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1
Mitchell, Angela M., and R. Jude Samulski. "Mechanistic Insights into the Enhancement of Adeno-Associated Virus Transduction by Proteasome Inhibitors." Journal of Virology 87, no. 23 (September 11, 2013): 13035–41. http://dx.doi.org/10.1128/jvi.01826-13.
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Proteasome inhibitors (e.g., bortezomib, MG132) are known to enhance adeno-associated virus (AAV) transduction; however, whether this results from pleotropic proteasome inhibition or off-target serine and/or cysteine protease inhibition remains unresolved. Here, we examined recombinant AAV (rAAV) effects of a new proteasome inhibitor, carfilzomib, which specifically inhibits chymotrypsin-like proteasome activity and no other proteases. We determined that proteasome inhibitors act on rAAV through proteasome inhibition and not serine or cysteine protease inhibition, likely through positive changes late in transduction.
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Avanzo, Petra, Jerica Sabotič, Sabina Anžlovar, Tatjana Popovič, Adrijana Leonardi, Roger H. Pain, Janko Kos, and Jože Brzin. "Trypsin-specific inhibitors from the basidiomycete Clitocybe nebularis with regulatory and defensive functions." Microbiology 155, no. 12 (December 1, 2009): 3971–81. http://dx.doi.org/10.1099/mic.0.032805-0.
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We have isolated serine protease inhibitors from the basidiomycete Clitocybe nebularis, CnSPIs, using trypsin affinity chromatography. Full-length gene and cDNA sequences were determined for one of them, named cnispin, and the recombinant protein was expressed in Escherichia coli at high yield. The primary structure and biochemical properties of cnispin are very similar to those of the Lentinus edodes serine protease inhibitor, until now the only member of the I66 family of protease inhibitors in the MEROPS classification. Cnispin is highly specific towards trypsin, with K i in the nanomolar range. It also exhibited weaker inhibition of chymotrypsin and very weak inhibition of subtilisin and kallikrein; other proteases were not inhibited. Inhibitory activity against endogenous proteases from C. nebularis revealed a possible regulatory role for CnSPIs in the endogenous proteolytic system. Another possible biological function in defence against predatory insects was indicated by the deleterious effect of CnSPIs on the development of larvae of Drosophila melanogaster. These findings, together with the biochemical and genetic characterization of cnispin, suggest a dual physiological role for this serine protease inhibitor of the I66 MEROPS family.
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Hudig, D., N. J. Allison, T. M. Pickett, U. Winkler, C. M. Kam, and J. C. Powers. "The function of lymphocyte proteases. Inhibition and restoration of granule-mediated lysis with isocoumarin serine protease inhibitors." Journal of Immunology 147, no. 4 (August 15, 1991): 1360–68. http://dx.doi.org/10.4049/jimmunol.147.4.1360.
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Abstract To kill other cells, lymphocytes can exocytose granules that contain serine proteases and pore-forming proteins (perforins). We report that mechanism-based isocoumarin inhibitors inhibited the proteases and inactivated lysis. When inhibited proteases were restored, lysis was also restored, indicating that the proteases were essential for lysis. We found three new lymphocyte protease activities, "Asp-ase,"Met-ase," and "Ser-ase," which in addition to ly-tryptase and ly-chymase, comprise five different protease activities in rat RNK-16 granules. The general serine protease inhibitor 3,4-dichloroisocoumarin (DCI) inhibited all five protease activities. Essentially all protease molecules were inactivated by DCI before lysis was reduced, as determined from DCI's second order inhibition rate constants for the proteases, the DCI concentrations, and the times of pretreatment needed to block lysis. The pH favoring DCI inhibition of lysis was the pH optimum for protease activity. Isocoumarin reagents acylate, and may sometimes secondarily alkylate, serine protease active sites. Granule proteases, inhibited by DCI acylation, were deacylated with hydroxylamine, restoring both the protease and lytic activities. Hydroxylamine does not restore alkylated proteases and did not restore the lytic activities after inhibition with 4-chloro-7-guanidino-3-(2-phenylethoxy)-isocoumarin, a more alkylating mechanism-based inhibitor designed to react with tryptases. It is improbable that isocoumarin reagents directly inactivated pore-forming proteins because 1) these reagents require protease activation, 2) their nonspecific effects are alkylating, and 3) alkylated proteins are not restored by hydroxylamine. We conclude that serine proteases participate in lysis when lysis is mediated by the complete assembly of granule proteins.
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Azouz, Nurit P., Andrea Klingler, Victoria Callahan, Ivan Akhrymuk, Katarina Elez, Lluís Raich, Brandon Henry, et al. "Alpha 1 Antitrypsin is an Inhibitor of the SARS-CoV-2–Priming Protease TMPRSS2." Pathogens and Immunity 6, no. 1 (April 26, 2021): 55–74. http://dx.doi.org/10.20411/pai.v6i1.408.
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Background: Host proteases have been suggested to be crucial for dissemination of MERS, SARS-CoV, and SARS-CoV-2 coronaviruses, but the relative contribution of membrane versus intracellular proteases remains controversial. Transmembrane serine protease 2 (TMPRSS2) is regarded as one of the main proteases implicated in the coronavirus S protein priming, an important step for binding of the S protein to the angiotensin-converting enzyme 2 (ACE2) receptor before cell entry. Methods: We developed a cell-based assay to identify TMPRSS2 inhibitors. Inhibitory activity was established in SARS-CoV-2 viral load systems. Results: We identified the human extracellular serine protease inhibitor (serpin) alpha 1 antitrypsin (A1AT) as a novel TMPRSS2 inhibitor. Structural modeling revealed that A1AT docked to an extracellular domain of TMPRSS2 in a conformation that is suitable for catalysis, resembling similar serine protease inhibitor complexes. Inhibitory activity of A1AT was established in a SARS-CoV-2 viral load system. Notably, plasma A1AT levels were associated with COVID-19 disease severity. Conclusions: Our data support the key role of extracellular serine proteases in SARS CoV-2 infections and indicate that treatment with serpins, particularly the FDA-approved drug A1AT, may be effective in limiting SARS-CoV-2 dissemination by affecting the surface of the host cells.
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Woodard, S. L., D. S. Jackson, A. S. Abuelyaman, J. C. Powers, U. Winkler, and D. Hudig. "Chymase-directed serine protease inhibitor that reacts with a single 30-kDa granzyme and blocks NK-mediated cytotoxicity." Journal of Immunology 153, no. 11 (December 1, 1994): 5016–25. http://dx.doi.org/10.4049/jimmunol.153.11.5016.
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Abstract Cytotoxic NK and T lymphocytes kill virally infected cells within minutes without causing damage to themselves or bystander cells. One mechanism of killing involves exocytosis of granules containing serine proteases and perforin. Serine protease inhibitors block killing of target cells mediated by the cytotoxic lymphocytes. There are at least five different serine protease activities in cytolytic granules. Ten different serine protease sequences have been identified with the use of cDNA-specific clones. It is not known whether only one or several of these serine proteases are essential for cytolytic activity. In this study we show that an irreversible serine protease inhibitor, biotinyl-Aca-Aca-Phe-Leu-PheP(OPh)2, selectively inhibits a chymotrypsin-like (chymase) serine protease activity of rat RNK-16 granule extracts. Under the same conditions, only one 30-kDa (reduced) band was detected on protein blots. Furthermore, only one of three chymase peaks separated by hydrophobic interaction chromatography was inhibited. When this granzyme was inhibited, granule-mediated lysis of erythrocytes was diminished. NK cell killing was completely blocked when biotinyl-Aca-Aca-Phe-Leu-PheP(OPh)2 was added to cytotoxicity assays at 200 microM with rat splenocytes as effectors. By confocal fluorescence microscopy, we show that this inhibitor localizes to distinct regions within RNK-16 cells and rat NK cells. Inhibitor treatment of intact cells inactivated the chymase activity and reduced lysis found in their dense organelles. Together these data indicate that biotinyl-Aca-Aca-Phe-Leu-PheP(OPh)2 inhibits a granule chymase that is essential to cytolytic activity of NK cells.
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Brüning, Mareke, Martina Lummer, Caterina Bentele, Marcel M. W. Smolenaars, Kees W. Rodenburg, and Hermann Ragg. "The Spn4 gene from Drosophila melanogaster is a multipurpose defence tool directed against proteases from three different peptidase families." Biochemical Journal 401, no. 1 (December 11, 2006): 325–31. http://dx.doi.org/10.1042/bj20060648.
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By alternative use of four RSL (reactive site loop) coding exon cassettes, the serpin (serine protease inhibitor) gene Spn4 from Drosophila melanogaster was proposed to enable the synthesis of multiple protease inhibitor isoforms, one of which has been shown to be a potent inhibitor of human furin. Here, we have investigated the inhibitory spectrum of all Spn4 RSL variants. The analyses indicate that the Spn4 gene encodes inhibitors that may inhibit serine proteases of the subtilase family (S8), the chymotrypsin family (S1), and the papain-like cysteine protease family (C1), most of them at high rates. Thus a cohort of different protease inhibitors is generated simply by grafting enzyme-adapted RSL sequences on to a single serpin scaffold, even though the target proteases contain different types and/or a varying order of catalytic residues and are descendents of different phylogenetic lineages. Since all of the Spn4 RSL isoforms are produced as intracellular residents and additionally as variants destined for export or associated with the secretory pathway, the Spn4 gene represents a versatile defence tool kit that may provide multiple antiproteolytic functions.
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Hong, Tran Thi, Ton That Huu Dat, Nguyen Phuong Hoa, Tran Thi Kim Dung, Vu Thi Thu Huyen, Le Minh Bui, Nguyen Thi Kim Cuc, and Pham Viet Cuong. "Expression and characterization of a new serine protease inhibitory protein in Escherichia coli." Biomedical Research and Therapy 7, no. 2 (February 29, 2020): 3633–44. http://dx.doi.org/10.15419/bmrat.v7i2.590.
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Introduction: Proteases are enzymes that catalyze the hydrolysis of peptide bonds and play an important role in almost all biological processes. However, excessive protein proteolysis can be implicated in several diseases, such as cancer, as well as cardiovascular, inflammatory, neurodegenerative, bacterial, viral and parasitic diseases. In these cases, protease inhibitors can be used as one of versatile tools for regulating proteolytic activity of target proteases as well as therapeutic applications. In this study, we expressed and characterized a new serine protease inhibitory protein (PI-QT) from the metagenome of sponge-associated microorganisms in Escherichia coli.
Methods: The gene PI-QT encoding for a new serine protease inhibitory protein was expressed in E. coli BL21(DE3). In addition, the expressed protein was purified and characterized.
Results: Optimization of expression of the recombinant protein PI-QT in E. coli showed that suitable conditions for expression of the protein were pre-induction cell density (OD600) of 0.6 - 0.7, IPTG concentration of 1 mM and temperature of 25oC. The protease inhibitory protein was also purified and identified by mass spectrometry LC-MS/MS. The recombinant protein showed inhibitory activity against trypsin anda-chymotrypsin with activity values of 97526 U/mg and 41714 U/mg, respectively. Maximum activity of the protease inhibitory protein was obtained at pH 7 and temperature 20-35oC. The inhibitor was stable over pH 4-9 and up to temperature 50oC. Addition of Zn2+, Mg2+ and Ca2+ enhanced inhibitory activity, whereas other metal ions, surfactants and oxidants reduced inhibitory activity of the protease inhibitor.
Conclusion: The recombinant protein PI-QT is a potential protease inhibitor for therapeutic applications.
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Cornwall, Gail A., Angus Cameron, Iris Lindberg, Daniel M. Hardy, Nathaly Cormier, and Nelson Hsia. "The Cystatin-Related Epididymal Spermatogenic Protein Inhibits the Serine Protease Prohormone Convertase 2." Endocrinology 144, no. 3 (March 1, 2003): 901–8. http://dx.doi.org/10.1210/en.2002-220997.
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The cystatin-related epididymal spermatogenic (CRES) protein is related to the family 2 cystatins of the cystatin superfamily of cysteine protease inhibitors. However, CRES lacks sequences important for cysteine protease inhibitory activity and is specifically expressed in reproductive and neuroendocrine tissues. Thus, CRES is distinct from cystatins and may perform unique tissue-specific functions. The purpose of the present study was to determine whether CRES functions as a protease inhibitor in in vitro assays. In contrast to mouse recombinant cystatin C, recombinant CRES did not inhibit the cysteine proteases papain and cathepsin B, suggesting that it probably does not function as a typical cystatin. CRES, however, inhibited the serine protease prohormone convertase 2 (PC2), a protease involved in prohormone processing in the neuroendocrine system, whereas cystatin C showed no inhibition. CRES did not inhibit subtilisin, trypsin, or the convertase family members, PC1 and furin, indicating that it selectively inhibits PC2. Kinetic analysis showed that CRES is a competitive inhibitor of PC2 with a Ki of 25 nm. The removal of N-terminal sequences from CRES decreased its affinity for PC2, suggesting that the N terminus may be important for CRES to function as an inhibitor. These studies suggest that CRES is a cross-class inhibitor that may regulate proprotein processing within the reproductive and neuroendocrine systems.
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Yoo Im, Sonia, Camila Ramalho Bonturi, Adriana Miti Nakahata, Clóvis Ryuichi Nakaie, Arnildo Pott, Vali Joana Pott, and Maria Luiza Vilela Oliva. "Differences in the Inhibitory Specificity Distinguish the Efficacy of Plant Protease Inhibitors on Mouse Fibrosarcoma." Plants 10, no. 3 (March 23, 2021): 602. http://dx.doi.org/10.3390/plants10030602.
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Metastasis, the primary cause of death from malignant tumors, is facilitated by multiple protease-mediated processes. Thus, effort has been invested in the development of protease inhibitors to prevent metastasis. Here, we investigated the effects of protease inhibitors including the recombinant inhibitors rBbKI (serine protease inhibitor) and rBbCI (serine and cysteine inhibitor) derived from native inhibitors identified in Bauhinia bauhinioides seeds, and EcTI (serine and metalloprotease inhibitor) isolated from the seeds of Enterolobium contortisiliquum on the mouse fibrosarcoma model (lineage L929). rBbKI inhibited 80% of cell viability of L929 cells after 48 h, while EcTI showed similar efficacy after 72 h. Both inhibitors acted in a dose and time-dependent manner. Conversely, rBbCI did not significantly affect the viability of L929 cells. Confocal microscopy revealed the binding of rBbKI and EcTI to the L929 cell surface. rBbKI inhibited approximately 63% of L929 adhesion to fibronectin, in contrast with EcTI and rBbCI, which did not significantly interfere with adhesion. None of the inhibitors interfered with the L929 cell cycle phases. The synthetic peptide RPGLPVRFESPL-NH2, based on the BbKI reactive site, inhibited 45% of the cellular viability of L929, becoming a promising protease inhibitor due to its ease of synthesis.
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Ó Cuív, Páraic, Rajesh Gupta, Hareshwar P. Goswami, and Mark Morrison. "Extending the Cellulosome Paradigm: the Modular Clostridium thermocellum Cellulosomal Serpin PinA Is a Broad-Spectrum Inhibitor of Subtilisin-Like Proteases." Applied and Environmental Microbiology 79, no. 19 (July 19, 2013): 6173–75. http://dx.doi.org/10.1128/aem.01912-13.
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ABSTRACTClostridium thermocellumencodes a cellulosomal, modular, and thermostable serine protease inhibitor (serpin), PinA. PinA stability but not inhibitory activity is affected by the Fn(III) and Doc(I) domains, and PinA is a broad inhibitor of subtilisin-like proteases and may play a key role in protecting the cellulosome from protease attack.
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Masler, Edward P. "Characterisation of the effects on proteases of Heterodera glycines and Meloidogyne incognita second-stage juveniles by inhibitors obtained from cysts of H. glycines." Nematology 20, no. 5 (2018): 461–70. http://dx.doi.org/10.1163/15685411-00003151.
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Summary The protease inhibitor component of Heterodera glycines cyst contents was explored using a battery of peptide substrates and H. glycines and Meloidogyne incognita second-stage juveniles as enzyme sources. Protease inhibitors were prepared by heat-denaturing H. glycines cyst-egg extract (hHglCE), which was used in all inhibition exploration. Eight substrates targeting four endoprotease groups (aspartic, cysteine, metallo- and serine proteases) revealed that protease inhibition by hHglCE varied significantly between H. glycines and M. incognita with seven of the eight substrates. Only cysteine protease activity was inhibited equally between H. glycines and M. incognita. Aspartic protease activity was inhibited more strongly in H. glycines and serine protease activity was inhibited more strongly in M. incognita. Digestion of five matrix metalloprotease (MMP) substrates was inhibited more strongly in H. glycines (two substrates) and M. incognita (three substrates). These variations were particularly intriguing given the potential association of MMP proteases with developing embryos. Inhibition of digestion of nematode FMRFamide-like peptides (FLPs) showed less variation between nematode species than the targeted substrates, but inhibition did vary significantly across substrates within each species. Digestion of FLP-6 was the least affected by hHglCE but was inhibited significantly more in M. incognita than in H. glycines. Residue differences between two FLP-14 sequences significantly affected inhibition of FLP-14 digestion in both H. glycines and M. incognita. RP-HPLC fractionation of hHglCE clearly demonstrated the presence of high (Fr No.5) and low (Fr No.14) polarity inhibitor components. Potency of inhibition of M. incognita serine protease activity, based upon IC50 values (1.68 and 2.78 hHglCEeq reaction−1 for Fr No.5 and Fr No.14, respectively), was reduced significantly from unfractionated hHglCE (IC50 = 0.61), suggesting inhibitor dilution, loss of component synergy, or both, due to fractionation.
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Chen, Xingchen, Darren Leahy, Jessica Van Haeften, Perry Hartfield, Peter J. Prentis, Chloé A. van der Burg, Joachim M. Surm, et al. "A Versatile and Robust Serine Protease Inhibitor Scaffold from Actinia tenebrosa." Marine Drugs 17, no. 12 (December 12, 2019): 701. http://dx.doi.org/10.3390/md17120701.
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Serine proteases play pivotal roles in normal physiology and a spectrum of patho-physiological processes. Accordingly, there is considerable interest in the discovery and design of potent serine protease inhibitors for therapeutic applications. This led to concerted efforts to discover versatile and robust molecular scaffolds for inhibitor design. This investigation is a bioprospecting study that aims to isolate and identify protease inhibitors from the cnidarian Actinia tenebrosa. The study isolated two Kunitz-type protease inhibitors with very similar sequences but quite divergent inhibitory potencies when assayed against bovine trypsin, chymostrypsin, and a selection of human sequence-related peptidases. Homology modeling and molecular dynamics simulations of these inhibitors in complex with their targets were carried out and, collectively, these methodologies enabled the definition of a versatile scaffold for inhibitor design. Thermal denaturation studies showed that the inhibitors were remarkably robust. To gain a fine-grained map of the residues responsible for this stability, we conducted in silico alanine scanning and quantified individual residue contributions to the inhibitor’s stability. Sequences of these inhibitors were then used to search for Kunitz homologs in an A. tenebrosa transcriptome library, resulting in the discovery of a further 14 related sequences. Consensus analysis of these variants identified a rich molecular diversity of Kunitz domains and expanded the palette of potential residue substitutions for rational inhibitor design using this domain.
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Higuchi, M., S. Singh, H. Chan, and BB Aggarwal. "Protease inhibitors differentially regulate tumor necrosis factor- induced apoptosis, nuclear factor-kappa B activation, cytotoxicity, and differentiation." Blood 86, no. 6 (September 15, 1995): 2248–56. http://dx.doi.org/10.1182/blood.v86.6.2248.bloodjournal8662248.
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We investigated the effect of various protease inhibitors on several tumor necrosis factor (TNF)-mediated cellular responses. Treatment of a human myelogenous leukemia cell line, ML-1a, with TNF in the presence of cycloheximide triggers endonucleolytic activity and apoptotic cell death within 90 minutes. The general serine protease inhibitor diisopropyl fluorophosphate (DFP) and the chymotrypsin-like protease inhibitor N-tosyl-L-lysyl chloromethyl ketone (TPCK) completely abrogated TNF-induced DNA fragmentation and the formation of apoptotic bodies. However, 13 other protease inhibitors, including serine protease inhibitors, did not. The addition of TPCK to cells 30 minutes after TNF treatment completely inhibited the cytokine action, indicating that TPCK-sensitive proteases are not involved in the early stages of signal transduction. TNF is cytotoxic and induces differentiation in ML-1a cells after a 3-day incubation. TPCK had no effect on the TNF-induced cytotoxicity and differentiation, indicating that TPCK-sensitive proteases are specific for DNA fragmentation. TPCK also blocked TNF-induced activation of nuclear factor (NF)-kappa B. The dose-response and the time-course of the inhibitor, however, indicated that the site of action of TPCK for NF-kappa B activation and for DNA fragmentation are quite distinct. Therefore, we conclude that TNF activates two distinct TPCK-sensitive pathways, one leading to apoptosis and the other to NF-kappa B activation.
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Kuznetsova, S. S., E. F. Kolesanova, A. V. Talanova, and A. V. Veselovsky. "Prospects for the design of new therapeutically significant protease inhibitors based on knottins and sunflower seed trypsin inhibitor (SFTI 1)." Biomeditsinskaya Khimiya 62, no. 4 (2016): 353–68. http://dx.doi.org/10.18097/pbmc20166204353.
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Plant seed knottins, mainly from the Cucurbitacea family, and sunflower seed trypsin inhibitor (SFTI 1) are the most low-molecular canonical peptide inhibitors of serine proteases. High efficiency of inhibition of various serine proteases, structure rigidity together with the possibility of limited variations of amino acid sequences, high chemical stability, lack of toxic properties, opportunity of production by either chemical synthesis or use of heterologous expression systems make these inhibitors attractive templates for design of new compounds for regulation of therapeutically significant serine protease activities. Hence the design of such compounds represents a prospective research field. The review considers structural characteristics of these inhibitors, their properties, methods of preparation and design of new analogs. Examples of successful employment of natural serine protease inhibitors belonging to knottin family and SFTI 1 as templates for the design of highly specific inhibitors of certain proteases are given.
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Otlewski, J., M. Jaskólski, O. Buczek, T. Cierpicki, H. Czapińska, D. Krowarsch, A. O. Smalas, D. Stachowiak, A. Szpineta, and M. Dadlez. "Structure-function relationship of serine protease-protein inhibitor interaction." Acta Biochimica Polonica 48, no. 2 (June 30, 2001): 419–28. http://dx.doi.org/10.18388/abp.2001_3926.
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We report our progress in understanding the structure-function relationship of the interaction between protein inhibitors and several serine proteases. Recently, we have determined high resolution solution structures of two inhibitors Apis mellifera chymotrypsin inhibitor-1 (AMCI-I) and Linum usitatissimum trypsin inhibitor (LUTI) in the free state and an ultra high resolution X-ray structure of BPTI. All three inhibitors, despite totally different scaffolds, contain a solvent exposed loop of similar conformation which is highly complementary to the enzyme active site. Isothermal calo- rimetry data show that the interaction between wild type BPTI and chymotrypsin is entropy driven and that the enthalpy component opposes complex formation. Our research is focused on extensive mutagenesis of the four positions from the protease binding loop of BPTI: P1, P1', P3, and P4. We mutated these residues to different amino acids and the variants were characterized by determination of the association constants, stability parameters and crystal structures of protease-inhibitor complexes. Accommodation of the P1 residue in the S1 pocket of four proteases: chymotrypsin, trypsin, neutrophil elastase and cathepsin G was probed with 18 P1 variants. High resolution X-ray structures of ten complexes between bovine trypsin and P1 variants of BPTI have been determined and compared with the cognate P1 Lys side chain. Mutations of the wild type Ala16 (P1') to larger side chains always caused a drop of the association constant. According to the crystal structure of the Leu16 BPTI-trypsin complex, introduction of the larger residue at the P1' position leads to steric conflicts in the vicinity of the mutation. Finally, mutations at the P4 site allowed an improvement of the association with several serine proteases involved in blood clotting. Conversely, introduction of Ser, Val, and Phe in place of Gly12 (P4) had invariably a destabilizing effect on the complex with these proteases.
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Panda, Subhamay, and Iman Ehsan. "MOLECULAR DOCKING STUDIES OF SNAKE VENOM SERINE PROTEASE OF SHARP-NOSED PIT VIPER WITH HESPERETIN." Asian Journal of Pharmaceutical and Clinical Research 11, no. 6 (June 7, 2018): 457. http://dx.doi.org/10.22159/ajpcr.2018.v11i6.25531.
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Objective: The management of snake bite envenomation is a global challenge affecting millions of people. Immunotherapy is still regarded as the treatment of choice; however, their subsequent adverse effects restrict their potential use for therapy against snake venom poisoning. In recent years, more attention has been given to the exploration of indigenous medicinal plants for their outstanding benefits for the treatment of several diseases and disorders, including snake venom poisoning. Hesperetin is a naturally occurring compound derived from a flavanone glycoside, hesperidin and is obtained from various citrus fruits. It is known to possess significant inhibitory activity against snake venom serine proteases. The aim of our present study was to investigate the significant inhibitory action of hesperetin on thrombin-like serine protease from sharp-nosed pit viper (Deinagkistrodon acutus) snake venom.Methods: We have employed molecular docking analysis by implementing the state-of-the-art docking program to validate the hypothesis of the prospective inhibitory properties of hesperetin on thrombin-like serine proteases of snake venom. AutoDock 4.2, InterProSurf, MGLTools, and MTiAutoDock were utilized for the molecular docking analysis of thrombin-like serine protease obtained from the snake venom of sharp-nosed pit viper with the natural compound hesperetin.Results: The results generated from in silico based approach reveals the significant inhibitory role of hesperetin against thrombin-like snake venom proteases, which might lead to the drug designing of the inhibitors of snake venom serine proteases.Conclusion: The implementation of molecular docking analysis by the employment of state-of-the-art docking program supports the potential of inhibitory activity of naturally obtained hesperetin compound on thrombin-like serine proteases of snake venom. The generated in silico results suggests that the novel structure hesperetin - flavanone might act as a potent inhibitor of thrombin-like snake venom proteases, and unlocks the possibilities for designing drugs of the inhibitors of snake venom serine proteases. Moreover, the investigation of the novel compound obtained from natural sources for their inhibitory activity against snake venom serine proteases would lead to the discovery of newer inhibitory compound from a highly uninvestigated research arena.
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Taggart, Clifford, Marcus A. Mall, Gilles Lalmanach, Didier Cataldo, Andreas Ludwig, Sabina Janciauskiene, Nicole Heath, et al. "Protean proteases: at the cutting edge of lung diseases." European Respiratory Journal 49, no. 2 (February 2017): 1501200. http://dx.doi.org/10.1183/13993003.01200-2015.
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Proteases were traditionally viewed as mere protein-degrading enzymes with a very restricted spectrum of substrates. A major expansion in protease research has uncovered a variety of novel substrates, and it is now evident that proteases are critical pleiotropic actors orchestrating pathophysiological processes. Recent findings evidenced that the net proteolytic activity also relies upon interconnections between different protease and protease inhibitor families in the protease web.In this review, we provide an overview of these novel concepts with a particular focus on pulmonary pathophysiology. We describe the emerging roles of several protease families including cysteine and serine proteases.The complexity of the protease web is exemplified in the light of multidimensional regulation of serine protease activity by matrix metalloproteases through cognate serine protease inhibitor processing. Finally, we will highlight how deregulated protease activity during pulmonary pathogenesis may be exploited for diagnosis/prognosis purposes, and utilised as a therapeutic tool using nanotechnologies.Considering proteases as part of an integrative biology perspective may pave the way for the development of new therapeutic targets to treat pulmonary diseases related to intrinsic protease deregulation.
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OFOSU, Frederick A., John FREEDMAN, Lori DEWAR, Yinqi SONG, and John W. FENTON. "A trypsin-like platelet protease propagates protease-activated receptor-1 cleavage and platelet activation." Biochemical Journal 336, no. 2 (December 1, 1998): 283–85. http://dx.doi.org/10.1042/bj3360283.
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Protease-activated receptor-1 (PAR-1) is a G-protein-linked receptor on platelets and perivascular cells activated by α-thrombin and the PAR-1-activating peptide, SFLLRN. α-Thrombin activates PAR-1 by cleaving it at R41–S42 to release the 41-residue peptide TR(1–41). Unexpectedly, platelet activation with SFLLRN was also associated with PAR-1 cleavage and the release of TR(1–41). Both PAR-1 cleavage and platelet activation resulting from SFLLRN addition to platelets were markedly inhibited by the serine protease inhibitor 4,2-(aminoethyl)-benzene sulphonylfluoride·HCl (pefabloc SC) and soybean trypsin inhibitor, but not by inhibitors of calpain, cysteine proteases or metalloproteases. Thus, a trypsin-like platelet protease propagates SFLLRN-dependent PAR-1 cleavage and platelet activation.
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Conners, Rebecca, Alexander V. Konarev, Jane Forsyth, Alison Lovegrove, Justin Marsh, Timothy Joseph-Horne, Peter Shewry, and R. Leo Brady. "An Unusual Helix-Turn-Helix Protease Inhibitory Motif in a Novel Trypsin Inhibitor from Seeds of Veronica (Veronica hederifolia L.)." Journal of Biological Chemistry 282, no. 38 (July 19, 2007): 27760–68. http://dx.doi.org/10.1074/jbc.m703871200.
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The storage tissues of many plants contain protease inhibitors that are believed to play an important role in defending the plant from invasion by pests and pathogens. These proteinaceous inhibitor molecules belong to a number of structurally distinct families. We describe here the isolation, purification, initial inhibitory properties, and three-dimensional structure of a novel trypsin inhibitor from seeds of Veronica hederifolia (VhTI). The VhTI peptide inhibits trypsin with a submicromolar apparent Ki and is expected to be specific for trypsin-like serine proteases. VhTI differs dramatically in structure from all previously described families of trypsin inhibitors, consisting of a helix-turn-helix motif, with the two α helices tightly associated by two disulfide bonds. Unusually, the crystallized complex is in the form of a stabilized acyl-enzyme intermediate with the scissile bond of the VhTI inhibitor cleaved and the resulting N-terminal portion of the inhibitor remaining attached to the trypsin catalytic serine 195 by an ester bond. A synthetic, truncated version of the VhTI peptide has also been produced and co-crystallized with trypsin but, surprisingly, is seen to be uncleaved and consequently forms a noncovalent complex with trypsin. The VhTI peptide shows that effective enzyme inhibitors can be constructed from simple helical motifs and provides a new scaffold on which to base the design of novel serine protease inhibitors.
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Shalamanova, Liliana, Bernd Kübler, Jens-Gerd Scharf, and Thomas Braulke. "MDCK cells secrete neutral proteases cleaving insulin-like growth factor-binding protein-2 to -6." American Journal of Physiology-Endocrinology and Metabolism 281, no. 6 (December 1, 2001): E1221—E1229. http://dx.doi.org/10.1152/ajpendo.2001.281.6.e1221.
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Proteolysis of insulin-like growth factor-binding proteins (IGFBPs) may be an important mechanism to regulate IGF availability and IGF-independent functions of IGFBPs. We analyzed the secretion of IGFBP proteases in Madin-Darby canine kidney (MDCK) cells. The results showed that several specific proteases were secreted, cleaving IGFBP-2 to -6 at neutral pH. The proteolytic activity against IGFBP-6 differed at least from IGFBP-5 protease activity in its sensitivity both to IGF-II and to the hydroxamic acid-based disintegrin metalloprotease inhibitor, as well as serine protease inhibitors. During partial purification steps, the serine protease inhibitor-sensitive fraction with IGFBP-6 protease activity was separated from fractions characterized by the presence of a 30-kDa disintegrin immunoreactive band. Whereas the IGFBP-4 and -6 proteases are predominantly secreted across the basolateral membrane, the majority of IGFBPs are sorted to the apical medium from filter-grown cells. These studies indicate that the side-specific secretion of several distinct IGFBP proteases with partially overlapping IGFBP specificities may be another level in the regulation of IGF-dependent epithelial functions.
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Pihl, Rasmus, Rasmus K. Jensen, Emil C. Poulsen, Lisbeth Jensen, Annette G. Hansen, Ida B. Thøgersen, József Dobó, et al. "ITIH4 acts as a protease inhibitor by a novel inhibitory mechanism." Science Advances 7, no. 2 (January 2021): eaba7381. http://dx.doi.org/10.1126/sciadv.aba7381.
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Inter-α-inhibitor heavy chain 4 (ITIH4) is a poorly characterized plasma protein that is proteolytically processed in multiple pathological conditions. However, no biological function of ITIH4 has been identified. Here, we show that ITIH4 is cleaved by several human proteases within a protease-susceptible region, enabling ITIH4 to function as a protease inhibitor. This is exemplified by its inhibition of mannan-binding lectin–associated serine protease-1 (MASP-1), MASP-2, and plasma kallikrein, which are key proteases for intravascular host defense. Mechanistically, ITIH4 acts as bait that, upon cleavage, forms a noncovalent, inhibitory complex with the executing protease that depends on the ITIH4 von Willebrand factor A domain. ITIH4 inhibits the MASPs by sterically preventing larger protein substrates from accessing their active sites, which remain accessible and fully functional toward small substrates. Thus, we demonstrate that ITIH4 functions as a protease inhibitor by a previously undescribed inhibitory mechanism.
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Castelo Branco, M. A., Y. N. T. C. Castelo Branco, F. J. Moraes Junior, F. N. Barros, F. P. S. Barçante, G. M. C. Carvalho, L. S. Melo Evangelista, A. L. Abreu-Silva, M. A. Sousa Filho, and J. A. T. Souza. "Plasminogen activator inhibitor 1 and Antipain preserve acrosome integrity of bovine spermatozoa during cryopreservation." Arquivo Brasileiro de Medicina Veterinária e Zootecnia 69, no. 5 (October 2017): 1114–24. http://dx.doi.org/10.1590/1678-4162-9252.
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ABSTRACT Seminal plasma contains serine proteases and serine protease inhibitor, which are involved in mammalian fertilization, and the inhibitors can be applied to prevent cold-induced sperm capacitation. The effects of different concentrations of two serine protease inhibitors were analyzed, Plasminogen activator inhibitor 1 - PAI-1 (70ƞg, 140ƞg and 210 ƞg) and Antipain (10µg, 50µg and 100µg) as supplementation to bovine semen cryopreservation extender. The effects of the inhibitors on the sperm parameters (sperm kinetics - CASA, acrosome integrity, plasma membrane integrity, mitochondrial membrane potential, sperm defects and acrosome reaction rate) were evaluated in the post-thaw semen. Cryopreservation of sperm with Antipain decreased post-thaw kinetic parameters of MP, VSL, LIN, SRT and the percentage of hyper-activated sperm while PAI-1 (210 ƞg) decreased VSL and LIN. Antipain and PAI-1 had no effect on the integrity parameters of the plasma membrane, mitochondrial membrane potential and sperm defects. Sperm cryopreserved in the presence of Antipain and PAI-1 (70 and 140 ƞg) preserved acrosome integrity, as they were able to complete the in vitro acrosome reaction. In conclusion, the serine protease inhibitors, Antipain and PAI-1 (70 and 140ƞg) are able to preserve the acrosome integrity of cryopreserved bovine sperm.
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Josephrajkumar, A., R. Chakrabarty, and G. Thomas. "Midgut proteases of the cardamom shoot and capsule borerConogethes punctiferalis(Lepidoptera: Pyralidae) and their interaction with aprotinin." Bulletin of Entomological Research 96, no. 1 (February 2006): 91–98. http://dx.doi.org/10.1079/ber2005403.
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AbstractProtease inhibitors cause mortality in a range of insects, and transgenic plants expressing protease inhibitors have been protected against pest attack, particularly internal feeders that are not amenable to control by conventional means. A study of luminal proteases inConogethes punctiferalisGuenée was performed to identify potential targets for proteinaceous biopesticides, such as protease inhibitors. The midgut protease profile of the gut lumen fromC. punctiferaliswas studied to determine the conditions for optimal protein hydrolysis. Optimum conditions for peptidase activity were found to be in 50 mm Tris-HCl, pH 10 containing 20 mm CaCl2; incubation for 30 min at 40°C. Four synthetic substrates, i.e. benzoyl-arg-p-nitroanilide, benzoyl-tyr-p-nitroanilide, succinyl-ala-ala-pro-leu-p-nitroanilide (SAAPLpNA) and leu-p-nitroanilide were hydrolysed byC. punctiferalisgut proteases in Tris-HCl buffer pH 10. Trypsin and elastase-like chymotrypsin were the prominent digestive proteases, and age-related modulation of midgut proteases existed for trypsin, chymotrypsin, elastase-like chymotrypsin and leucine aminopeptidase. Serine protease inhibitors such as aprotinin, soybean trypsin inhibitor and phenylmethanesulfonyl fluoride inhibited peptidase activity. Some metal ions such as Ca2+, Mg2+, Pb2+and Co2+enhanced BApNA-ase activity whereas others like Mn2+, Zn2+, Cu2+, Fe2+and Hg2+were inhibitory at 6 mm concentration. Trypsin and elastase-like chymotrypsin were significantly inhibited by 94% and 29%, respectively, by aprotinin (150 nm) underin vitroconditions. A possible incorporation of protease inhibitors into transgenic plants is discussed.
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Guedes, Herbert L. M., João M. Rezende Neto, Mayra A. Fonseca, Cristiane M. C. Salles, Bartira Rossi-Bergmann, and Giovanni De-Simone. "Identification of Serine Proteases from Leishmania braziliensis." Zeitschrift für Naturforschung C 62, no. 5-6 (June 1, 2007): 373–81. http://dx.doi.org/10.1515/znc-2007-5-610.
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Leishmania (V.) braziliensis is one of the most important ethiologic agents of the two distinct forms of American tegumentary leishmaniasis (cutaneous and mucosal). The drugs of choice used in leishmaniasis therapy are significantly toxic, expensive and are associated with frequent refractory infections. Among the promising new targets for anti-protozoan chemotherapy are the proteases. In this study, serine proteases were partially purified from aqueous, detergent and extracellular extracts of Leishmania braziliensis promastigotes by aprotinin-agarose affinity chromatography. By zymography, the enzymes purified from the aqueous extract showed apparent activity bands of 60 kDa and 45 kDa; of 130 kDa, 83 kDa, 74 kDa and 30 kDa from the detergent extract; and of 62 kDa, 59 kDa, 57 kDa, 49 kDa and 35 kDa from the extracellular extract. All purified proteases exhibited esterase activity against Nα-benzoyl-l-arginine ethyl ester hydrochloride and Nα-p-tosyl-l-arginine methyl ester hydrochloride (serine protease substrates) and optimal activity at pH 8. 0. Proteases purified from the aqueous and extracellular extracts were effectively inhibited by benzamidine (trypsin inhibitor) and those from the detergent extract were inhibited by N-tosyl-l-phenylalanine chloromethyl ketone (chymotrypsin inhibitor) indicating that all these enzymes are serine proteases. These findings indicate that L. braziliensis serine proteases display some biochemical similarities with L. amazonensis serine proteases, demonstrating a conservation of this enzymatic class in the Leishmania genus. This is the first study to report the purification of a serine protease from Leishmania braziliensis.
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Waxman, Lloyd, and Paul L. Darke. "The Herpesvirus Proteases as Targets for Antiviral Chemotherapy." Antiviral Chemistry and Chemotherapy 11, no. 1 (February 2000): 1–22. http://dx.doi.org/10.1177/095632020001100101.
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Viruses of the family Herpesviridae are responsible for a diverse set of human diseases. The available treatments are largely ineffective, with the exception of a few drugs for treatment of herpes simplex virus (HSV) infections. For several members of this DNA virus family, advances have been made recently in the biochemistry and structural biology of the essential viral protease, revealing common features that may be possible to exploit in the development of a new class of anti-herpesvirus agents. The herpesvirus proteases have been identified as belonging to a unique class of serine protease, with a Ser-His-His catalytic triad. A new, single domain protein fold has been determined by X-ray crystallography for the proteases of at least three different herpesviruses. Also unique for serine proteases, dimerization has been shown to be required for activity of the cytomegalovirus and HSV proteases. The dimerization requirement seriously impacts methods needed for productive, functional analysis and inhibitor discovery. The conserved functional and catalytic properties of the herpesvirus proteases lead to common considerations for this group of proteases in the early phases of inhibitor discovery. In general, classical serine protease inhibitors that react with active site residues do not readily inactivate the herpesvirus proteases. There has been progress however, with activated carbonyls that exploit the selective nucleophilicity of the active site serine. In addition, screening of chemical libraries has yielded novel structures as starting points for drug development. Recent crystal structures of the herpesvirus proteases now allow more direct interpretation of ligand structure—activity relationships. This review first describes basic functional aspects of herpesvirus protease biology and enzymology. Then we discuss inhibitors identified to date and the prospects for their future development.
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Mueller, Niklaus H., Nagarajan Pattabiraman, Camilo Ansarah-Sobrinho, Prasanth Viswanathan, Theodore C. Pierson, and R. Padmanabhan. "Identification and Biochemical Characterization of Small-Molecule Inhibitors of West Nile Virus Serine Protease by a High-Throughput Screen." Antimicrobial Agents and Chemotherapy 52, no. 9 (July 7, 2008): 3385–93. http://dx.doi.org/10.1128/aac.01508-07.
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ABSTRACT West Nile virus and dengue virus are mosquito-borne flaviviruses that cause a large number of human infections each year. No vaccines or chemotherapeutics are currently available. These viruses encode a serine protease that is essential for polyprotein processing, a required step in the viral replication cycle. In this study, a high-throughput screening assay for the West Nile virus protease was employed to screen ∼32,000 small-molecule compounds for identification of inhibitors. Lead inhibitor compounds with three distinct core chemical structures (1 to 3) were identified. In a secondary screening of selected compounds, two compounds, belonging to the 8-hydroxyquinoline family (compounds A and B) and containing core structure 1, were identified as potent inhibitors of the West Nile virus protease, with K i values of 3.2 ± 0.3 μM and 3.4 ± 0.6 μM, respectively. These compounds inhibited the dengue virus type 2 protease with K i values of 28.6 ± 5.1 μM and 30.2 ± 8.6 μM, respectively, showing some selectivity in the inhibition of these viral proteases. However, the compounds show no inhibition of cellular serine proteases, trypsin, or factor Xa. Kinetic analysis and molecular docking of compound B onto the known crystal structure of the West Nile virus protease indicate that the inhibitor binds in the substrate-binding cleft. Furthermore, compound B was capable of inhibiting West Nile virus RNA replication in cultured Vero cells (50% effective concentration, 1.4 ± 0.4 μM; selectivity index, 100), presumably by inhibition of polyprotein processing.
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van Pel, Melissa, Ronald van Os, Gerjo A. Velders, Henny Hagoort, Ivan J. Lindley, Roelof Willemze, and Willem E. Fibbe. "Alpha-1 Antitrypsin Is a Potent Inhibitor of Cytokine-Induced Hematopoietic Stem Cell Mobilization." Blood 104, no. 11 (November 16, 2004): 1185. http://dx.doi.org/10.1182/blood.v104.11.1185.1185.
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Abstract Previously, we have shown that IL-8 and G-CSF-induced hematopoietic stem cell (HSC) mobilization is inhibited in mice that underwent low dose (0.5 Gy) total body irradiation (TBI), whereas the number of progenitor cells in the bone marrow remained similar in all groups. The mechanism underlying this inhibition remains unknown. Since the release of granular proteases by neutrophils is well known to play a role in HSC mobilization, we also considered a possible role for serine protease inhibitors in the induction of HSC mobilization. Serine proteases, such as elastase and cathepsin G, are irreversibly inhibited by serine protease inhibitors including alpha-1 antitrypsin (alpha-1 AT) and alpha2-macroglobulin. In-vitro tests revealed that addition of bone marrow extracellular extracts, that were obtained from murine femurs 24 hours following low dose (0.5 Gy) TBI, inhibited the activity of exogenous elastase in a chromogenic substrate conversion assay up to 78.1 % compared to extracts obtained from sham irradiated controls (p<0.05). Since elastase inhibition by alpha2-macroglobulin cannot be detected in a chromogenic substrate conversion assay, alpha-1 AT was considered as the primary candidate serine protease inhibitor to inhibit elastase activity in our in-vitro system. Quantitative PCR of total bone marrow cells revealed that alpha-1 AT mRNA was 20-fold increased relative to the housekeeping gene ß-actin and 7-fold relative to the housekeeping genes HPRT and GAPDH at 24 hours following low dose (0.5 Gy) TBI. In addition, Western blot analysis indicated that alpha-1 AT protein concentrations were significantly (p<0.01) increased in bone marrow extracellular extracts derived from low dose (0.5 Gy) irradiated mice, compared to extracts obtained from sham-irradiated controls (5.1 ± 0.6 scanning units [SU] vs. 3.9 ± 0.7 SU for 0.5 Gy;n=8 vs. 0 Gy; n=6 respectively). To further substantiate a possible in-vivo role of alpha-1 AT in the inhibition of HSC mobilization, we administered alpha-1 AT (300 μg/mouse i.p.) at 2 hours and at 5 minutes prior to IL-8 injection (30 μg/mouse i.p.). Administration of alpha-1 AT prior to IL-8 injection completely (p<0.05) inhibited IL-8-induced HSC mobilization (472.9 ± 289.5 CFU-GM per ml blood for IL-8; n=5 vs. 44.8 ± 35.5 CFU-GM per ml blood for alpha-1 AT/IL-8; n =11). These results indicate that 1) alpha-1 AT is a potent inhibitor of IL-8-induced HSC mobilization and 2) in-vivo induced alpha-1 AT contributes to the inhibition of HSC mobilization after low-dose (0.5 Gy) TBI. We hypothesize that a critical balance between serine proteases and serine protease inhibitors plays an important role in cytokine-induced HSC mobilization.
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Uchimura, Kohei, Yutaka Kakizoe, Tomoaki Onoue, Manabu Hayata, Jun Morinaga, Rika Yamazoe, Miki Ueda, et al. "In vivo contribution of serine proteases to the proteolytic activation of γENaC in aldosterone-infused rats." American Journal of Physiology-Renal Physiology 303, no. 7 (October 1, 2012): F939—F943. http://dx.doi.org/10.1152/ajprenal.00705.2011.
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Aldosterone plays an important role in the regulation of blood pressure by modulating the activity of the epithelial sodium channel (ENaC) that consists of α-, β-, and γ-subunits. Aldosterone induces a molecular weight shift of γENaC from 85 to 70 kDa that is necessary for the channel activation. In vitro experiments demonstrated that a dual cleavage mechanism is responsible for this shift. It has been postulated that furin executes the primary cleavage in the Golgi and that the second cleavage is provided by other serine proteases such as prostasin or plasmin at the plasma membrane. However, the in vivo contribution of serine proteases to this cleavage remains unclear. To address this issue, we administered the synthetic serine protease inhibitor camostat mesilate (CM) to aldosterone-infused rats. CM decreased the abundance of the 70-kDa form of ENaC and led to a new 75-kDa form with a concomitant increase in the urinary Na-to-K ratio. Because CM inhibits the protease activity of serine proteases such as prostasin and plasmin, but not furin, our findings strongly indicate that CM inhibited the second cleavage of γENaC and subsequently suppressed ENaC activity. The results of our current studies also suggest the possibility that the synthetic serine protease inhibitor CM might represent a new strategy for the treatment of salt-sensitive hypertension in humans.
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Murakami, Yoji, Yoshihiro Wada, Hidetomo Kobayashi, Atsushi Irie, Makoto Hasegawa, Hiroyasu Yamanaka, Keinosuke Okamoto, Masatoshi Eto, and Takahisa Imamura. "Inhibition of Aeromonas sobria serine protease (ASP) by α2-macroglobulin." Biological Chemistry 393, no. 10 (October 1, 2012): 1193–200. http://dx.doi.org/10.1515/hsz-2012-0117.
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Abstract ASP is a serine protease secreted by Aeromonas sobria. ASP cleaves various plasma proteins, which is associated with onset of sepsis complications, such as shock and blood coagulation disorder. To investigate a host defense mechanism against this virulence factor, we examined the plasma for ASP inhibitor(s). Human plasma inhibited ASP activity for azocasein, which was almost completely abolished by treating plasma with methylamine, which inactivates α2-macroglobulin (α2-MG). The ASP-inhibitor complex in ASP-added plasma was not detected by immunoblotting using anti-ASP antibody; however, using gel filtration of the plasma ASP activity for an oligopeptide, the ASP substrate was eluted in the void fraction (Mw>200 000), suggesting ASP trapping by α2-MG. Indeed, human α2-MG inhibited ASP azocaseinolytic activity in a dose-dependent manner, rapidly forming a complex with the ASP. Fibrinogen degradation by ASP was completely inhibited in the presence of α2-MG. α1-Protease inhibitor, antithrombin, and α2-plasmin inhibitor neither inhibited ASP activity nor formed a complex with ASP. Surprisingly, ASP degraded these plasma serine protease inhibitors. Thus, α2-MG is the major ASP inhibitor in the human plasma and can limit ASP virulence activities in A. sobria infection sites. However, as shown by fluorescence correlation spectroscopy, slow ASP inhibition by α2-MG in plasma may indicate insufficient ASP control in vivo.
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Szałapata, Katarzyna, Monika Osińska-Jaroszuk, Justyna Kapral-Piotrowska, Bożena Pawlikowska-Pawlęga, Rafał Łopucki, Robert Mroczka, and Anna Jarosz-Wilkołazka. "Serine Protease Inhibitors—New Molecules for Modification of Polymeric Biomaterials." Biomolecules 10, no. 1 (January 4, 2020): 82. http://dx.doi.org/10.3390/biom10010082.
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Three serine protease inhibitors (AEBSF, soy inhibitor, α1-antitrypsin) were covalently immobilized on the surface of three polymer prostheses with the optimized method. The immobilization efficiency ranged from 11 to 51%, depending on the chosen inhibitor and biomaterial. The highest activity for all inhibitors was observed in the case of immobilization on the surface of the polyester Uni-Graft prosthesis, and the preparations obtained showed high stability in the environment with different pH and temperature values. Modification of the Uni-Graft prosthesis surface with the synthetic AEBSF inhibitor and human α1-antitrypsin inhibited the adhesion and multiplication of Staphylococcus aureus subs. aureus ATCC® 25923TM and Candida albicans from the collection of the Department of Genetics and Microbiology, UMCS. Optical profilometry analysis indicated that, after the immobilization process on the surface of AEBSF-modified Uni-Graft prostheses, there were more structures with a high number of protrusions, while the introduction of modifications with a protein inhibitor led to the smoothing of their surface.
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Hua, Ya, Guohua Xi, Richard F. Keep, Jimin Wu, Yajun Jiang, and Julian T. Hoff. "Plasminogen Activator Inhibitor-1 Induction after Experimental Intracerebral Hemorrhage." Journal of Cerebral Blood Flow & Metabolism 22, no. 1 (January 2002): 55–61. http://dx.doi.org/10.1097/00004647-200201000-00007.
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Serine proteases, such as thrombin and tissue-type plasminogen activator, play an important role in brain injury after intracerebral hemorrhage and other neurologic disorders. Plasminogen activator inhibitor-1 is one of the serine protease inhibitors, or serpins. The balance between serine proteases and serpins may affect the outcome of intracerebral hemorrhage. The purpose of this study was to determine whether plasminogen activator inhibitor-1 and tissue-type plasminogen activator are upregulated after intracerebral hemorrhage and the role that thrombin plays in that induction. Plasminogen activator inhibitor-1 protein levels were upregulated after intracerebral hemorrhage. Brain plasminogen activator inhibitor-1 content also increased after thrombin infusion in a dose-dependent manner. Hirudin, a specific thrombin inhibitor, blocked the upregulation of plasminogen activator inhibitor-1 after intracerebral hemorrhage. Time courses showed that plasminogen activator inhibitor-1 levels around the hematoma peaked at the first day. Plasminogen activator inhibitor-1–positive cells were detected in the perihematomal area and the ipsilateral basal ganglia after thrombin infusion, but not in the contralateral hemisphere. Plasminogen activator inhibitor-1 messenger RNA levels were increased at 24 hours after intracerebral hemorrhage and after thrombin infusion. However, tissue-type plasminogen activator protein levels were the same in the control, whole-blood, and thrombin-infusion groups. In conclusion, intracerebral hemorrhage and thrombin infusion stimulate plasminogen activator inhibitor-1 but not tissue-type plasminogen activator production in the brain. The upregulation of plasminogen activator inhibitor-1 may be neuroprotective by limiting thrombin or other serine protease-induced toxicity.
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Karlsson, Anna, Patricia Saravia-Otten, Karin Tegmark, Eva Morfeldt, and Staffan Arvidson. "Decreased Amounts of Cell Wall-Associated Protein A and Fibronectin-Binding Proteins in Staphylococcus aureus sarA Mutants due to Up-Regulation of Extracellular Proteases." Infection and Immunity 69, no. 8 (August 1, 2001): 4742–48. http://dx.doi.org/10.1128/iai.69.8.4742-4748.2001.
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ABSTRACT Data have been presented indicating that Staphylococcus aureus cell surface protein can be degraded by extracellular proteases produced by the same bacterium. We have found that insarA mutant cells, which produce high amounts of four major extracellular proteases (staphylococcal serine protease [V8 protease] [SspA], cysteine protease [SspB], aureolysin [metalloprotease] [Aur], and staphopain [Scp]), the levels of cell-bound fibronectin-binding proteins (FnBPs) and protein A were very low compared to those of wild-type cells, in spite of unaltered or increased transcription of the corresponding genes. Cultivation ofsarA mutant cells in the presence of the global protease inhibitor α2-macroglobulin resulted in a 16-fold increase in cell-bound FnBPs, indicating that extracellular proteases were responsible for the decreased amounts of FnBPs in sarAmutant cells. The protease inhibitor E64 had no effect on the level of FnBPs, indicating that cysteine proteases were not involved. Inactivation of either ssp or aur in the prototype S. aureus strain 8325-4 resulted in a threefold increase in the amount of cell-bound FnBPs. Inactivation of the same protease genes in a sarA mutant of 8325-4 resulted in a 10- to 20-fold increase in cell-bound protein A. As the serine protease requires aureolysin to be activated, it can thus be concluded that the serine protease is the most important protease in the release of cell-bound FnBPs and protein A.
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Heskamp, M. L., and W. Barz. "Characterization of Proteases from Rhizopus Species after Growth on Soybean Protein." Zeitschrift für Naturforschung C 52, no. 9-10 (October 1, 1997): 595–604. http://dx.doi.org/10.1515/znc-1997-9-1006.
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Culture filtrates of different fungi of the genus Rhizopus forming tempe (i.e. traditional Indonesian food) were grown on a soybean protein-raffinose-phytate medium and investigated for protease activity using soyprotein as substrate. All isolates belonging to the species R. oryzae, R. stolonifer, R. oligosporus, and R. microsporus var. chinensis, formed the wellknown Rhizopus-pepsin (aspartic proteinase, 35 kD, isoelectric points: 5.9, 5.0, <4) and an additional protease mainly active under alkaline conditions. The new protease (33 kD, isoelectric points: variable and isolate specific) was purified approximately 300-fold and shown to be a serine protease (inhibitor studies). During fungal culture (12 -135 h) the aspartic proteinase is expressed first followed by the serine protease. Both proteases are insensitive to the soybean Kunitz and Bowman-Birk inhibitors. The best rate of soyprotein degradation is achieved by the coordinate action of both proteases at pH 6.5. The examined Rhizopus isolates differ in the time course and intensity of the expression of the alkaline protease
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Reihill, James A., Xuan Ouyang, Zhixuan Yang, Lisa E. J. Douglas, Mei Zhou, Tianbao Chen, and S. Lorraine Martin. "A Novel Serine Protease Inhibitor PE-BBI Ameliorates Cockroach Extract-Mediated Airway Epithelial Barrier Dysfunction." Biomolecules 10, no. 4 (March 28, 2020): 515. http://dx.doi.org/10.3390/biom10040515.
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Epithelial barrier dysfunction, characteristic of allergic airway disease may be, at least in part, due to the action of allergen-associated protease activities. Cockroach allergy is a major global health issue, with cockroaches containing considerable serine trypsin-like protease (TLP) activity. The present study sought to evaluate two novel protease inhibitors (PE-BBI and pLR-HL), recently isolated from amphibian skin secretions, for their potential to neutralise cockroach TLP activity and to determine any protective effect on cockroach-induced airway epithelial barrier disruption. Inhibitor potencies against the cockroach-associated activities were determined using a fluorogenic peptide substrate-based activity assay. 16HBE14o- cells (16HBE; a bronchial epithelial cell line) were treated with cockroach extract (CRE) in the presence or absence of the compounds in order to assess cell viability (RealTime Glo luminescent assay) and epithelial barrier disruption (transepithelial resistance and paracellular dextran flux). PE-BBI potently and selectively inhibited CRE TLP activity (pIC50 -8), but not host (16HBE) cell surface activity, which conferred protection of 16HBE cells from CRE-induced cell damage and barrier disruption. Novel protease inhibitor strategies such as PE-BBI may be useful for the treatment of allergic airway disease caused by cockroach proteases.
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Zeerleder, Sacha. "C1-Inhibitor: More Than a Serine Protease Inhibitor." Seminars in Thrombosis and Hemostasis 37, no. 04 (June 2011): 362–74. http://dx.doi.org/10.1055/s-0031-1276585.
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Lu, Fengxin, Pedro Mejia, and Alvin Davis. "C1 inhibitor, a multi-functional serine protease inhibitor." Thrombosis and Haemostasis 104, no. 11 (2010): 886–93. http://dx.doi.org/10.1160/th10-01-0073.
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SummaryC1 inhibitor (C1INH) is a serpin that regulates both complement and contact (kallikrein-kinin) system activation. It consists of a serpin domain that is highly homologous to other serpins and an amino terminal non-serpin mucin-like domain. Deficiency of C1INH results in hereditary angioedema, a disease characterised by episodes of angioedema of the skin or the mucosa of the gastrointestinal tract or the oropharynx. Although early data suggested that angioedema was mediated via complement system activation, the preponderance of the data indicate that bradykinin is the mediator. In the past few years, it has become apparent that C1INH has additional anti-inflammatory functions independent of protease inhibition. These include interactions with leukocytes that may result in enhanced phagocytosis, with endothelial cells via Eand P-selectins that interfere with leukocyte rolling and in turn results in suppression of transmigration of leukocytes across the endothelium, and interactions with extracellular matrix components that may serve to concentrate C1INH at sites of inflammation. In addition, C1INH suppresses gram negative sepsis and endotoxin shock, partly via direct interaction with endotoxin that interferes with its interaction with macrophages, thereby suppressing tumour necrosis factor-α and other inflammatory mediators. C1INH treatment improves outcome in a number of disease models, including sepsis and other bacterial infections, possibly malaria, ischaemia-reperfusion injury (intestinal, hepatic, muscle, cardiac, brain), hyper-acute transplant rejection, and other inflammatory disease models. Recent data suggest that this effectiveness is the result of mechanisms that do not require protease inhibition, in addition to both complement and contact system activation.
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Lavergne, Marion, Audrey Guillon-Munos, Woodys Lenga Ma Bonda, Sylvie Attucci, Thomas Kryza, Aurélia Barascu, Thierry Moreau, et al. "Tissue factor pathway inhibitor 2 is a potent kallikrein-related protease 12 inhibitor." Biological Chemistry 402, no. 10 (May 12, 2021): 1257–68. http://dx.doi.org/10.1515/hsz-2020-0389.
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Abstract The protease activities are tightly regulated by inhibitors and dysregulation contribute to pathological processes such as cancer and inflammatory disorders. Tissue factor pathway inhibitor 2 (TFPI-2) is a serine proteases inhibitor, that mainly inhibits plasmin. This protease activated matrix metalloproteases (MMPs) and degraded extracellular matrix. Other serine proteases are implicated in these mechanisms like kallikreins (KLKs). In this study, we identified for the first time that TFPI-2 is a potent inhibitor of KLK5 and 12. Computer modeling showed that the first Kunitz domain of TFPI-2 could interact with residues of KLK12 near the catalytic triad. Furthermore, like plasmin, KLK12 was able to activate proMMP-1 and -3, with no effect on proMMP-9. Thus, the inhibition of KLK12 by TFPI-2 greatly reduced the cascade activation of these MMPs and the cleavage of cysteine-rich 61, a matrix signaling protein. Moreover, when TFPI-2 bound to extracellular matrix, its classical localisation, the KLK12 inhibition was retained. Finally, TFPI-2 was downregulated in human non-small-cell lung tumour tissue as compared with non-affected lung tissue. These data suggest that TFPI-2 is a potent inhibitor of KLK12 and could regulate matrix remodeling and cancer progression mediated by KLK12.
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Barile, Elisa, Carlo Baggio, Luca Gambini, Sergey A. Shiryaev, Alex Y. Strongin, and Maurizio Pellecchia. "Potential Therapeutic Targeting of Coronavirus Spike Glycoprotein Priming." Molecules 25, no. 10 (May 22, 2020): 2424. http://dx.doi.org/10.3390/molecules25102424.
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Processing of certain viral proteins and bacterial toxins by host serine proteases is a frequent and critical step in virulence. The coronavirus spike glycoprotein contains three (S1, S2, and S2′) cleavage sites that are processed by human host proteases. The exact nature of these cleavage sites, and their respective processing proteases, can determine whether the virus can cross species and the level of pathogenicity. Recent comparisons of the genomes of the highly pathogenic SARS-CoV2 and MERS-CoV, with less pathogenic strains (e.g., Bat-RaTG13, the bat homologue of SARS-CoV2) identified possible mutations in the receptor binding domain and in the S1 and S2′ cleavage sites of their spike glycoprotein. However, there remains some confusion on the relative roles of the possible serine proteases involved for priming. Using anthrax toxin as a model system, we show that in vivo inhibition of priming by pan-active serine protease inhibitors can be effective at suppressing toxicity. Hence, our studies should encourage further efforts in developing either pan-serine protease inhibitors or inhibitor cocktails to target SARS-CoV2 and potentially ward off future pandemics that could develop because of additional mutations in the S-protein priming sequence in coronaviruses.
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Wan, Hu, Bo Yeon Kim, Kwang Sik Lee, Hyung Joo Yoon, Kyung Yong Lee, and Byung Rae Jin. "A bumblebee (Bombus ignitus) venom serine protease inhibitor that acts as a microbial serine protease inhibitor." Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology 167 (January 2014): 59–64. http://dx.doi.org/10.1016/j.cbpb.2013.10.002.
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Safavi, Farinaz, Zichen Li, Limei Wang, Bogoljub Ciric, Javad Rasouli, Guang-Xian Zhang, and Abdolmohamad Rostami. "Bowman birk protease inhibitor suppresses GM-CSF, Th17 cells and CNS autoimmune inflammatory demyelination." Journal of Immunology 196, no. 1_Supplement (May 1, 2016): 187.8. http://dx.doi.org/10.4049/jimmunol.196.supp.187.8.
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Abstract Serine proteases play numerous roles in immune system and their inhibition modulates immune and autoimmune responses. Bowman Birk inhibitor (BBI), a soybean-derived serine protease inhibitor potently suppresses experimental autoimmune encephalitis (EAE) after oral administration. In this study, we show that BBI inhibits development of Th17 cells and reduces Th17 cell encephalitogenicity by suppressing their GM-CSF production and decreasing their numbers in the CNS of EAE mice. We also demonstrate that BBI induces IFN-γ production in Th17 cell culture, and that lack of IFN-γ or IFN-γR abrogates Th17 suppressive effect of BBI. In addition, BBI requires IL-27R, Stat-1, T-bet, and IL-10 to inhibit Th17 development. Taken together, our data demonstrate that BBI suppresses pathogenic Th17 cells through IFN- γ dependent pathway and ameliorates EAE. This suggests a novel immunomodulatory effect for serine protease inhibitors in immunity. In addition, BBI has potential to be safe oral therapy for autoimmune diseases, such as multiple sclerosis.
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Bosnic, Olivera, Kristina Gopcevic, Miroslav Vrvic, and Ivanka Karadzic. "Inhibition of trypsin by heparin and dalteparin, a low molecular weight heparin." Journal of the Serbian Chemical Society 74, no. 4 (2009): 379–88. http://dx.doi.org/10.2298/jsc0904379b.
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The interaction between trypsin, a prototype S1 serine protease, with heparin and its low molecular weight derivative dalteparin were investigated. Direct inhibition of the proteolytic activity of trypsin by heparin and dalteparin, used in concentrations typical for their clinical application, was detected. The half-maximum inhibition of the trypsin activity was achieved at 15.25?1.22 ?g/mL for heparin and was estimated to be at 58.47?15.20 ?g/mL for dalteparin. Kinetic analyses showed that heparin and its low molecular weight derivative dalteparin inhibited trypsin by occupation of an exosite, producing noncompetitive and mixed inhibition, respectively. Heparin as a noncompetitive inhibitor with constant of inhibition Ki1,2 = 0.151?0.019 ?M and dalteparin with Ki1 = 0.202?0.030 ?M and Ki2 = 0.463?0.069 ?M in mixed inhibition both represent moderate inhibitors of serine protease trypsin. The obtained constants of inhibition indicate that under the clinically applied concentrations of heparin and dalteparin, trypsins and their homolog S1 serine proteases could be directly inhibited, influencing the delicate control of proteolytic reactions in homeostasis.
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Song, Xiao-yu, Li Zeng, Wenwen Jin, John Thompson, Diane E. Mizel, Ke-jian Lei, R. C. Billinghurst, A. Robin Poole, and Sharon M. Wahl. "Secretory Leukocyte Protease Inhibitor Suppresses the Inflammation and Joint Damage of Bacterial Cell Wall–Induced Arthritis." Journal of Experimental Medicine 190, no. 4 (August 16, 1999): 535–42. http://dx.doi.org/10.1084/jem.190.4.535.
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Disruption of the balance between proteases and protease inhibitors is often associated with pathologic tissue destruction. To explore the therapeutic potential of secretory leukocyte protease inhibitor (SLPI) in erosive joint diseases, we cloned, sequenced, and expressed active rat SLPI, which shares the protease-reactive site found in human SLPI. In a rat streptococcal cell wall (SCW)-induced model of inflammatory erosive polyarthritis, endogenous SLPI was unexpectedly upregulated at both mRNA and protein levels in inflamed joint tissues. Systemic delivery of purified recombinant rat SLPI inhibited joint inflammation and cartilage and bone destruction. Inflammatory pathways as reflected by circulating tumor necrosis factor α and nuclear factor κB activation and cartilage resorption detected by circulating levels of type II collagen collagenase-generated cleavage products were all diminished by SLPI treatment in acute and chronic arthritis, indicating that the action of SLPI may extend beyond inhibition of serine proteases.
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Sarin, A., D. H. Adams, and P. A. Henkart. "Protease inhibitors selectively block T cell receptor-triggered programmed cell death in a murine T cell hybridoma and activated peripheral T cells." Journal of Experimental Medicine 178, no. 5 (November 1, 1993): 1693–700. http://dx.doi.org/10.1084/jem.178.5.1693.
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The hypothesis that cytoplasmic proteases play a functional role in programmed cell death was tested by examining the effect of protease inhibitors on the T cell receptor-mediated death of the 2B4 murine T cell hybridoma and activated T cells. The cysteine protease inhibitors trans-epoxysuccininyl-L-leucylamido-(4-guanidino) butane (E-64) and leupeptin, the calpain selective inhibitor acetyl-leucyl-leucyl-normethional, and the serine protease inhibitors diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride, all showed dose-dependent blocking of the 2B4 death response triggered by the T cell receptor complex and by anti-Thy-1. These protease inhibitors enhanced rather than inhibited IL-2 secretion triggered by T cell receptor cross-linking, showing that they did not act by preventing signal transduction. Growth inhibition induced by cross-linking the 2B4 T cell receptor, measured by inhibition of thymidine incorporation, was not generally blocked by these protease inhibitors. All five of these protease inhibitors enhanced rather than blocked 2B4 cell death triggered by dexamethasone, an agent previously shown to have a death pathway antagonistic with that of the TCR. 2B4 cytolysis by the cytotoxic agents staphylococcal alpha-toxin and dodecyl imidazole, and that caused by hypotonic conditions, was not significantly affected by the five protease inhibitors tested. The selected protease inhibitors blocked both the apoptotic nuclear morphology changes and DNA fragmentation induced by T cell receptor cross-linking, and enhanced both these properties induced by dexamethasone in 2B4 cells. The T cell receptor-induced death of activated murine lymph node T cells and human peripheral blood CD4+ T cells was blocked by both cysteine and serine protease inhibitors, showing that the protease-dependent death pathway also operates in these systems.
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Torquato, Ricardo J. S., Stephen Lu, Nadia Helena Martins, Aparecida S. Tanaka, and Pedro José Barbosa Pereira. "High-resolution structure of a Kazal-type serine protease inhibitor from the dengue vectorAedes aegypti." Acta Crystallographica Section F Structural Biology Communications 73, no. 8 (July 26, 2017): 469–75. http://dx.doi.org/10.1107/s2053230x17010007.
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Blood-feeding exoparasites are rich sources of protease inhibitors, and the mosquitoAedes aegypti, which is a vector ofDengue virus,Yellow fever virus,Chikungunya virusandZika virus, is no exception. AaTI is a single-domain, noncanonical Kazal-type serine proteinase inhibitor fromA. aegyptithat recognizes both digestive trypsin-like serine proteinases and the central protease in blood clotting, thrombin, albeit with an affinity that is three orders of magnitude lower. Here, the 1.4 Å resolution crystal structure of AaTI is reported from extremely tightly packed crystals (∼22% solvent content), revealing the structural determinants for the observed inhibitory profile of this molecule.
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Bestle, Dorothea, Miriam Ruth Heindl, Hannah Limburg, Thuy Van Lam van, Oliver Pilgram, Hong Moulton, David A. Stein, et al. "TMPRSS2 and furin are both essential for proteolytic activation of SARS-CoV-2 in human airway cells." Life Science Alliance 3, no. 9 (July 23, 2020): e202000786. http://dx.doi.org/10.26508/lsa.202000786.
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The novel emerged SARS-CoV-2 has rapidly spread around the world causing acute infection of the respiratory tract (COVID-19) that can result in severe disease and lethality. For SARS-CoV-2 to enter cells, its surface glycoprotein spike (S) must be cleaved at two different sites by host cell proteases, which therefore represent potential drug targets. In the present study, we show that S can be cleaved by the proprotein convertase furin at the S1/S2 site and the transmembrane serine protease 2 (TMPRSS2) at the S2′ site. We demonstrate that TMPRSS2 is essential for activation of SARS-CoV-2 S in Calu-3 human airway epithelial cells through antisense-mediated knockdown of TMPRSS2 expression. Furthermore, SARS-CoV-2 replication was also strongly inhibited by the synthetic furin inhibitor MI-1851 in human airway cells. In contrast, inhibition of endosomal cathepsins by E64d did not affect virus replication. Combining various TMPRSS2 inhibitors with furin inhibitor MI-1851 produced more potent antiviral activity against SARS-CoV-2 than an equimolar amount of any single serine protease inhibitor. Therefore, this approach has considerable therapeutic potential for treatment of COVID-19.
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Morris, Carl A., Linda D. Morris, Ann R. Kennedy, and H. Lee Sweeney. "Attenuation of skeletal muscle atrophy via protease inhibition." Journal of Applied Physiology 99, no. 5 (November 2005): 1719–27. http://dx.doi.org/10.1152/japplphysiol.01419.2004.
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Skeletal muscle atrophy in response to a number of muscle wasting conditions, including disuse, involves the induction of increased protein breakdown, decreased protein synthesis, and likely a variable component of apoptosis. The increased activation of specific proteases in the atrophy process presents a number of potential therapeutic targets to reduce muscle atrophy via protease inhibition. In this study, mice were provided with food supplemented with the Bowman-Birk inhibitor (BBI), a serine protease inhibitor known to reduce the proteolytic activity of a number of proteases, such as chymotrypsin, trypsin, elastase, cathepsin G, and chymase. Mice fed the BBI diet were suspended for 3–14 days, and the muscle mass and function were then compared with those of the suspended mice on a normal diet. The results indicate that dietary supplementation with BBI significantly attenuates the normal loss of muscle mass and strength following unloading. Furthermore, the data reveal the existence of yet uncharacterized serine proteases that are important contributors to the evolution of disuse atrophy, since BBI inhibited serine protease activity that was elevated following hindlimb unloading and also slowed the loss of muscle fiber size. These results demonstrate that targeted reduction of protein degradation can limit the severity of muscle mass loss following hindlimb unloading. Thus BBI is a candidate therapeutic agent to minimize skeletal muscle atrophy and loss of strength associated with disuse, cachexia, sepsis, weightlessness, or the combination of age and inactivity.
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SIMONET, Gert, Bert BREUGELMANS, Paul PROOST, Ilse CLAEYS, Jozef VAN DAMME, Arnold DE LOOF, and Jozef VANDEN BROECK. "Characterization of two novel pacifastin-like peptide precursor isoforms in the desert locust (Schistocerca gregaria): cDNA cloning, functional analysis and real-time RT-PCR gene expression studies." Biochemical Journal 388, no. 1 (May 10, 2005): 281–89. http://dx.doi.org/10.1042/bj20041414.
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In the last decade, a new serine protease inhibitor family has been described in arthropods. Eight members of the family were purified from locusts and share a conserved cysteine array (Cys-Xaa9–12-Cys-Asn-Xaa-Cys-Xaa-Cys-Xaa2–3-Gly-Xaa3–6-Cys-Thr-Xaa3-Cys) with nine inhibitory domains of the light chain of the crayfish protease inhibitor, pacifastin (PLDs; pacifastin light chain domains). Using cDNA cloning, several pacifastin-related precursors have been identified, encoding additional PLD-related peptides in different insect species. In the present study, two isoforms of a novel pacifastin-related precursor (SGPP-4) have been identified in the desert locust, predicting the previously identified SGPI-5 (Schistocerca gregaria PLD-related inhibitor-5) peptide and two novel PLD-related peptide sequences. One novel peptide (SGPI-5A) was synthesized chemically, and its inhibitory activity was assessed in vitro. Although proteases from a locust midgut extract were very sensitive to SGPI-5A, the same peptide proved to be a relatively poor inhibitor of bovine trypsin. By an in silico datamining approach, a novel pacifastin-related precursor with seven PLD-related domains was identified in the mosquito, Aedes aegypti. As in other insect pacifastin-related precursors, the Aedes precursor showed a particular domain architecture that is not encountered in other serine protease inhibitor families. Finally, a comparative real-time RT-PCR analysis of SGPP-4 transcripts in different tissues of isolated- (solitarious) and crowded-reared (gregarious) locusts was performed. This showed that SGPP-4 mRNA levels are higher in the brain, testes and fat body of gregarious males than of solitarious males. These results have been compared with data from a similar study on SGPP-1–3 transcripts and discussed with respect to a differential regulation of serine-protease-dependent pathways as a possible mechanism underlying locust phase polymorphism.
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Swystun, Veronica A., Bernard Renaux, France Moreau, Shoubin Wen, Michael A. Peplowski, Morley D. Hollenberg, and Wallace K. MacNaughton. "Serine proteases decrease intestinal epithelial ion permeability by activation of protein kinase Cζ." American Journal of Physiology-Gastrointestinal and Liver Physiology 297, no. 1 (July 2009): G60—G70. http://dx.doi.org/10.1152/ajpgi.00096.2009.
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Epithelial permeability to ions and larger molecules in the gut is essential for fluid balance, and its dysregulation contributes to intestinal pathology. We investigated the effect of digestive serine proteases on epithelial paracellular permeability. Trypsin, chymotrypsin, and elastase elicited sustained increases in transepithelial resistance (RTE) in polarized monolayers of three intestinal epithelial cell lines. This effect was reflected by decreases in paracellular conductances of Na+ and Cl− and a concomitant decrease in permeability to 3,000 molecular weight dextran. The enzyme activities of the proteases were required, yet activators of known protease-activated receptors (PARs) did not reproduce the effect of these proteases on RTE. PKCζ isoform-specific inhibitor significantly reduced the trypsin-induced increase in RTE whereas PKCζ activity was increased in cells treated with trypsin and chymotrypsin compared with control cells; this activity was reduced to control levels in the presence of PKCζ-specific inhibitor. Ca2+ chelators and pharmacological inhibitors of cell signaling support the role for PKCζ in the protease-induced effect. Finally, we showed that treatment with the serine proteases increased occludin immunostaining and zonula occludin-1 coimmunoprecipitation with occludin in the detergent-insoluble fraction of cell lysates, and these increases were ablated by pretreatment with PKCζ-specific inhibitor. This finding indicates increased insertion of occludin into the cell junctional complex. These data demonstrate a role for serine proteases in the facilitation of epithelial barrier function through a mechanism that is independent of PARs and is mediated by activation of PKCζ.
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Sellami, S., and K. Jamoussi. "Investigation of larvae digestive β-glucosidase and proteases of the tomato pest Tuta absoluta for inhibiting the insect development." Bulletin of Entomological Research 106, no. 3 (February 22, 2016): 406–14. http://dx.doi.org/10.1017/s0007485316000079.
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AbstractThe tomato leaf miner Tuta absoluta is one of the most devastating pests for tomato crops. Digestive proteases and β-glucosidase enzymes were investigated using general and specific substrates and inhibitors. Maximal β-glucosidase and proteolytic activities occurred at temperature and pH optima of 30 and 40°C, 5 and 10–11 unit of pH, respectively. Zymogram analysis showed the presence of distinguished β-glucosidase exhibiting a specific activity of about 183 ± 15 µmol min−1 mg−1. In vitro inhibition experiments suggested that serine proteases were the primary gut proteases. Gel based protease inhibition assays demonstrated that the 28 and 73 kDa proteases might be trypsin-like and chymotrypsin-like enzymes, respectively. Overall gut trypsin-like and chymotrypsin-like activities were evaluated to be about 27.2 ± 0.84 and 1.68 ± 0.03 µmol min−1 mg−1, respectively. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed that T. absoluta gut serine proteases are responsible for Bacillus thuringiensis Cry insecticidal proteins proteolysis. Additionally, bioassays showed that T. absoluta larvae development was more affected by the β-glucosidases inhibitor (D-glucono-δ-lactone) than the serine proteases inhibitor (soybean trypsin inhibitor). These results are of basic interest since they present interesting data of β-glucosidases and gut serine proteases of T. absoluta larvae.
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Lu, Liangjun, Tami J. Pilot-Matias, Kent D. Stewart, John T. Randolph, Ron Pithawalla, Wenping He, Peggy P. Huang, Larry L. Klein, Hongmei Mo, and Akhteruzzaman Molla. "Mutations Conferring Resistance to a Potent Hepatitis C Virus Serine Protease Inhibitor In Vitro." Antimicrobial Agents and Chemotherapy 48, no. 6 (June 2004): 2260–66. http://dx.doi.org/10.1128/aac.48.6.2260-2266.2004.
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ABSTRACT BILN 2061 is a novel, specific hepatitis C virus (HCV) NS3 serine protease inhibitor discovered by Boehringer Ingelheim that has shown potent activity against HCV replicons in tissue culture and is currently under clinical investigation for the treatment of HCV infection. The poor fidelity of the HCV RNA-dependent RNA polymerase will likely lead to the development of drug-resistant viruses in treated patients. The development of resistance to BILN 2061 was studied by the in vitro passage of HCV genotype 1b replicon cells in the presence of a fixed concentration of the drug. Three weeks posttreatment, four colonies were expanded for genotypic and phenotypic characterization. The 50% inhibitory concentrations of BILN 2061 for these colonies were 72- to 1,228-fold higher than that for the wild-type replicon. Sequencing of the individual colonies identified several mutations in the NS3 serine protease gene. Molecular clones containing the single amino acid substitution A156T, R155Q, or D168V resulted in 357-fold, 24-fold, and 144-fold reductions in susceptibility to BILN 2061, respectively, compared to the level of susceptibility shown by the wild-type replicon. Modeling studies indicate that all three of these residues are located in close proximity to the inhibitor binding site. These findings, in addition to the three-dimensional structure analysis of the NS3/NS4A serine protease inhibitor complex, provide a strategic guide for the development of next-generation inhibitors of HCV NS3/NS4A serine protease.