven T. Olson. « The heparin-binding site of antithrombin is crucial for antiangiogenic activity ». Blood 106, no 5 (1 septembre 2005) : 1621–28. http://dx.doi.org/10.1182/blood-2005-02-0547.
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Abstract The heparin-binding site of antithrombin is shown here to play a crucial role in mediating the antiangiogenic activity of conformationally altered cleaved and latent forms of the serpin. Blocking the heparin-binding site of cleaved or latent antithrombin by complexation with a high-affinity heparin pentasaccharide abolished the serpin's ability to inhibit proliferation, migration, capillary-like tube formation, basic fibroblast growth factor (bFGF) signaling, and perlecan gene expression in bFGF-stimulated human umbilical vein endothelial cells. Mutation of key heparin binding residues, when combined with modifications of Asn-linked carbohydrate chains near the heparin-binding site, also could abrogate the anti-proliferative activity of the cleaved serpin. Surprisingly, mutation of Lys114, which blocks anticoagulant activation of antithrombin by heparin, caused the native protein to acquire antiproliferative activity without the need for conformational change. Together, these results indicate that the heparin-binding site of antithrombin is of crucial importance for mediating the serpin's antiangiogenic activity and that heparin activation of native antithrombin constitutes an antiangiogenic switch that is responsible for turning off the antiangiogenic activity of the native serpin.
6
O'Reilly, M. S. « Antiangiogenic Activity of the Cleaved Conformation of the Serpin Antithrombin ». Science 285, no 5435 (17 septembre 1999) : 1926–28. http://dx.doi.org/10.1126/science.285.5435.1926.
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7
Wells, Michael, William Sheffield et Morris Blajchman. « The Clearance of Thrombin-antithrombin and Related Serpin-enzyme Complexes from the Circulation : Role of Various Hepatocyte Receptors ». Thrombosis and Haemostasis 81, no 03 (1999) : 325–37. http://dx.doi.org/10.1055/s-0037-1614472.
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IntroductionPeptide bond cleavage can herald the end of a protein’s active life, or its transformation from an inactive precursor to an active enzyme. If the newly activated protein is a proteinase, even a highly specific proteinase, then its activity must be regulated in order that unbridled cleavage and damage to the host organism do not ensue. Such regulation for many of the key serine proteinases of the coagulation, fibrinolytic, complement, and inflammatory pathways is provided by the inhibitory proteins of the serpin family.The serpins are a large family of over 100 proteins (1). Many are plasma proteins such as antithrombin (AT), α1-proteinase inhibitor (α-PI), α1-antichymotrypsin (α-AC), heparin cofactor II (HCII), plasminogen activator inhibitors (PAI) I and II, α2-antiplasmin (α2-AP) and proteinase nexin I (PN-1). While some serpins are readily recognizable as family members, solely by virtue of homology, others have been characterized in detail, particularly those that are suicide inhibitors of their cognate proteinases; enzymes that recognize and attack the reactive centre loop of the inhibitory serpins. The resulting serpin-enzyme complex (SEC) is comprised of the inhibitor, which is irreversibly inactivated by virtue of the cleavage of its reactive centre peptide bond, and the enzyme, which is reversibly inactivated by the formation of an acyl ester linkage between its active site serine and a serpin side chain. Thus, a stable, covalent, and stoichiometric complex resistant to denaturation is formed (2, 3).The reversible nature of the proteinase’s inactivation in the SEC means that while substantial regulation of the proteinase has been achieved, the organism has only prolonged the inevitable by forming the SEC. Because the SEC is only kinetically but not thermodynamically stable, given sufficient time it will break down, releasing cleaved serpin and active enzyme (4, 5). To prevent this, receptor-mediated mechanisms have evolved to effectively remove SECs from the circulation. Since the initial studies of Ohlsson, who investigated the clearance of α-PI-trypsin complexes in the circulation of dogs (6, 7), a large body of evidence has accumulated to indicate that SECs are cleared from the circulation more rapidly than their constituent serpins. This accelerated clearance seals the fate of the serpin-complexed proteinases, and prevents their release from SECs by sequestering the SECs inside cells, where they are catabolized. In this article, we review the available data with respect to the mechanisms involved in SEC removal from the circulation. Specifically, we address those proteins or molecules that have been reported to act as cellular receptors for SEC removal, and propose a model for SEC removal which includes several of the available candidate receptors. Where possible, we have focussed on the thrombin-antithrombin (TAT) complex, both because of our laboratory’s longstanding interest in antithrombin, and because of thrombin’s key role in haemostasis and thrombosis (8).
8
Ellisdon, Andrew M., Qingwei Zhang, Michelle A. Henstridge, Travis K. Johnson, Coral G. Warr, Ruby HP Law et James C. Whisstock. « High resolution structure of cleaved Serpin 42 Da from Drosophila melanogaster ». BMC Structural Biology 14, no 1 (2014) : 14. http://dx.doi.org/10.1186/1472-6807-14-14.
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9
Onda, Maki, Kazuyo Nakatani, Sayaka Takehara, Mika Nishiyama, Nobuyuki Takahashi et Masaaki Hirose. « Cleaved Serpin Refolds into the Relaxed State via a Stressed Conformer ». Journal of Biological Chemistry 283, no 25 (7 avril 2008) : 17568–78. http://dx.doi.org/10.1074/jbc.m709262200.
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10
Hejgaard, Jørn, William A. Laing, Salla Marttila, Andrew P. Gleave et Thomas H. Roberts. « Serpins in fruit and vegetative tissues of apple (Malus domestica) : expression of four serpins with distinct reactive centres and characterisation of a major inhibitory seed form, MdZ1b ». Functional Plant Biology 32, no 6 (2005) : 517. http://dx.doi.org/10.1071/fp04220.
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Most serpins irreversibly inhibit serine proteinases of the chymotrypsin family using a suicide-substrate-based mechanism. Serpins are present in all domains of life, but physiological functions in the plant kingdom are yet to be elucidated. Inhibitory properties of many abundant cereal grain serpins are well characterised, but serpins have not been identified in eudicot seeds. In apple (Malus domestica Borkh.), the origin of 88 serpin expressed sequence tags (ESTs) identified among 160 000 ESTs from 30 cultivar-, tissue- and time-specific libraries showed that serpin genes are expressed in a wide variety of tissues, including developing and mature fruits, seeds and vegetative buds as well as developing, mature and senescing leaves. Analysis of 46 sequences, most full-length, identified serpins with four distinct reactive centres belonging to two subfamilies (MdZ1 and MdZ2) with ~85% amino acid sequence identity. MdZ1 included three molecular forms with identical reactive centre loop (RCL) sequences except for three different, but related, residues at P2 (Asp, Asn or Glu). A major seed serpin, MdZ1b, with P2–P1′ Glu–Arg–Arg was purified from decorticated seeds and characterised kinetically. MdZ1b was a fast inhibitor of bovine and porcine trypsin (second-order association rate constant k a ~4 × 106 m –1 s–1 and stoichiometry of inhibition SI = 1). Human plasmin and urokinase-type plasminogen activator (u-PA), but not thrombin, were inhibited at lower rates (k a ~104 m –1 s–1). Chymotrypsin was inhibited at the same site (k a~4 × 103 m –1 s–1), but a significant part of MdZ1b was cleaved as substrate (SI > 2). Unexpectedly, the MdZ1b-trypsin complex was relatively short-lived with a first-order dissociation rate constant k d in the order of 10−4 s–1. The bulk of mature seed MdZ1b was localised to the cotyledons. The content of MdZ1b in ripe apples was 5–26 µg per seed, whereas MdZ1b could not be detected in the cortex or skin. Localisation and inhibitory specificity of serpins in monocot and eudicot plants are compared and putative functions are discussed.
11
Stein, P. E., D. A. Tewkesbury et R. W. Carrell. « Ovalbumin and angiotensinogen lack serpin S–R conformational change ». Biochemical Journal 262, no 1 (15 août 1989) : 103–7. http://dx.doi.org/10.1042/bj2620103.
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Cleavage of ovalbumin and angiotensinogen at sites homologous to the reactive centre loop of alpha 1-antitrypsin is not accompanied by the increase in heat-stability associated with the transition from the native stressed (S) structure to a cleaved relaxed (R) form that is typical of other serpins. Failure to undergo the S-R change in ovalbumin is not due to phosphorylation of Ser-344 near the sites of cleavage on the loop. The suggested explanation is the unique presence of bulky side chains at the P10-P12 site in ovalbumin and angiotensinogen.
12
GRASBERGER, Helmut, Henriette M. B. GOLCHER, Anja FINGERHUT et Onno E. JANSSEN. « Loop variants of the serpin thyroxine-binding globulin : implications for hormone release upon limited proteolysis ». Biochemical Journal 365, no 1 (1 juillet 2002) : 311–16. http://dx.doi.org/10.1042/bj20020014.
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Thyroxine-binding globulin (TBG) and corticosteroid-binding globulin are unique among non-inhibitory members of the superfamily of serine-proteinase inhibitors (serpins) in undergoing a dramatic increase in stability [stressed-to-relaxed (S→R) transition] after proteolytic cleavage within their exposed reactive-site-loop (RSL) equivalent. This structural rearrangement involves the insertion of the cleaved loop as a new strand into the β-sheet A and is accompanied by a decrease in hormone binding. To define the mechanism that leads to disruption of hormone binding of TBG after proteolytic cleavage, the effect of partial loop deletions and replacements by the α1-proteinase inhibitor homologues of TBG were evaluated. Unexpectedly, deletion of the loop's C-terminus, thought to be important for thyroxine binding, improved the binding affinity over that of normal TBG. Proteolytic cleavage of this variant revealed an intact S→R transition and reduced its binding activity to that of cleaved TBG. In contrast, a chimaera with C-terminal loop extension mimicked the decreased binding affinity of cleaved TBG and had a thermal stability intermediate between that of native and cleaved serpins. This variant was still susceptible to loop cleavage and underwent an S→R transition, yet without changing its binding affinity. Our data exclude a direct involvement of loop residues in thyroxine binding of native TBG. Limited insertion of the RSL into β-sheet A appears to trigger hormone release after proteolytic cleavage. In support of this concept, residues within the hinge region of the TBG loop are phylogenetically highly conserved, suggestive of their physiological role as a functional switch in vivo.
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Schreuder, Herman A., Bijtske de Boer, Rein Dijkema, John Mulders, Henri J. M. Theunissen, Peter D. J. Grootenhuis et Wim G. J. Hol. « The intact and cleaved human antithrombin III complex as a model for serpin–proteinase interactions ». Nature Structural & ; Molecular Biology 1, no 1 (janvier 1994) : 48–54. http://dx.doi.org/10.1038/nsb0194-48.
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Mahon, Brian P., Sriram Ambadapadi, Jordan R. Yaron, Carrie L. Lomelino, Melissa A. Pinard, Shahar Keinan, Igor Kurnikov et al. « Crystal Structure of Cleaved Serp-1, a Myxomavirus-Derived Immune Modulating Serpin : Structural Design of Serpin Reactive Center Loop Peptides with Improved Therapeutic Function ». Biochemistry 57, no 7 (6 février 2018) : 1096–107. http://dx.doi.org/10.1021/acs.biochem.7b01171.
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ARII, Yasuhiro, et Masaaki HIROSE. « Probing the serpin structural-transition mechanism in ovalbumin mutant R339T by proteolytic-cleavage kinetics of the reactive-centre loop ». Biochemical Journal 363, no 2 (8 avril 2002) : 403–9. http://dx.doi.org/10.1042/bj3630403.
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A mutant ovalbumin (R339T), but not the wild-type protein, is transformed into the canonical loop-inserted, thermostabilized form after the P1—P1′ cleavage [Yamasaki, Arii, Mikami and Hirose (2002) J. Mol. Biol. 315, 113–120]. The loop-insertion mechanism in the ovalbumin mutant was investigated by proteolytic-cleavage kinetics. The nature of the inserted loop prevented futher cleavage of the P1—P1′ pre-cleaved R339T mutant by subtilisin, which cleaved the second P8—P7 loop site in the P1—P1′ pre-cleaved wild-type protein. After subtilisin proteolysis of the intact R339T, however, two final products that corresponded to the single P1—P1′ and double P1—P1′/P8—P7 cleavages were generated with variable ratios depending on the proteolysis conditions. This was accounted for by the occurrence of two mutually competitive reactions: the loop-insertion reaction and the proteolytic cleavage of the second P8—P7 site in the immediate intermediate after the P1—P1′ cleavage. The competitive nature of the two reactions enabled us to establish a kinetic method to determine the rate constants of the reactions. The first-order rate constant for the loop insertion was determined to be 4.0×10−3/s in the R339T mutant. The second-order rate constant for the P8—P7 cleavage in the immediate P1—P1′ cleavage product for the R339T mutant was >10 times compared with that for its wild-type counterpart. This highly accessible loop nature may play a crucial role in the loop-insertion mechanism for R339T mutant ovalbumin.
16
Zhang, Weiqing, Yung-Jen Chuang, Richard Swanson, Juan Li, Kyunga Seo, Lawrence Leung, Lester F. Lau et Steven T. Olson. « Antiangiogenic antithrombin down-regulates the expression of the proangiogenic heparan sulfate proteoglycan, perlecan, in endothelial cells ». Blood 103, no 4 (15 février 2004) : 1185–91. http://dx.doi.org/10.1182/blood-2003-08-2920.
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Abstract Antithrombin, a key serpin family regulator of blood coagulation proteases, is transformed into a potent antiangiogenic factor by limited proteolysis or mild heating. Here, we show by cDNA microarray, semiquantitative reverse transcriptase–polymerase chain reaction (RT-PCR), Northern blotting, and immunoblotting analyses that the expression of the proangiogenic heparan sulfate proteoglycan (HSPG), perlecan, but not other HSPGs, is dramatically down-regulated in human umbilical vein endothelial cells (HUVECs) treated with antiangiogenic cleaved and latent forms of antithrombin but not with the native form. Down-regulation of perlecan expression by cleaved and latent antithrombins was observed in both basic fibroblast growth factor (bFGF)–stimulated and unstimulated cells, whereas the antiangiogenic antithrombins inhibited the proliferation of only bFGF-stimulated HUVECs by arresting cells at the G1 cell cycle phase. The importance of perlecan expression levels in mediating the antiproliferative effect of the antiangiogenic antithrombins was suggested by the finding that transforming growth factor-β1, a potent stimulator of perlecan expression in endothelial cells, blocked the down-regulation of perlecan expression and antiproliferative activity of cleaved antithrombin on endothelial cells. The previously established key role of perlecan in mediating bFGF stimulation of endothelial cell proliferation and angiogenesis suggests that a primary mechanism by which antiangiogenic antithrombins exert their effects is through the down-regulation of perlecan expression.
17
Lechowicz, Urszula, Stefan Rudzinski, Aleksandra Jezela-Stanek, Sabina Janciauskiene et Joanna Chorostowska-Wynimko. « Post-Translational Modifications of Circulating Alpha-1-Antitrypsin Protein ». International Journal of Molecular Sciences 21, no 23 (2 décembre 2020) : 9187. http://dx.doi.org/10.3390/ijms21239187.
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Alpha-1-antitrypsin (AAT), an acute-phase protein encoded by the SERPINA1 gene, is a member of the serine protease inhibitor (SERPIN) superfamily. Its primary function is to protect tissues from enzymes released during inflammation, such as neutrophil elastase and proteinase 3. In addition to its antiprotease activity, AAT interacts with numerous other substances and has various functions, mainly arising from the conformational flexibility of normal variants of AAT. Therefore, AAT has diverse biological functions and plays a role in various pathophysiological processes. This review discusses major molecular forms of AAT, including complex, cleaved, glycosylated, oxidized, and S-nitrosylated forms, in terms of their origin and function.
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PIMENTA, Daniel C., Julie CHAO, Lee CHAO, Maria A. JULIANO et Luiz JULIANO. « Specificity of human tissue kallikrein towards substrates containing Phe–Phe pair of amino acids ». Biochemical Journal 339, no 2 (8 avril 1999) : 473–79. http://dx.doi.org/10.1042/bj3390473.
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We have explored in detail the determinants of specificity for the hydrolysis by human tissue kallikrein (hK1) of substrates containing the Phe–Phe amino acid pair, after which hK1 cleaves kallistatin (human kallikrein-binding protein), a specific serpin for this protease, as well as somatostatin 1–14. Internally quenched fluorogenic peptides were synthesized with the general structure Abz-peptidyl-EDDnp [Abz, o-aminobenzoic acid; EDDnp, N-(2,4-dinitrophenyl)ethylenediamine], based on the natural reactive-centre loop sequence of kallistatin from P9 to P´13, and the kinetic parameters of their hydrolysis by hK1 were determined. All these peptides were cleaved after the Phe–Phe pair. For comparison, we have also examined peptides containing the reactive-centre loop sequences of human protein-C inhibitor (PCI) and rat kallikrein-binding protein, which were hydrolysed after Phe–Arg and Leu–Lys bonds, respectively. Hybrid peptides containing kallistatin–PCI sequences showed that the efficiency of hK1 activity on the peptides containing kallistatin and PCI sequences depended on both the nature of the P1 amino acid as well as on residues at the P- and P´-sides. Moreover, we have made systematic modifications on the hydrophobic pair Phe–Phe, and on Lys and Ile at the P3 and P4 positions according to the peptide substrate, Abz-AIKFFSRQ-EDDnp. All together, we concluded that tissue kallikrein was very effective on short substrates that are cleaved after the Phe–Arg pair; however, hydrolysis after Phe–Phe or other hydrophobic pairs of amino acids was more restrictive, requiring additional enzyme–substrate interaction and/or particular substrate conformations.
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Carrell, Robin W., et James A. Huntington. « How serpins change their fold for better and for worse ». Biochemical Society Symposia 70 (1 septembre 2003) : 163–78. http://dx.doi.org/10.1042/bss0700163.
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The serpins differ from the many other families of serine protease inhibitors in that they undergo a profound change in topology in order to entrap their target protease in an irreversible complex. The solving of the structure of this complex has now provided a video depiction of the changes involved. Cleavage of the exposed reactive centre of the serpin triggers an opening of the five-stranded A-sheet of the molecule, with insertion of the cleaved reactive loop as an additional strand in the centre of the sheet. The drastic displacement of the acyl-linked protease grossly disrupts its active site and gives an overall loss of 40% of ordered structure. This ability to provide effectively irreversible inhibition explains the selection of the serpins to control the proteolytic cascades of higher organisms. The conformational mechanism provides another advantage in its potential to modulate activity. Sequential crystallographic structures now provide clear depictions of the way antithrombin is activated on binding to the heparans of the microcirculation, and how evolution has utilized this mobile mechanism for subtle variations in activity. The complexity of these modulatory mechanisms is exemplified by heparin cofactor II, where the change in fold is seen to trigger multiple allosteric effects. The downside of the mobile mechanism of the serpins is their vulnerability to aberrant intermolecular ϐ-linkages, resulting in various disorders from cirrhosis to thrombosis. These provide a well defined structural prototype for the new entity of the conformational diseases, including the common dementias, as confirmed by the recent identification of the familial neuroserpin dementias.
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Ni, Hongyu, Balwant Chauhan et Steven Olson. « Effect of Native and Cleaved Forms of Antithrombin on Nuclear Factor κB Activation in Endothelial Cells. » Blood 104, no 11 (16 novembre 2004) : 3923. http://dx.doi.org/10.1182/blood.v104.11.3923.3923.
Texte intégralRésumé :
Abstract Antithrombin (AT) is a single chain plasma glycoprotein that belongs to a superfamily of serine protease inhibitors (Serpin). AT is the major inhibitor of the serine proteases of the coagulation cascade, notably thrombin and factor Xa. In addition, AT has also been demonstrated to have anti-inflammatory properties. Recently, cleaved and latent forms of AT have been shown to function as antiangiogenic agents. However, the molecular mechanisms by which native AT and cleaved AT exert their anti-inflammatory and antiangiogenic effects remains unknown. In this study, we have investigated the effects of both native and cleaved forms of AT on endothelial cell proliferation and nuclear factor κB (NF-κB) activation in the endothelial cells. Bovine pulmonary artery endothelial cells (BPAE) was used in the study. Human α-antithrombin was purified from plasma by affinity chromatography on heparin Sepharose, followed by anion-exchange chromatography. Cleaved form of AT was prepared by digestion of purified human AT with human neutrophil elastase, followed by chromatography on a heparin Sepharose column. The endothelial cells were stimulated with basic fibroblast growth factor (bFGF) or tumor necrosis factor-α (TNF-α) in culture and incubated with either native AT or cleaved AT. Endothelial cell proliferation was measured by the MTT (methylthiazolyldiphenyl-tetrazolium bromide) cell proliferation assay as well as by counting cell numbers before and after the treatment. The results demonstrate that both native AT and cleaved AT could inhibit bovine pulmonary artery endothelial cell proliferation. These results contrast with those reported with other types of endothelial cells such as human umbilical vein endothelial cells (HUVEC) in which only cleaved form but not native form of AT inhibits cell proliferation. NF-κB activation was detected by the ELISA-based assay using antibodies specific for the activated form of p50 and p65 subunit of the NF-κB. In native AT, the inhibitory activity of endothelial cell proliferation was associated with down-regulation of NF-κB as measured by decreased nuclear p65. However, the cleaved AT showed minimal effect on the NF-κB activation. Our results suggest that although both native and cleaved AT inhibit endothelial cell proliferation, they might use different signal transduction pathways. Moreover, our findings suggest that native and cleaved AT may have differential effects on different types of endothelial cells.
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PEDERSEN, Katrine E., Anja P. EINHOLM, Anni CHRISTENSEN, Lotte SCHACK, Troels WIND, John M. KENNEY et Peter A. ANDREASEN. « Plasminogen activator inhibitor-1 polymers, induced by inactivating amphipathic organochemical ligands ». Biochemical Journal 372, no 3 (15 juin 2003) : 747–55. http://dx.doi.org/10.1042/bj20021868.
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Negatively charged organochemical inactivators of the anti-proteolytic activity of plasminogen activator inhibitor-1 (PAI-1) convert it to inactive polymers. As investigated by native gel electrophoresis, the size of the PAI-1 polymers ranged from dimers to multimers of more than 20 units. As compared with native PAI-1, the polymers exhibited an increased resistance to temperature-induced unfolding. Polymerization was associated with specific changes in patterns of digestion with non-target proteases. During incubation with urokinase-type plasminogen activator, the polymers were slowly converted to reactive centre-cleaved monomers, indicating substrate behaviour of the terminal PAI-1 molecules in the polymers. A quadruple mutant of PAI-1 with a retarded rate of latency transition also had a retarded rate of polymerization. Studying a number of serpins by native gel electrophoresis, ligand-induced polymerization was observed only with PAI-1 and heparin cofactor II, which were also able to copolymerize. On the basis of these results, we suggest that the binding of ligands in a specific region of PAI-1 leads to so-called loop–sheet polymerization, in which the reactive centre loop of one molecule binds to β-sheet A in another molecule. Induction of serpin polymerization by small organochemical ligands is a novel finding and is of protein chemical interest in relation to pathological protein polymerization in general.
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Nawata, Shugo, Kazuyuki Nakamura, Hiroshi Hirakawa, Kotaro Sueoka, Tomoko Emoto, Akihiro Murakami, Kenji Umayahara et al. « Electrophoretic analysis of the cleaved form of serpin, squamous cell carcinoma antigen-1 in normal and malignant squamous epithelial tissues ». ELECTROPHORESIS 24, no 14 (juillet 2003) : 2277–82. http://dx.doi.org/10.1002/elps.200305501.
Texte intégral
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van Meijer, Marja, Annelies Smilde, Guido Tans, Michael E. Nesheim, Hans Pannekoek et Anton J. G. Horrevoets. « The Suicide Substrate Reaction Between Plasminogen Activator Inhibitor 1 and Thrombin Is Regulated by the Cofactors Vitronectin and Heparin ». Blood 90, no 5 (1 septembre 1997) : 1874–82. http://dx.doi.org/10.1182/blood.v90.5.1874.
Texte intégralRésumé :
AbstractThe interaction of thrombin with plasminogen activator inhibitor 1 (PAI-1) is shown to result in the simultaneous formation of both cleaved PAI-1 and a sodium dodecyl sulfate-stable thrombin-PAI-1 complex. The kinetics of this reaction can be described by a “suicide substrate” mechanism that includes a branched reaction pathway, which terminates in either the stable inhibitor-enzyme complex or the cleaved inhibitor plus free enzyme. Because of the branched pathway, approximately three moles of PAI-1 are needed to completely inhibit one mole of thrombin. Heparin and vitronectin enhance the rate of inhibition from 9.8 × 102 L mol−1 s−1 to 6.2 × 104 L mol−1 s−1 and 2.1 × 105 L mol−1 s−1, respectively, under optimal conditions. In addition to enhancing the rate of inhibition, both cofactors increase the apparent stoichiometry of the PAI-1–thrombin interaction, with cofactor concentration dependencies similar to the inhibition reaction. Thus, at 37°C approximately six cleavage reactions occur per inhibition reaction. Therefore, thrombin will efficiently inactivate PAI-1 in the presence of either vitronectin or heparin, unless a sufficient excess of the inhibitor is present. These results show that physiological cofactors are able to switch a protease-serpin inhibition reaction to a substrate reaction, depending on the local concentrations of each of the components.
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Saito, A., et H. Sinohara. « Rabbit α-1-antiproteinase E : a novel recombinant serpin which does not inhibit proteinases ». Biochemical Journal 307, no 2 (15 avril 1995) : 369–75. http://dx.doi.org/10.1042/bj3070369.
Texte intégralRésumé :
A cDNA coding for the E isoform of alpha-1-antiproteinase (also called alpha-1-antitrypsin or alpha-1-proteinase inhibitor) was isolated by oligonucleotide hybridization following immunochemical screening of the rabbit liver cDNA library. The deduced amino acid sequence of the E isoform showed 96.4% identity in 413 residues of the F and S-1 isoforms of rabbit alpha-1-antiproteinase. The N-terminal half of the amino acid residues of the three isoforms was almost identical, but the putative reactive-site loop structure (P8-P′8) was significantly different in the various forms, the P1 site of the E form being glutamic acid. Interaction of the recombinant E form with the various proteinases was investigated by SDS/PAGE, followed by immunoblot analysis. The recombinant protein and trypsin formed a 62 kDa equimolar complex, which gradually became graded to the 37 kDa fragment through several intermediates. The E form also formed a complex of a similar size with elastase and became degraded to the 31 kDa fragment. Several proteinases which cleaved the E form without forming a detectable complex on SDS/PAGE are chymotrypsin, protease V8, pancreas kallikrein, thermolysin, papain and ficin. Other proteinases, with a stringent substrate specificity, such as thrombin, factor Xa, plasmin, plasma kallikrein and cathepsin G, did not attack the E form. Unlike the F and S-1 forms of rabbit plasma alpha-1-antiproteinase, the recombinant E form did not inhibit the amidolytic and proteolytic activities of trypsin. Neither elastase nor protease V8 was inhibited by the E form. Thus the change in the amino acid residues in the reactive-site loop, probably in the P1 site, is responsible for the loss of inhibitory activity of rabbit alpha-1-antiproteinase E. The novel character of the E form could provide a new insight into the interaction of serpin and proteinases.
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Leu, Chia-Hsing, Mei-Lin Yang, Nai-Hui Chung, Yen-Jang Huang, Yu-Chu Su, Yi-Cheng Chen, Chia-Cheng Lin et al. « Kallistatin Ameliorates Influenza Virus Pathogenesis by Inhibition of Kallikrein-Related Peptidase 1-Mediated Cleavage of Viral Hemagglutinin ». Antimicrobial Agents and Chemotherapy 59, no 9 (6 juillet 2015) : 5619–30. http://dx.doi.org/10.1128/aac.00065-15.
Texte intégralRésumé :
ABSTRACTProteolytic cleavage of the hemagglutinin (HA) of influenza virus by host trypsin-like proteases is required for viral infectivity. Some serine proteases are capable of cleaving influenza virus HA, whereas some serine protease inhibitors (serpins) inhibit the HA cleavage in various cell types. Kallikrein-related peptidase 1 (KLK1, also known as tissue kallikrein) is a widely distributed serine protease. Kallistatin, a serpin synthesized mainly in the liver and rapidly secreted into the circulation, forms complexes with KLK1 and inhibits its activity. Here, we investigated the roles of KLK1 and kallistatin in influenza virus infection. We show that the levels of KLK1 increased, whereas those of kallistatin decreased, in the lungs of mice during influenza virus infection. KLK1 cleaved H1, H2, and H3 HA molecules and consequently enhanced viral production. In contrast, kallistatin inhibited KLK1-mediated HA cleavage and reduced viral production. Cells transduced with the kallistatin gene secreted kallistatin extracellularly, which rendered them more resistant to influenza virus infection. Furthermore, lentivirus-mediated kallistatin gene delivery protected mice against lethal influenza virus challenge by reducing the viral load, inflammation, and injury in the lung. Taking the data together, we determined that KLK1 and kallistatin contribute to the pathogenesis of influenza virus by affecting the cleavage of the HA peptide and inflammatory responses. This study provides a proof of principle for the potential therapeutic application of kallistatin or other KLK1 inhibitors for influenza. Since proteolytic activation also enhances the infectivity of some other viruses, kallistatin and other kallikrein inhibitors may be explored as antiviral agents against these viruses.
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Jiang, Qijiao, Neill A. Gingles, Marc A. Olivier, Lindsey A. Miles et Robert J. Parmer. « The anti-fibrinolytic SERPIN, plasminogen activator inhibitor 1 (PAI-1), is targeted to and released from catecholamine storage vesicles ». Blood 117, no 26 (30 juin 2011) : 7155–63. http://dx.doi.org/10.1182/blood-2010-05-287672.
Texte intégralRésumé :
Recent studies suggest a crucial role for plasminogen activator inhibitor-1 (PAI-1) in mediating stress-induced hypercoagulability and thrombosis. However, the mechanisms by which PAI-1 is released by stress are not well-delineated. Here, we examined catecholaminergic neurosecretory cells for expression, trafficking, and release of PAI-1. PAI-1 was prominently expressed in PC12 pheochromocytoma cells and bovine adrenomedullary chromaffin cells as detected by Northern blotting, Western blotting, and specific PAI-1 ELISA. Sucrose gradient fractionation studies and immunoelectron microscopy demonstrated localization of PAI-1 to catecholamine storage vesicles. Secretogogue stimulation resulted in corelease of PAI-1 with catecholamines. Parallel increases in plasma PAI-1 and catecholamines were observed in response to acute sympathoadrenal activation by restraint stress in mice in vivo. Reverse fibrin zymography demonstrated free PAI-1 in cellular releasates. Detection of high molecular weight complexes by Western blotting, consistent with PAI-1 complexed with t-PA, as well as bands consistent with cleaved PAI-1, suggested that active PAI-1 was present. Modulation of PAI-1 levels by incubating PC12 cells with anti–PAI-1 IgG caused a marked decrease in nicotine-mediated catecholamine release. In summary, PAI-1 is expressed in chromaffin cells, sorted into the regulated pathway of secretion (into catecholamine storage vesicles), and coreleased, by exocytosis, with catecholamines in response to secretogogues.
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Jairajpuri, Mohamad Aman, et Shoyab Ansari. « Using serpins cysteine protease cross-specificity to possibly trap SARS-CoV-2 Mpro with reactive center loop chimera ». Clinical Science 134, no 17 (1 septembre 2020) : 2235–41. http://dx.doi.org/10.1042/cs20200767.
Texte intégralRésumé :
Abstract Human serine protease inhibitors (serpins) are the main inhibitors of serine proteases, but some of them also have the capability to effectively inhibit cysteine proteases. Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) main protease (Mpro) is a chymotrypsin-type cysteine protease that is needed to produce functional proteins essential for virus replication and transcription. Serpin traps its target proteases by presenting a reactive center loop (RCL) as protease-specific cleavage site, resulting in protease inactivation. Mpro target sites with its active site serine and other flanking residues can possibly interact with serpins. Alternatively, RCL cleavage site of serpins with known evidence of inhibition of cysteine proteases can be replaced by Mpro target site to make chimeric proteins. Purified chimeric serpin can possibly inhibit Mpro that can be assessed indirectly by observing the decrease in ability of Mpro to cleave its chromogenic substrate. Chimeric serpins with best interaction and active site binding and with ability to form 1:1 serpin–Mpro complex in human plasma can be assessed by using SDS/PAGE and Western blot analysis with serpin antibody. Trapping SARS-CoV-2 Mpro cysteine protease using cross-class serpin cysteine protease inhibition activity is a novel idea with significant therapeutic potential.
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Smith, K. F., R. A. Harrison et S. J. Perkins. « Structural comparisons of the native and reactive-centre-cleaved forms of α1-antitrypsin by neutron- and X-ray-scattering in solution ». Biochemical Journal 267, no 1 (1 avril 1990) : 203–12. http://dx.doi.org/10.1042/bj2670203.
Texte intégralRésumé :
alpha 1-Antitrypsin is the best-characterized member of the serpin (serine-proteinase inhibitor) superfamily. Its solution structure was studied by high-flux neutron-scattering and synchrotron X-ray-scattering. Neutron data show that its absorption coefficient A1% 280,1cm is 5.4. The neutron radius of gyration RG at infinite contrast for native alpha 1-antitrypsin is 2.61 nm, characteristic of a moderately elongated structure, and its cross-sectional RG is 1.34 nm. The internal inhomogeneity of scattering densities within alpha 1-antitrypsin is high at 29 x 10(-5). The X-ray RG is 2.91 nm, in good agreement with the neutron RG of 2.82 nm in 1H2O. This RG is unchanged in reactive-centre-cleaved alpha 1-antitrypsin. These parameters are also unchanged at pH 8 in sodium/potassium phosphate buffers up to 0.6 M. The neutron and X-ray curves for native alpha 1-antitrypsin were compared with Debye simulation based on the crystal structure of reactive-centre-cleaved (papain) alpha 1-antitrypsin. After allowance for residues not visible in the crystallographic electron-density map, and rejoining the proteolysed site between Met-358 and Ser-359 by means of a relatively minor conformational re-arrangement, good agreement to a structural resolution of 4 nm is obtained with the neutron data in two contrasts and with the X-ray data. The structures of the native and cleaved forms of alpha 1-antitrypsin are thus similar within the resolution of solution scattering. This places an upper limit on the magnitude of the presumed conformational changes that occur in alpha 1-antitrypsin on reactive-centre cleavage, as indicated in earlier spectroscopic investigations of the Met-358-Ser-359 peptide-bond cleavage. Methods for scattering-curve simulations from crystal structures are critically assessed. The RG data lead to dimensions of 7.8 nm x 4.9 nm x 2.2 nm for native alpha 1-antitrypsin. The high internal inhomogeneity and the asymmetric shorter semi-axes of 4.9 nm and 2.2 nm suggest that the three oligosaccharide chains of alpha 1-antitrypsin are essentially freely extended into solvent in physiological conditions. This conclusion is also supported by the Debye simulations, and by modelling based on hydrodynamic parameters.
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Turner, Peter C., M. Carmen Sancho, S. R. Thoennes, A. Caputo, R. C. Bleackley et Richard W. Moyer. « Myxoma Virus Serp2 Is a Weak Inhibitor of Granzyme B and Interleukin-1β-Converting Enzyme In Vitro and Unlike CrmA Cannot Block Apoptosis in Cowpox Virus-Infected Cells ». Journal of Virology 73, no 8 (1 août 1999) : 6394–404. http://dx.doi.org/10.1128/jvi.73.8.6394-6404.1999.
Texte intégralRésumé :
ABSTRACT The Serp2 protein encoded by the leporipoxvirus myxoma virus is essential for full virulence (F. Messud-Petit, J. Gelfi, M. Delverdier, M. F. Amardeilh, R. Py, G. Sutter, and S. Bertagnoli, J. Virol. 72:7830–7839, 1998) and, like crmA of cowpox virus (CPV), is reported to inhibit the interleukin-1β-converting enzyme (ICE, caspase-1) (F. Petit, S. Bertagnoli, J. Gelfi, F. Fassy, C. Boucraut-Baralon, and A. Milon, J. Virol. 70:5860–5866, 1996). Serp2 and CrmA both contain Asp at the P1 position within the serpin reactive site loop and yet are only 35% identical overall. Serp2 protein was cleaved by ICE but, unlike CrmA, did not form a stable complex with ICE that was detectable by native gel electrophoresis. Attempts to covalently cross-link ICE-serpin inhibitory complexes were successful with CrmA, but no complex between ICE and Serp2 was visible after cross-linking. Purified His10-tagged Serp2 protein was a relatively poor inhibitor of ICE, with aKi of 80 nM compared to 4 pM for CrmA. Serp2 protein resembled CrmA in that a stable complex with the serine proteinase granzyme B was detectable after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, Serp2 was less effective at inhibiting granzyme B activity (Ki = 420 nM) than CrmA (Ki = 100 nM). Finally, Serp2 was tested for the ability to replace CrmA and inhibit apoptosis in LLC-PK1 cells infected with a CPV recombinant deleted for CrmA but expressing Serp2. Unlike wild-type-CPV-infected cells, apoptosis was readily observed in cells infected with the recombinant virus, as indicated by the induction of both nuclear fragmentation and caspase-mediated cleavage of DEVD-AMC [acetyl-Asp-Glu-Val-Asp-(amino-4-methyl coumarin)]. These results indicate that Serp2 is unable to functionally substitute for CrmA within the context of CPV and that the inhibition spectra for Serp2 and CrmA are distinct.
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Yuasa, Hiroyuki, Hitoshi Tanaka, Tatsuya Hayashi, Toshiaki Wakita, Hideaki Nakamura, Junji Nishioka, Yoshifumi Kawarada et Koji Suzuki. « Bovine Protein C Inhibitor Has a Unique Reactive Site and Can Transiently Inhibit Plasmin ». Thrombosis and Haemostasis 83, no 02 (2000) : 262–67. http://dx.doi.org/10.1055/s-0037-1613797.
Texte intégralRésumé :
SummaryProtein C inhibitor (PCI) regulates the anticoagulant protein C pathway by neutralizing activated protein C and thrombin-thrombomodulin complex in the human hemostatic system. In this study, we cloned a full-length bovine PCI cDNA encoding a putative 19-residue signal peptide and a 385-residue mature protein; this showed 70.6%, 70.6%, 57.5% and 59.6% amino acid sequence homology with the human, rhesus monkey, rat and mouse PCIs, respectively. Bovine PCI mRNA (2.1 kb in size) was expressed strongly in the liver, and moderately in the kidney and testis, but not in other tissues tested. Bovine PCI has a putative reactive site peptide bond, Lys-Ser, that is different from the reactive site sequence (Arg-Ser) of other species’ PCI. We found that bovine PCI transiently inhibits bovine plasmin, but not human plasmin. Western blot analysis showed that the reactive site of bovine PCI is cleaved during the course of complex formation with bovine plasmin; degraded PCI is released from the complex gradually concomitant with the recovery of plasmin activity. These findings suggest that bovine PCI plays a role not only in the protein C pathway but also in the fibrinolytic activity of bovine hemostatic system. Abbreviations: PCI, protein C inhibitor, Serpin, serine protease inhibitor, APC, activated protein C, TM, thrombomodulin.
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Huang, Xin, Yahui Yan, Yizheng Tu, Jeffrey Gatti, George J. Broze, Aiwu Zhou et Steven T. Olson. « Structural basis for catalytic activation of protein Z–dependent protease inhibitor (ZPI) by protein Z ». Blood 120, no 8 (23 août 2012) : 1726–33. http://dx.doi.org/10.1182/blood-2012-03-419598.
Texte intégralRésumé :
Abstract The anticoagulant serpin, protein Z-dependent protease inhibitor (ZPI), is catalytically activated by its cofactor, protein Z (PZ), to regulate the function of blood coagulation factor Xa on membrane surfaces. The X-ray structure of the ZPI-PZ complex has shown that PZ binds to a unique site on ZPI centered on helix G. In the present study, we show by Ala-scanning mutagenesis of the ZPI-binding interface, together with native PAGE and kinetic analyses of PZ binding to ZPI, that Tyr240 and Asp293 of ZPI are crucial hot spots for PZ binding. Complementary studies with protein Z–protein C chimeras show the importance of both pseudocatalytic and EGF2 domains of PZ for the critical ZPI interactions. To understand how PZ acts catalytically, we analyzed the interaction of reactive loop–cleaved ZPI (cZPI) with PZ and determined the cZPI X-ray structure. The cZPI structure revealed changes in helices A and G of the PZ-binding site relative to native ZPI that rationalized an observed 6-fold loss in PZ affinity and PZ catalytic action. These findings identify the key determinants of catalytic activation of ZPI by PZ and suggest novel strategies for ameliorating hemophilic states through drugs that disrupt the ZPI-PZ interaction.
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ATTUCCI, Sylvie, Brice KORKMAZ, Luiz JULIANO, Eric HAZOUARD, Catherine GIRARDIN, Michèle BRILLARD-BOURDET, Sophie RÉHAULT, Philippe ANTHONIOZ et Francis GAUTHIER. « Measurement of free and membrane-bound cathepsin G in human neutrophils using new sensitive fluorogenic substrates ». Biochemical Journal 366, no 3 (15 septembre 2002) : 965–70. http://dx.doi.org/10.1042/bj20020321.
Texte intégralRésumé :
Activated human polymorphonuclear neutrophils at inflammatory sites release the chymotrypsin-like protease cathepsin G, together with elastase and proteinase 3 (myeloblastin), from their azurophil granules. The low activity of cathepsin G on synthetic substrates seriously impairs studies designed to clarify its role in tissue inflammation. We have solved this problem by producing new peptide substrates with intramolecularly quenched fluorescence. These substrates were deduced from the sequence of putative protein targets of cathepsin G, including the reactive loop sequence of serpin inhibitors and the N-terminal domain of the protease-activated receptor of thrombin, PAR-1. Two substrates were selected, Abz-TPFSGQ-EDDnp and Abz-EPFWEDQ-EDDnp, that are cleaved very efficiently by cathepsin G but not by neutrophil elastase or proteinase 3, with specificity constants (kcat/Km) in the 105M-1·s-1 range. They can be used to measure subnanomolar concentrations of free enzyme in vitro and at the surface of neutrophils purified from fresh human blood. Purified neutrophils express 0.02—0.7pg of cathepsin G/cell (n = 15) at their surface. This means that about 104 purified cells may be enough to record cathepsin G activity within minutes. This may be most important for investigating the role of cathepsin G as an inflammatory agent, especially in bronchoalveolar lavage fluids from patients with pulmonary inflammatory disorders.
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Young, Laura, Neil Van de Water, Paul L. Harper, Paul A. Ockelford, Anita Horvath, Paul Coughlin et Peter Browett. « Protein Z Dependent Protease Inhibitor Inhibition of Factor XIa Is Accelerated by Unfractionated Heparin in the Presence and Absence of Protein Z : Further Evidence of the Potential Significance of XIa Inhibitory Activity. » Blood 112, no 11 (16 novembre 2008) : 2032. http://dx.doi.org/10.1182/blood.v112.11.2032.2032.
Texte intégralRésumé :
Abstract Protein Z dependent protease inhibitor (ZPI) is a human plasma serpin that inhibits factors Xa and XIa in the coagulation pathway. Kinetic studies have shown that ZPI is an efficient inhibitor of factor Xa. This interaction is accelerated by protein Z in the presence of calcium and phospholipid. The inhibition of factor XIa is by an alternative mechanism that does not require phospholipid but is influenced by both heparin and protein Z; heparin increases inhibition whereas protein Z /calcium delays inhibition. The latter maybe physiologically relevant to XIa inhibitory activity as ZPI and protein Z circulate in complex in plasma, with ZPI in slight excess. The kinetics of ZPI/XIa/heparin interactions have not been fully characterised. In this study we report the kinetic analysis of the interaction between factor XIa and recombinant ZPI expressed in Escherichia coli, in the presence of unfractionated heparin, pentasaccharide and protein Z. Results: Recombinant ZPI (rZPI) had characteristics similar to native protein as previously described; serpin-protease complexes were non-covalent, and reactive centre loop proteolytic cleavage of ZPI was noted during this reaction. On SDS gel rZPI had a molecular weight of 50kDa. Circular dichroism of rZPI showed an unfolding transition on thermal denaturation typical of a serpin in native conformation with a Tm of 58.8°C. In contrast loop cleaved rZPI had increased thermal stability as expected for a serpin. A discontinuous method was used to measure the rate of inhibition of target proteases by rZPI; the first order rate constant was measured over time under pseudo first order conditions, and was used to derive the second order rate constant (ka) as shown in Table 1. Table 1: Second order rate constants (ka) Xa (Ms−1) XIa (Ms−1) * And calcium/phospholipid vesicles ZPI 1.9 x 103 9.0 x 104 ZPI + protein Z* 6.6 x 105 1.8 x 104 ZPI + unfractionated heparin (1IU/mL) - 1.0 x 106 ZPI + pentasaccharide (500nM) - 1.0 x 105 ZPI + protein Z* + unfractionated heparin (1IU/mL) - 6.0 x 105 In the presence of calcium and phospholipid vesicles the ka for Xa in the absence and presence of protein Z was similar to previous reports. The ka for ZPI inhibition of XIa increased 11 fold in the presence of unfractionated heparin. Heparin titration curves suggest that a template mechanism is likely; the heparin pentasaccharide had no impact on inhibition rate consistent with this observation. It has previously been reported that protein Z impedes the ZPI inhibition of XIa; our results support this with a 5-fold reduction in ka. However the addition of heparin to ZPI in the presence of Protein Z leads to a significant 33-fold increase in inhibitory activity with a rate similar to the inhibitory activity of ZPI against Xa in the presence of protein Z. Conclusions: There are several physiological inhibitors of factor XIa. In previous reports ZPI inhibition only increased 2-fold in the presence of heparin. Our results confirm that heparin increases the ZPI inhibition of XIa but with a significantly higher increase than previously reported. The increase in activity is even more marked in the presence of protein Z. Although protein Z alone inhibits ZPI activity against XIa the combination with heparin achieves a 33 fold increase in activity. Of all the serpin inhibitors of XIa, antithrombin is considered a major inhibitor in the presence of heparin. Our results show that the ka for FXIa inhibition in the presence of ZPI, protein Z and heparin is more than 20 fold higher than the ka reported for the antithrombin/heparin inhibition of XIa (2-3x104Ms−1). We have previously reported a higher incidence of venous thrombosis in patients with nonsense mutations in the ZPI gene suggesting the clinical significance of this protein. The suggestion that the inhibition of XIa maybe more relevant than that of Xa has been raised in recent murine work where ZPI knockouts have a more severe phenotype than protein Z knockouts. ZPI inhibition of XIa in the presence of endogenous heparins maybe an important contributor to its physiological significance.
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Moon, Kristin B., Peter C. Turner et Richard W. Moyer. « SPI-1-Dependent Host Range of Rabbitpox Virus and Complex Formation with Cathepsin G Is Associated with Serpin Motifs ». Journal of Virology 73, no 11 (1 novembre 1999) : 8999–9010. http://dx.doi.org/10.1128/jvi.73.11.8999-9010.1999.
Texte intégralRésumé :
ABSTRACT Serpins are a superfamily of serine proteinase inhibitors which function to regulate a number of key biological processes including fibrinolysis, inflammation, and cell migration. Poxviruses are the only viruses known to encode functional serpins. While some poxvirus serpins regulate inflammation (myxoma virus SERP1 and cowpox virus [CPV] crmA/SPI-2) or apoptosis (myxoma virus SERP2 and CPV crmA/SPI-2), the function of other poxvirus serpins remains unknown. The rabbitpox virus (RPV) SPI-1 protein is 47% identical to crmA and shares all of the serpin structural motifs. However, no serpin-like activity has been demonstrated for SPI-1 to date. Earlier we showed that RPV with the SPI-1 gene deleted, unlike wild-type virus, fails to grow on A549 or PK15 cells (A. Ali, P. C. Turner, M. A. Brooks, and R. W. Moyer, Virology 202:306–314, 1994). Here we demonstrate that in the absence of a functional SPI-1 protein, infected nonpermissive cells which exhibit the morphological features of apoptosis fail to activate terminal caspases or cleave the death substrates PARP or lamin A. We show that SPI-1 forms a stable complex in vitro with cathepsin G, a member of the chymotrypsin family of serine proteinases, consistent with serpin activity. SPI-1 reactive-site loop (RSL) mutations of the critical P1 and P14 residues abolish this activity. Viruses containing the SPI-1 RSL P1 or P14 mutations also fail to grow on A549 or PK15 cells. These results suggest that the full virus host range depends on the serpin activity of SPI-1 and that in restrictive cells SPI-1 inhibits a proteinase with chymotrypsin-like activity and may function to inhibit a caspase-independent pathway of apoptosis.
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Cunningham, Michael, Varsha Bhakta et William Sheffield. « Altering Heparin Cofactor II at VAL439 (P6) either Impairs Inhibition of Thrombin or Confers Elastase Resistance ». Thrombosis and Haemostasis 88, no 07 (2002) : 89–97. http://dx.doi.org/10.1055/s-0037-1613159.
Texte intégralRésumé :
SummaryHeparin cofactor II (HCII) is plasma glycoprotein and thrombin inhibitor of the serpin type previously shown to inhibit thrombin in the absence of its N-terminal 74 amino acids, and to be cleaved by neutrophil elastase (NE) at two sites: I66-F67 and V439-G440, the P6-P5 bond of the reactive center loop. We examined the contribution of Val439 to the reaction of HCII with thrombin and NE. Hexahistidine-tagged HCII proteins lacking residues 1-66 (H6Δ66HCII) containing either the wild-type Val 439 or one of six substitutions were expressed in E. coli. The rates of heparin-catalyzed thrombin inhibition of the V439L, C, or R variants were reduced at least 80-fold compared to wild-type H6Δ66HCII, while those of the F, S, or W variants were largely unchanged. Following controlled exposure to NE in the presence of heparin, these latter variants retained 3.5to 4.5-fold more residual anti-thrombin activity than wild-type H6Δ66HCII treated in the same manner. This resistance arose due to deflection of NE attack from V439-G440 to secondary sites. The F, S, or W V439 variants exhibited a similar or greater degree of NE resistance when re-expressed as full-length hexahistidine-tagged HCII proteins, suggesting that the I66-F67 NE site is not well recognized in non-glycosylated HCII. Of these full-length variants, the V439F was the most active, exhibiting only a 2-fold reduction in its heparin-catalyzed rate of thrombin inhibition. HCII can therefore be made NE-resistant without severely compromising its capacity to inhibit thrombin.
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Wang, Yang, Fan Yang, Xiaolong Cao, Zhen Zou, Zhiqiang Lu, Michael R. Kanost et Haobo Jiang. « Hemolymph protease-5 links the melanization and Toll immune pathways in the tobacco hornworm,Manduca sexta ». Proceedings of the National Academy of Sciences 117, no 38 (8 septembre 2020) : 23581–87. http://dx.doi.org/10.1073/pnas.2004761117.
Texte intégralRésumé :
Proteolytic activation of phenoloxidase (PO) and the cytokine Spätzle during immune responses of insects is mediated by a network of hemolymph serine proteases (HPs) and noncatalytic serine protease homologs (SPHs) and inhibited by serpins. However, integration and conservation of the system and its control mechanisms are not fully understood. Here we present biochemical evidence that PO-catalyzed melanin formation, Spätzle-triggered Toll activation, and induced synthesis of antimicrobial peptides are stimulated via hemolymph (serine) protease 5 (HP5) inManduca sexta. Previous studies have demonstrated a protease cascade pathway in which HP14 activates proHP21; HP21 activates proPAP2 and proPAP3, which then activate proPO in the presence of a complex of SPH1 and SPH2. We found that both HP21 and PAP3 activate proHP5 by cleavage at ESDR176*IIGG. HP5 then cleaves proHP6 at a unique site of LDLH112*ILGG. HP6, an ortholog ofDrosophilaPersephone, activates both proHP8 and proPAP1. HP8 activates proSpätzle-1, whereas PAP1 cleaves and activates proPO. HP5 is inhibited byManduca sextaserpin-4, serpin-1A, and serpin-1J to regulate its activity. In summary, we have elucidated the physiological roles of HP5, a CLIPB with unique cleavage specificity (cutting after His) that coordinates immune responses in the caterpillar.
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Han, Xin, Ryan Fiehler et George J. Broze. « Characterization of the protein Z–dependent protease inhibitor ». Blood 96, no 9 (1 novembre 2000) : 3049–55. http://dx.doi.org/10.1182/blood.v96.9.3049.
Texte intégralRésumé :
Abstract Protein Z-dependent protease inhibitor (ZPI) is a 72-kd member of the serpin superfamily of proteinase inhibitors that produces rapid inhibition of factor Xa in the presence of protein Z (PZ), procoagulant phospholipids, and Ca++ (t1/2 less than 10 seconds). The rate of factor Xa inhibition by ZPI is reduced more than 1000-fold in the absence of PZ. The factor Xa–ZPI complex is not stable to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, but is detectable by alkaline–polyacrylamide gel electrophoresis. The combination of PZ and ZPI dramatically delays the initiation and reduces the ultimate rate of thrombin generation in mixtures containing prothrombin, factor V, phospholipids, and Ca++. In similar mixtures containing factor Va, however, PZ and ZPI do not inhibit thrombin generation. Thus, the major effect of PZ and ZPI is to dampen the coagulation response prior to the formation of the prothrombinase complex. Besides factor Xa, ZPI also inhibits factor XIa in the absence of PZ, phospholipids, and Ca++. Heparin (0.2 U/mL) enhances the rate (t1/2 = 25 seconds vs 50 seconds) and the extent (99% vs 93% at 30 minutes) of factor XIa inhibition by ZPI. During its inhibitory interaction with factor Xa and factor XIa, ZPI is proteolytically cleaved with the release of a 4.2-kd peptide. The N-terminal amino acid sequence of this peptide (SMPPVIKVDRPF) establishes Y387 as the P1 residue at the reactive center of ZPI. ZPI activity is consumed during the in vitro coagulation of plasma through a proteolytic process that involves the actions of factor Xa with PZ and factor XIa.
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Han, Xin, Ryan Fiehler et George J. Broze. « Characterization of the protein Z–dependent protease inhibitor ». Blood 96, no 9 (1 novembre 2000) : 3049–55. http://dx.doi.org/10.1182/blood.v96.9.3049.h8003049_3049_3055.
Texte intégralRésumé :
Protein Z-dependent protease inhibitor (ZPI) is a 72-kd member of the serpin superfamily of proteinase inhibitors that produces rapid inhibition of factor Xa in the presence of protein Z (PZ), procoagulant phospholipids, and Ca++ (t1/2 less than 10 seconds). The rate of factor Xa inhibition by ZPI is reduced more than 1000-fold in the absence of PZ. The factor Xa–ZPI complex is not stable to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, but is detectable by alkaline–polyacrylamide gel electrophoresis. The combination of PZ and ZPI dramatically delays the initiation and reduces the ultimate rate of thrombin generation in mixtures containing prothrombin, factor V, phospholipids, and Ca++. In similar mixtures containing factor Va, however, PZ and ZPI do not inhibit thrombin generation. Thus, the major effect of PZ and ZPI is to dampen the coagulation response prior to the formation of the prothrombinase complex. Besides factor Xa, ZPI also inhibits factor XIa in the absence of PZ, phospholipids, and Ca++. Heparin (0.2 U/mL) enhances the rate (t1/2 = 25 seconds vs 50 seconds) and the extent (99% vs 93% at 30 minutes) of factor XIa inhibition by ZPI. During its inhibitory interaction with factor Xa and factor XIa, ZPI is proteolytically cleaved with the release of a 4.2-kd peptide. The N-terminal amino acid sequence of this peptide (SMPPVIKVDRPF) establishes Y387 as the P1 residue at the reactive center of ZPI. ZPI activity is consumed during the in vitro coagulation of plasma through a proteolytic process that involves the actions of factor Xa with PZ and factor XIa.
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Tong, Yu K., Rossa WK Chiu, Tak Y. Leung, Chunming Ding, Tze K. Lau, Tse N. Leung et YM Dennis Lo. « Detection of Restriction Enzyme–Digested Target DNA by PCR Amplification Using a Stem-Loop Primer : Application to the Detection of Hypomethylated Fetal DNA in Maternal Plasma ». Clinical Chemistry 53, no 11 (1 novembre 2007) : 1906–14. http://dx.doi.org/10.1373/clinchem.2007.092619.
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Abstract Background: The discovery of cell-free fetal DNA in maternal plasma has opened up new possibilities for noninvasive prenatal diagnosis and monitoring. Among the fetal markers that have been described, methylation markers are sex and polymorphism independent. Methylation-sensitive restriction endonucleases are commonly used to digest hypomethylated DNA molecules, and the hypermethylated molecules remain intact for detection. The positive detection of the cleaved hypomethylated molecules would be useful for certain targets but has not been reported. Methods: The use of a stem-loop primer in microRNA detection has previously been described. In this study, DNA assays were designed and performed on maternal plasma, which contained the hypomethylated placental serpin peptidase inhibitor, clade B (ovalbumin), member 5 (SERPINB5; maspin) gene in an excess background of hypermethylated maternal SERPINB5. Detection of the enzyme-digested placenta-derived hypomethylated SERPINB5 molecules was achieved by performing stem-loop extension followed by real-time PCR on maternal plasma. The placental origin of the stem-loop–extended SERPINB5 molecules was confirmed by genotyping. Results: From the real-time PCR results on maternal plasma, stem-loop–extended SERPINB5 promoter sequences were detectable in all 11 enzyme-digested predelivery maternal plasma samples. Postpartum clearance was demonstrated. In 9 cases in which the fetal and maternal SERPINB5 genotypes were distinguishable, the placental-specific genotypes were detected in all predelivery maternal plasma samples. Conclusion: Detection of restriction enzyme-digested hypomethylated placental DNA molecules in maternal plasma by the use of a stem-loop primer represents a novel approach in fetal epigenetic marker detection. The analytical approach may also be generally applicable to the detection of restriction enzyme-digested nucleic acid fragments.
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Egelund, R., S. Jensen, K. W. Rodenburg et P. A. Andreasen. « Solvent Effects on Activity and Conformation of Plasminogen Activator Inhibitor-1 ». Thrombosis and Haemostasis 81, no 03 (1999) : 407–14. http://dx.doi.org/10.1055/s-0037-1614487.
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SummaryWe have studied effects of the solvent composition on the activity and the conformation of human plasminogen activator inhibitor-1 (PAI-1) from HT-1080 fibrosarcoma cells. Non-ionic detergents, including Triton X-100, reduced the inhibitory activity of PAI-1 more than 20-fold at 0° C, but less than 2-fold at 37° C, while glycerol partly prevented the detergent-induced activity-loss at 0° C. The activity-loss was associated with an increase in PAI-1 substrate behaviour. Evaluating the PAI-1 conformation by proteolytic susceptibility of specific peptide bonds, we found that the V8-proteinase susceptibility of the Glu332-Ser333 (P17-P16) bond, part of the hinge between the reactive centre loop (RCL) and β-strand 5A, and the endoproteinase Asp-N susceptibility of several bonds in the β-strand 2A-α-helix E region were increased by detergents at both 0 and 37° C. The susceptibility of the Gln321-Ala322 and the Lys325-Val326 bonds in β-strand 5A to papain and trypsin, respectively, was increased by detergents at 0° C, but not at 37° C, showing a strict correlation between proteinase susceptibility of β-strand 5A and activity-loss at 0° C. Since the β-strand 2A-α-helix E region also showed differential susceptibility to endoproteinase Asp-N in latent, active, and reactive centre-cleaved PAI-1, we propose that a detergent-induced conformational change of the β-strand 2A-α-helix E region influences the movements of β-sheet A, resulting in a cold-induced conformational change of β-strand 5A and thereby an increased substrate behaviour at low temperatures. These results provide new information about the structural basis for serpin substrate behaviour.
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Sim, R. B., et S. A. Tsiftsoglou. « Proteases of the complement system ». Biochemical Society Transactions 32, no 1 (1 février 2004) : 21–27. http://dx.doi.org/10.1042/bst0320021.
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The complement system is a group of about 35 soluble and cell-surface proteins which interact to recognize, opsonize and clear or kill invading micro-organisms or altered host cells (e.g. apoptotic or necrotic cells). Complement is a major part of the innate immune system. Recognition proteins such as C1q, MBL (mannan-binding lectin) and ficolins bind to targets via charge or sugar arrays. Binding causes activation of a series of serine protease proenzymes, such as C1r, C1s and MASP2 (MBL-associated serine protease 2), which in turn activate the atypical serine proteases factor B and C2, which then activate the major opsonin of the system, C3. Activated C3 binds covalently to targets, and is recognized by receptors on phagocytic cells. Two of the complement proteases, factors D and I, circulate not as proenzymes, but in activated form, and they have no natural inhibitors; their substrates are transient protein complexes (e.g. C3bB and C3bH) which form during complement activation. Factor B and C2 also have no natural inhibitor; they are active only when proteolytically cleaved and bound in an unstable, short-lived complex with C3b or C4b. C1r, C1s and the MASPs, in contrast, are regulated more conventionally by the natural serpin, C1-inhibitor. Complement proteases in general have very narrow specificity, and low substrate turnover with both natural and synthetic substrates. Excessive activation of complement is inflammatory, and causes tissue damage (e.g. in rheumatoid arthritis, or in ischaemia/reperfusion injury). Substances that regulate complement activation are likely to be useful in the regulation of inflammation. Complement activation might potentially be controlled at many different steps. Much attention has been focused on controlling the formation or activity of the protease complexes C3bBb and C4b2a (containing activated factor B and C2 respectively), as these generate the inflammatory peptides C3a and C5a.
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Fortenberry, Yolanda, et Jared Damare. « Inactivation of Plasminogen Activator Inhibitor-1 by RNA Aptamer Molecules ». Blood 120, no 21 (16 novembre 2012) : 1107. http://dx.doi.org/10.1182/blood.v120.21.1107.1107.
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Abstract Abstract 1107 Introduction: The serine protease inhibitor (serpin) plasminogen activator inhibitor-1 (PAI-1), binds and inhibits the following plasminogen activators: tissue-type plasminogen activator (tPA), and urokinase-type plasminogen activator (uPA). This decreases plasmin production and triggers the dissolution of fibrin clots. Elevated levels of PAI-1 have been correlated with an increased risk for cardiovascular disease, as well as obesity and metabolic syndrome. Consequently, pharmacologically suppressing PAI-1 might prevent, or successfully treat vascular disease. Several PAI-1 small molecule inhibitors have recently been studied (PAI-039 is the best characterized). Since PAI-1 is a multifunctional protein, completely inhibiting PAI-1 may hinder its other functions. Therefore, it is important to independently develop inhibitors to the various regions of PAI-1. This can be accomplished by using small RNA molecules (aptamers) that bind with high affinity and specificity to individual protein domains. We recently published a paper showing how PAI-1 specific RNA aptamers bind to the heparin/vitronectin binding site of PAI-1 (Blake et al., 2009). We demonstrated that PAI-1 specific aptamers prevent cancer cells from detaching from vitronectin (in the presence of PAI-1), resulting in increased cell adhesion. These aptamers had no effect on PAI-1's other functions, particularly its antiproteolytic activity. Objective: This study's goal was to develop RNA aptamers to the active site of PAI-1; thereby, preventing the ability of PAI-1 to interact with plasminogen activators (tPA and uPA). Methods: The aptamers were generated by the systematic evolution of ligands by exponential enrichment (SELEX). Adopting the SELEX in vitro selection technique ensures the creation of nuclease-resistant RNA molecules that will bind to target proteins. We used in vitroassays to determine the effect of the aptamers on the interaction of PAI-1 with both tPA and uPA. Results: We isolated a family of aptamers that bind to wild-type PAI-1 with affinities in the nanomolar range. From this family, two aptamer clones (10–2 and 10–4) exhibited reduced binding to elastase cleaved PAI-1 and the PAI-1/tPA complex. This suggests that they bind to, or in the vicinity of, the active site. Using a chromogenic assay, we showed that the aptamer clone 10–4, and (to a lesser extent) the aptamer clone 10–2, inhibited PAI-1's antiproteolytic activity against tPA, further suggesting that these clones bind to PAI-1 within its active site region. Interestingly, neither clone was able to prevent PAI-1 from inhibiting uPA activity. Both aptamer clones disrupted PAI-1's ability to form a stable covalent complex with tPA. Increasing aptamer concentrations positively correlated with an increase in cleaved PAI-1, suggesting that these aptamer clones convert PAI-1 from an inhibitor to a substrate. Furthermore, we showed that both aptamer clones are able to inhibit PAI-1's activity in the presence of vitronectin. Conclusions: We have shown that we are able to inhibit one of PAI-1's functions without hindering its other functions. To our knowledge, this is the first report of an RNA molecule that is able to inhibit the antiproteolytic activity of PAI-1. We have generated two specific RNA aptamer molecules that hinder the ability of PAI-1 to interact with tPA, which has the potential to be used as an antithrombotic agent. Disclosures: No relevant conflicts of interest to declare.
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Perkins, Stephen J., Kathryn F. Smith, Adam S. Nealis, Parvez I. Haris, Dennis Chapman, Christopher J. Bauer et Richard A. Harrison. « Secondary structure changes stabilize the reactive-centre cleaved form of SERPINs ». Journal of Molecular Biology 228, no 4 (décembre 1992) : 1235–54. http://dx.doi.org/10.1016/0022-2836(92)90329-i.
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Modha, J., et M. J. Doenhoff. « Schistosoma mansoni host–parasite relationship : interaction of contrapsin with adult worms ». Parasitology 109, no 4 (novembre 1994) : 487–95. http://dx.doi.org/10.1017/s0031182000080744.
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SUMMARYContrapsin, a serine protease inhibitor (serpin) present in mouse serum, was compared with that found in adult Schistosoma mansoni worm homogenates, which although immunologically identical to contrapsin in mouse serum, had a higher molecular weight in Western blotting. Immunolocalization studies demonstrated parasite-associated contrapsin on the surface and interstitial cells of adult male worms. After extraction of these parasites with Triton X-114, contrapsin was found in the aqueous phase of the detergent, suggesting it is unlikely to be an integral membrane protein. Treatment of adult worms with deoxycholate resulted in a change in the electrophoretic behaviour of worm-derived contrapsin. Parallel studies with trypsin suggested this was due to interaction of the serpin with a protease. Using porcine pancreatic trypsin as a model for a putative schistosome protease reacting with contrapsin, we have shown that trypsin, following complex formation with contrapsin, loses immunogenicity. Thus, when contrapsin–trypsin complexes were used as immunogen, the resulting antisera contained antibodies to contrapsin and contrapsin–trypsin complexes only, and none to native trypsin. Thus, epitopes characterizing native trypsin were presumably either masked following complex formation with contrapsin, or their processing and presentation to antigen presenting cells was suppressed, so that an antibody response was not mounted against them. These observations encourage speculation that S. mansoni may be elaborating an immune evasion strategy whereby immunologically sensitive proteases are first complexed with host serpins, which would render them immunogenically inert, and then cleared from the circulation by the host's reticulo-endothelial system. In this way the immune system would be unable to ‘see’ sensitive parasite proteases sufficiently to mount a response against the parasite.
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Karoopongse, Ekapun, Sabina Janciauskiene, Charles A. Dinarello, H. Joachim Deeg et A. Mario Q. Marcondes. « α1 Anti-Trypsin (AAT) Mitigates Hematopoietic Injury and Enhances Bone Marrow Recovery After Total Body Irradiation (TBI) ». Blood 120, no 21 (16 novembre 2012) : 4142. http://dx.doi.org/10.1182/blood.v120.21.4142.4142.
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Abstract Abstract 4142 Background: Injury to healthy (non-target) tissues still is a major limitation of radiation therapy, at least in part related to release of cytokines, including TNFα and IL-1β, which amplify tissue damage. Cytokine release is particularly prominent in patients who receive total body irradiation (TBI) before hematopoietic cell transplantation (HCT), as interactions of allogeneic donor cells with patient tissue contribute to the resulting “cytokine storm” and the development of graft versus-host-disease (GVHD). In murine models, administration of alpha-1 anti-trypsin (AAT) in the peri-transplant period decreased GVHD incidence and mortality. AAT, a member of the serine protease inhibitor (serpin) family, is a major protective protein in the circulation and has been used successfully in the clinic for other indications. Methods: We were interested in determining the potential benefits of AAT in preventing toxicity related to the transplant conditioning regimen, specifically TBI. AAT inhibits proteinase-3 (PR3) which, among other targets, cleaves IL-32 thereby leading to activation of TNFa and enhancing the cytokine storm. Since others have also suggested that AAT, via enhanced expression of heme oxygenase-1 (HMOX-1), leads to activation of Nrf2, thereby enhancing transcription of anti-inflammatory cytokines such as IL10 and the IL-1 receptor antagonist (IL1Ra), we determined the overall shift in the cytokine milieu and cell death. Thus, we irradiated male C3H/HeN mice (n = 5 mice per group; 6–8 weeks old) with sub-lethal doses (500, 600 and 700 cGy) of TBI from a 137Cs source. AAT (300 μg/animal) was administered intra-peritoneally 1 hour before and every 48 hours after TBI for a total of 6 doses. The effect of TBI/AAT on hematopoiesis was analyzed by growing granulocyte-macrophage colony forming units (GM-CFU) from bone marrow cells, on days 3, 7 and 14 after TBI. Results: Results were compared to those with cells from albumin-treated controls. Marrows (days 3 and 7) from AAT-treated mice generated higher GM-CFU counts than those from controls (mean = 25 vs. 5 colonies per 25 × 103 cells plated after 500cGy, and 15 vs. 3 colonies per 25 × 103 cells after 600 cGy). Peripheral blood and unsorted bone marrow from AAT-treated mice showed up-regulation of HMOX-1 and Nrf2 (30 and 50 log2 increase, respectively), and enhanced transcription of IL-10 and IL-1Ra (50 and 10 log2, respectively) compared to albumin treated donors. PR3 and TNFα, in contrast, were down-regulated (5 log2 and 7 log2 decrease in comparison to albumin treated controls). The cytokine mitigating effects of AAT were accompanied by attenuation of ATM-p53-dependent DNA damage responses in bone marrow cells (determined on days 3, 7, and 14), showing a 3-fold decrease in p53 protein levels, and a 6-fold decrease in phospho-ATM in comparison to albumin treated controls (peak day 7). In addition, protein levels of caspase 3 and caspase 9 were decreased (2-fold and 5-fold, respectively; peak on day 7) in unsorted marrow cells and spleen lysates of AAT treated mice in comparison to albumin treated controls. Bone marrow histopathology revealed normo-cellularity and a decrease in cleaved caspase 3 staining in AAT-treated mice compared to albumin treated animals. Summary and conclusions: AAT treatment significantly mitigated the hematopoietic toxicity induced by sub-lethal TBI. The mechanism involves cytokine suppression, associated with attenuation of ATM-p53 mediated DNA damage response. Taken together, these data suggest that AAT treatment pre-TBI provides protection against radiation injury. Disclosures: No relevant conflicts of interest to declare.
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Wright, H. Tonie, et Morris A. Blajcliman. « Proteolytically cleaved mutant antithrombin-Hamilton has high stability to denaturation characteristic of wild type inhibitor serpins ». FEBS Letters 348, no 1 (4 juillet 1994) : 14–16. http://dx.doi.org/10.1016/0014-5793(94)00568-0.
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Baumann, U., R. Huber, W. Bode, D. Grosse, M. Lesjak et C. B. Laurell. « Crystal structure of cleaved human α1-antichymotrypsin at 2.7 å resolution and its comparison with other serpins ». Journal of Molecular Biology 218, no 3 (avril 1991) : 595–606. http://dx.doi.org/10.1016/0022-2836(91)90704-a.
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Hernández-Espinosa, David, Adriana Ordóñez, Vicente Vicente et Javier Corral. « Factors with conformational effects on haemostatic serpins : Implications in thrombosis ». Thrombosis and Haemostasis 98, no 09 (2007) : 557–63. http://dx.doi.org/10.1160/th07-02-0152.
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SummarySerpins are key actors of systems involving proteolytic reactions, such as the haemostatic system, as they are irreversible suicide inhibitors of serine proteases. The structural flexibility and physical properties of serpins that are required for their efficient inhibitory mechanism also make them especially vulnerable to even minor factors that induce conformational changes in the native form of these molecules, leading to a number of inactive conformations, such as latent, cleaved or polymers. Increasing numbers of conformational mutations affecting haemostatic serpins, mainly antithrombin, the main endogenous anticoagulant, have been described. These mutations cause circulating deficiencies of the molecules, in most cases due to intracellular retention, which may be associated with a hyper-coagulable state. Indeed, conformational mutations in antithrombin have been identified in patients with severe venous thrombosis,which has led to the hypothesis that these disorders might be included in the group of conformational diseases. Moreover,we have recently demonstrated that other factors,including both drugs,such as the treatment with L-asparaginase,or environmental factors, such as high temperatures or hyperlipidemia, may also have conformational consequences on hepatic antithrombin,thus resulting in intracellular aggregation and plasma deficiency, which may increase the risk of thrombosis. In this study,we review the causes of deficiency of haemostatic serpins that may be explained by conformational mechanisms, and their association with an increased risk of venous thrombosis.
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ANNAND, Robert R., Jeffrey R. DAHLEN, Cindy A. SPRECHER, Piet DE DREU, Donald C. FOSTER, John A. MANKOVICH, Robert V. TALANIAN, Walter KISIEL et David A. GIEGEL. « Caspase-1 (interleukin-1β-converting enzyme) is inhibited by the human serpin analogue proteinase inhibitor 9 ». Biochemical Journal 342, no 3 (5 septembre 1999) : 655–65. http://dx.doi.org/10.1042/bj3420655.
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The regulation of caspases, cysteine proteinases that cleave their substrates after aspartic residues, is poorly understood, even though they are involved in tightly regulated cellular processes. The recently discovered serpin analogue proteinase inhibitor 9 (PI9) is unique among human serpin analogues in that it has an acidic residue in the putative specificity-determining position of the reactive-site loop. We measured the ability of PI9 to inhibit the amidolytic activity of several caspases. The hydrolysis of peptide substrates by caspase-1 (interleukin-1β-converting enzyme), caspase-4 and caspase-8 is inhibited by PI9 in a time-dependent manner. The rate of reaction of caspase-1 with PI9, as well as the rate of substrate hydrolysis of the initial caspase-PI9 complex, shows a hyperbolic dependence on the concentration of PI9, indicative of a two-step kinetic mechanism for inhibition with an apparent second-order rate constant of 7×102 M-1˙s-1. The hydrolysis of a tetrapeptide substrate by caspase-3 is not inhibited by PI9. The complexes of caspase-1 and caspase-4 with PI9 can be immunoprecipitated but no complex with caspase-3 can be detected. No complex can be immunoprecipitated if the active site of the caspase is blocked with a covalent inhibitor. These results show that PI9 is an inhibitor of caspase-1 and to a smaller extent caspase-4 and caspase-8, but not of the more distantly related caspase-3. PI9 is the first example of a human serpin analogue that inhibits members of this class of cysteine proteinases.
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Rustgi, Sachin, Edouard Boex-Fontvieille, Christiane Reinbothe, Diter von Wettstein et Steffen Reinbothe. « Serpin1 and WSCP differentially regulate the activity of the cysteine protease RD21 during plant development in Arabidopsis thaliana ». Proceedings of the National Academy of Sciences 114, no 9 (8 février 2017) : 2212–17. http://dx.doi.org/10.1073/pnas.1621496114.
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Proteolytic enzymes (proteases) participate in a vast range of physiological processes, ranging from nutrient digestion to blood coagulation, thrombosis, and beyond. In plants, proteases are implicated in host recognition and pathogen infection, induced defense (immunity), and the deterrence of insect pests. Because proteases irreversibly cleave peptide bonds of protein substrates, their activity must be tightly controlled in time and space. Here, we report an example of how nature evolved alternative mechanisms to fine-tune the activity of a cysteine protease dubbed RD21 (RESPONSIVE TO DESICCATION-21). One mechanism in the model plant Arabidopsis thaliana studied here comprises irreversible inhibition of RD21’s activity by Serpin1, whereas the other mechanism is a result of the reversible inhibition of RD21 activity by a Kunitz protease inhibitor named water-soluble chlorophyll-binding protein (WSCP). Activity profiling, complex isolation, and homology modeling data revealed unique interactions of RD21 with Serpin1 and WSCP, respectively. Expression studies identified only partial overlaps in Serpin1 and WSCP accumulation that explain how RD21 contributes to the innate immunity of mature plants and arthropod deterrence of seedlings undergoing skotomorphogenesis and greening.