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1
Sampson, M. T., and A. K. Kakkar. "Coagulation proteases and human cancer." Biochemical Society Transactions 30, no. 2 (April 1, 2002): 201–7. http://dx.doi.org/10.1042/bst0300201.
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Tumours are capable of activating blood coagulation through the expression of procoagulant molecules such as tissue factor, cancer procoagulant and hepsin. Initiation of the clotting cascade results in the generation of the activated serine proteases factor VIIa, factor Xa and thrombin. These proteases act via protease-activated receptors and tissue factor to alter gene expression, thereby modulating tumour cell growth, invasion, metastasis and angiogenesis.
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Weksler, BB, EA Jaffe, MS Brower, and OF Cole. "Human leukocyte cathepsin G and elastase specifically suppress thrombin- induced prostacyclin production in human endothelial cells." Blood 74, no. 5 (October 1, 1989): 1627–34. http://dx.doi.org/10.1182/blood.v74.5.1627.1627.
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Abstract Polymorphonuclear leukocytes (PMN) when activated release products that can potentially injure endothelial cells or alter endothelial function. Exposure of cultured human umbilical vein endothelial cells to cathepsin G and elastase isolated from human PMN at concentrations reached in vivo (100 ng/mL to 10 micrograms/mL) selectively inhibited thrombin-induced prostacyclin production and the thrombin-induced rise in cytosolic free calcium ([Ca++]i) concentration. These proteases also blocked thrombin-induced release of arachidonic acid from prelabeled endothelial cells (EC). In contrast, induction of prostacyclin (PGI2) production by arachidonate, histamine, or the calcium ionophore A23187 was not altered by treatment of EC with these proteases. The effects of the proteases were concentration-dependent, were blocked by serum or serum protease inhibitors, and were reversed when the endothelial cells were further cultured for 24 hours in the absence of the proteases. Elastase, but not cathepsin G, also produced detachment of endothelial cells. Thus, the major leukocyte proteases selectively suppress thrombin-induced prostacyclin production by human vascular endothelial cells and may alter the hemostatic balance at sites of PMN activation.
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Weksler, BB, EA Jaffe, MS Brower, and OF Cole. "Human leukocyte cathepsin G and elastase specifically suppress thrombin- induced prostacyclin production in human endothelial cells." Blood 74, no. 5 (October 1, 1989): 1627–34. http://dx.doi.org/10.1182/blood.v74.5.1627.bloodjournal7451627.
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Polymorphonuclear leukocytes (PMN) when activated release products that can potentially injure endothelial cells or alter endothelial function. Exposure of cultured human umbilical vein endothelial cells to cathepsin G and elastase isolated from human PMN at concentrations reached in vivo (100 ng/mL to 10 micrograms/mL) selectively inhibited thrombin-induced prostacyclin production and the thrombin-induced rise in cytosolic free calcium ([Ca++]i) concentration. These proteases also blocked thrombin-induced release of arachidonic acid from prelabeled endothelial cells (EC). In contrast, induction of prostacyclin (PGI2) production by arachidonate, histamine, or the calcium ionophore A23187 was not altered by treatment of EC with these proteases. The effects of the proteases were concentration-dependent, were blocked by serum or serum protease inhibitors, and were reversed when the endothelial cells were further cultured for 24 hours in the absence of the proteases. Elastase, but not cathepsin G, also produced detachment of endothelial cells. Thus, the major leukocyte proteases selectively suppress thrombin-induced prostacyclin production by human vascular endothelial cells and may alter the hemostatic balance at sites of PMN activation.
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Avidano, Michael A., Cheryl S. Cotter, Scott P. Stringer, and Gregory S. Schultz. "Analysis of protease activity in human otitis media." Otolaryngology–Head and Neck Surgery 119, no. 4 (October 1998): 346–51. http://dx.doi.org/10.1016/s0194-5998(98)70076-2.
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Cronic otitis media is a common problem associated with a nonintact tympanic membrane frequently involving Staphylococcus aureus and Pseudomonas aeruginosa. The virulence of Pseudomonas bacteria is related to the production of two matrix metalloproteinases, elastase and alkaline protease. Serine proteases, such as neutrophil elastase, are produced by the host inflammatory response. These proteases are thought to contribute to tissue destruction and assist bacterial invasion during infection. This preliminary study was done to identify protease activity in otorrhea samples from patients with otitis media and a nonintact tympanic membrane and to examine the ability of selective protease inhibitors to decrease protease activity. Ilomostat (galardin) is a synthetic, specific inhibitor of matrix metalloproteinases including P. aeruginosa elastase and alkaline protease, where-as α1-antitrypsin inhibits serine proteases including neutrophil elastase. Samples were collected and cultured from 20 patients with otorrhea resulting from tympanic membrane perforations or pressure-equalization tubes. A protease assay that used azocasein as the substrate was used to quantify protease activity, with and without addition of selective protease inhibitors. Cultures revealed P. aeruginosa alone in 7 samples, P. aeruginosa plus other organisms in 10, and S. aureus alone in 3. Protease activity was detected in 15 (75%) of the samples. A statistically significant ( p < 0.05) decrease in protease activity was seen with the addition of α1-antitrypsin or Ilomostat plus α1-antitrypsin, but not with Ilomostat alone. Analyzing the 10 samples with the highest protease activity, a statistically significant decrease in activity was seen with Ilomostat or αα1-antitrypsin alone and with both Ilomostat and α1-antitrypsin together. Bacteriologic type, source of sample, age and gender of the subject, and duration of infection were not significantly related to protease activity. This is the first study to quantify protease activity and inhibition by selective protease inhibitors in human otorrhea. Protease inhibitors effectively decrease protease activity in most cases and in addition to standard antibiotic therapy might prove beneficial in the treatment of otitis media with a nonintact tympanic membrane. This study supports future clinical investigations into the role of proteases and inhibition of protease activity in the treatment of otitis media.
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Duvezin-Caubet, Stéphane, Mirko Koppen, Johannes Wagener, Michael Zick, Lars Israel, Andrea Bernacchia, Ravi Jagasia, et al. "OPA1 Processing Reconstituted in Yeast Depends on the Subunit Composition of the m-AAA Protease in Mitochondria." Molecular Biology of the Cell 18, no. 9 (September 2007): 3582–90. http://dx.doi.org/10.1091/mbc.e07-02-0164.
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The morphology of mitochondria in mammalian cells is regulated by proteolytic cleavage of OPA1, a dynamin-like GTPase of the mitochondrial inner membrane. The mitochondrial rhomboid protease PARL, and paraplegin, a subunit of the ATP-dependent m-AAA protease, were proposed to be involved in this process. Here, we characterized individual OPA1 isoforms by mass spectrometry, and we reconstituted their processing in yeast to identify proteases involved in OPA1 cleavage. The yeast homologue of OPA1, Mgm1, was processed both by PARL and its yeast homologue Pcp1. Neither of these rhomboid proteases cleaved OPA1. The formation of small OPA1 isoforms was impaired in yeast cells lacking the m-AAA protease subunits Yta10 and Yta12 and was restored upon expression of murine or human m-AAA proteases. OPA1 processing depended on the subunit composition of mammalian m-AAA proteases. Homo-oligomeric m-AAA protease complexes composed of murine Afg3l1, Afg3l2, or human AFG3L2 subunits cleaved OPA1 with higher efficiency than paraplegin-containing m-AAA proteases. OPA1 processing proceeded normally in murine cell lines lacking paraplegin or PARL. Our results provide evidence for different substrate specificities of m-AAA proteases composed of different subunits and reveal a striking evolutionary switch of proteases involved in the proteolytic processing of dynamin-like GTPases in mitochondria.
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Menou, Awen, JanWillem Duitman, Pauline Flajolet, Jean-Michel Sallenave, Arnaud André Mailleux, and Bruno Crestani. "Human airway trypsin-like protease, a serine protease involved in respiratory diseases." American Journal of Physiology-Lung Cellular and Molecular Physiology 312, no. 5 (May 1, 2017): L657—L668. http://dx.doi.org/10.1152/ajplung.00509.2016.
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More than 2% of all human genes are coding for a complex system of more than 700 proteases and protease inhibitors. Among them, serine proteases play extraordinary, diverse functions in different physiological and pathological processes. The human airway trypsin-like protease (HAT), also referred to as TMPRSS11D and serine 11D, belongs to the emerging family of cell surface proteolytic enzymes, the type II transmembrane serine proteases (TTSPs). Through the cleavage of its four major identified substrates, HAT triggers specific responses, notably in epithelial cells, within the pericellular and extracellular environment, including notably inflammatory cytokine production, inflammatory cell recruitment, or anticoagulant processes. This review summarizes the potential role of this recently described protease in mediating cell surface proteolytic events, to highlight the structural features, proteolytic activity, and regulation, including the expression profile of HAT, and discuss its possible roles in respiratory physiology and disease.
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Puente, X. S., L. M. Sánchez, A. Gutiérrez-Fernández, G. Velasco, and C. López-Otín. "A genomic view of the complexity of mammalian proteolytic systems." Biochemical Society Transactions 33, no. 2 (April 1, 2005): 331–34. http://dx.doi.org/10.1042/bst0330331.
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Proteolytic enzymes play an essential role in different physiological processes, including development, reproduction and host defence, as well as in numerous pathologies, like inflammatory diseases, neurological disorders or cancer. The completion of the human genome sequence allowed us to determine that more than 2% of all human genes are proteases or protease inhibitors, reflecting the importance of proteolysis in human biology. To understand better the complexity of proteases in human and other model organisms, we have used the available genome sequences of different mammalian organisms, including mouse, rat and chimpanzee, to identify and compare their degradomes, the complete set of protease genes in these species. Surprisingly, the rodent protease complement is more complex when compared with that of primates, mainly due to the expansion of protease families implicated in reproduction and host defence. Similarly, most differences between human and chimpanzee proteases are found in genes implicated in the immune system, which might explain some of the differences between both organisms. We have also found several genes implicated in reproduction, nutrition and the immune system, which are functional in rat, mouse or chimpanzee, but have been inactivated by mutations in the human lineage. These findings suggest that pseudogenization of specific protease genes has been a mechanism contributing to the evolution of the human genome. Finally, we found that proteases implicated in human hereditary diseases, and especially in neurodegenerative disorders, are highly conserved among mammals.
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Szabo, Roman, Qingyu Wu, Robert Dickson, Sarah Netzel-Arnett, Toni Antalis, and Thomas Bugge. "Type II transmembrane serine proteases." Thrombosis and Haemostasis 90, no. 08 (2003): 185–93. http://dx.doi.org/10.1160/th03-02-0071.
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SummaryThe recent availability of human and mouse genome sequences and expressed sequence tag databases facilitated the identification of a large new family of membrane anchored serine proteases, the type II transmembrane serine proteases or TTSPs. Analyses of human inherited disorders and gene targeting studies in mice have revealed that several members of this new protease family have critical functions in development and health. Preliminary studies also suggest that aberrant expression of type II transmembrane serine proteases may be linked to disease progression. The knowledge gathered thus far of the genetics, physiology, and pathology of this interesting new serine protease family will be reviewed here in brief.
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Aimes, Ronald, Andries Zijlstra, John Hooper, Steven Ogbourne, Mae-Le Sit, Simone Fuchs, David Gotley, James Quigley, and Toni Antalis. "Endothelial cell serine proteases expressed during vascular morphogenesis and angiogenesis." Thrombosis and Haemostasis 89, no. 03 (2003): 561–72. http://dx.doi.org/10.1055/s-0037-1613388.
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SummaryMany serine proteases play important regulatory roles in complex biological systems, but only a few have been linked directly with capillary morphogenesis and angiogenesis. Here we provide evidence that serine protease activities, independent of the plasminogen activation cascade, are required for microvascular endothelial cell reorganization and capillary morphogenesis in vitro. A homology cloning approach targeting conserved motifs present in all serine proteases, was used to identify candidate serine proteases involved in these processes, and revealed 5 genes (acrosin, testisin, neurosin, PSP and neurotrypsin), none of which had been associated previously with expression in endothelial cells. A subsequent gene-specific RT-PCR screen for 22 serine proteases confirmed expression of these 5 genes and identified 7 additional serine protease genes expressed by human endothelial cells, urokinase-type plasminogen activator, protein C, TMPRSS2, hepsin, matriptase/ MT-SP1, dipeptidylpeptidase IV, and seprase. Differences in serine protease gene expression between microvascular and human umbilical vein endothelial cells (HUVECs) were identified and several serine protease genes were found to be regulated by the nature of the substratum, ie. artificial basement membrane or fibrillar type I collagen. mRNA transcripts of several serine protease genes were associated with blood vessels in vivo by in situ hybridization of human tissue specimens. These data suggest a potential role for serine proteases, not previously associated with endothelium, in vascular function and angiogenesis.
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Ehrhardt, Katrin, Natalie Steck, Reinhild Kappelhoff, Stephanie Stein, Florian Rieder, Ilyssa O. Gordon, Erin C. Boyle, et al. "Persistent Salmonella enterica Serovar Typhimurium Infection Induces Protease Expression During Intestinal Fibrosis." Inflammatory Bowel Diseases 25, no. 10 (May 9, 2019): 1629–43. http://dx.doi.org/10.1093/ibd/izz070.
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AbstractBackgroundIntestinal fibrosis is a common and serious complication of Crohn’s disease characterized by the accumulation of fibroblasts, deposition of extracellular matrix, and formation of scar tissue. Although many factors including cytokines and proteases contribute to the development of intestinal fibrosis, the initiating mechanisms and the complex interplay between these factors remain unclear.MethodsChronic infection of mice with Salmonella enterica serovar Typhimurium was used to induce intestinal fibrosis. A murine protease-specific CLIP-CHIP microarray analysis was employed to assess regulation of proteases and protease inhibitors. To confirm up- or downregulation during fibrosis, we performed quantitative real-time polymerase chain reaction (PCR) and immunohistochemical stainings in mouse tissue and tissue from patients with inflammatory bowel disease. In vitro infections were used to demonstrate a direct effect of bacterial infection in the regulation of proteases.ResultsMice develop severe and persistent intestinal fibrosis upon chronic infection with Salmonella enterica serovar Typhimurium, mimicking the pathology of human disease. Microarray analyses revealed 56 up- and 40 downregulated proteases and protease inhibitors in fibrotic cecal tissue. Various matrix metalloproteases, serine proteases, cysteine proteases, and protease inhibitors were regulated in the fibrotic tissue, 22 of which were confirmed by quantitative real-time PCR. Proteases demonstrated site-specific staining patterns in intestinal fibrotic tissue from mice and in tissue from human inflammatory bowel disease patients. Finally, we show in vitro that Salmonella infection directly induces protease expression in macrophages and epithelial cells but not in fibroblasts.ConclusionsIn summary, we show that chronic Salmonella infection regulates proteases and protease inhibitors during tissue fibrosis in vivo and in vitro, and therefore this model is well suited to investigating the role of proteases in intestinal fibrosis.
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Capel, Elena, Glòria Martrus, Mariona Parera, Bonaventura Clotet, and Miguel Angel Martínez. "Evolution of the human immunodeficiency virus type 1 protease: effects on viral replication capacity and protease robustness." Journal of General Virology 93, no. 12 (December 1, 2012): 2625–34. http://dx.doi.org/10.1099/vir.0.045492-0.
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The rapid spread of human immunodeficiency virus type 1 (HIV-1) in humans has been accompanied by continuous extensive genetic diversification of the virus. The aim of this study was to investigate the impact of HIV-1 diversification on HIV-1 replication capacity (RC) and mutational robustness. Thirty-three HIV-1 protease sequences were amplified from three groups of viruses: two naïve sample groups isolated 15 years apart plus a third group of protease inhibitor-(PI) resistant samples. The amplified proteases were recombined with an HXB2 infectious clone and RC was determined in MT-4 cells. RC was also measured in these three groups after random mutagenesis in vitro using error-prone PCR. No significant RC differences were observed between recombinant viruses from either early or recent naïve isolates (P = 0.5729), even though the proteases from the recent isolates had significantly lower sequence conservation scores compared with a subtype B ancestral sequence (P<0.0001). Randomly mutated recombinant viruses from the three groups exhibited significantly lower RC values than the corresponding wild-type viruses (P<0.0001). There was no significant difference regarding viral infectivity reduction between viruses carrying randomly mutated naïve proteases from early or recent sample isolates (P = 0.8035). Interestingly, a significantly greater loss of RC was observed in the PI-resistant protease group (P = 0.0400). These results demonstrate that protease sequence diversification has not affected HIV-1 RC or protease robustness and indicate that proteases carrying PI resistance substitutions are less robust than naïve proteases.
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Bastos, Paulo, Fábio Trindade, Rita Ferreira, Mercedes Arguello Casteleiro, Robert Stevens, Julie Klein, and Rui Vitorino. "Unveiling antimicrobial peptide–generating human proteases using PROTEASIX." Journal of Proteomics 171 (January 2018): 53–62. http://dx.doi.org/10.1016/j.jprot.2017.02.016.
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Jairajpuri, Mohamad Aman, and 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 (September 1, 2020): 2235–41. http://dx.doi.org/10.1042/cs20200767.
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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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Blusch, Jürgen H., Sigrid Seelmeir, and Klaus von der Helm. "Molecular and Enzymatic Characterization of the Porcine Endogenous Retrovirus Protease." Journal of Virology 76, no. 15 (August 1, 2002): 7913–17. http://dx.doi.org/10.1128/jvi.76.15.7913-7917.2002.
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ABSTRACT The protease of the porcine endogenous retrovirus (PERV) subtypes A/B and C was recombinantly expressed in Escherichia coli as proteolytically active enzyme and characterized. The PERV Gag precursor was also recombinantly produced and used as the substrate in an in vitro enzyme assay in parallel with synthetic nonapeptide substrates designed according to cleavage site sequences identified in the PERV Gag precursor. The proteases of all PERV subtypes consist of 127 amino acid residues with an M r of 14,000 as revealed by determining the protease N and C termini. The PERV proteases have a high specificity for PERV substrates and do not cleave human immunodeficiency virus (HIV)-specific substrates, nor are they inhibited by specific HIV protease inhibitors. Among the known retroviral proteases, the PERV proteases resemble most closely the protease of the murine leukemia retrovirus.
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Bagossi, Péter, Tamás Sperka, Anita Fehér, János Kádas, Gábor Zahuczky, Gabriella Miklóssy, Péter Boross, and József Tözsér. "Amino Acid Preferences for a Critical Substrate Binding Subsite of Retroviral Proteases in Type 1 Cleavage Sites." Journal of Virology 79, no. 7 (April 1, 2005): 4213–18. http://dx.doi.org/10.1128/jvi.79.7.4213-4218.2005.
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ABSTRACT The specificities of the proteases of 11 retroviruses representing each of the seven genera of the family Retroviridae were studied using a series of oligopeptides with amino acid substitutions in the P2 position of a naturally occurring type 1 cleavage site (Val-Ser-Gln-Asn-Tyr↓Pro-Ile-Val-Gln; the arrow indicates the site of cleavage) in human immunodeficiency virus type 1 (HIV-1). This position was previously found to be one of the most critical in determining the substrate specificity differences of retroviral proteases. Specificities at this position were compared for HIV-1, HIV-2, equine infectious anemia virus, avian myeloblastosis virus, Mason-Pfizer monkey virus, mouse mammary tumor virus, Moloney murine leukemia virus, human T-cell leukemia virus type 1, bovine leukemia virus, human foamy virus, and walleye dermal sarcoma virus proteases. Three types of P2 preferences were observed: a subgroup of proteases preferred small hydrophobic side chains (Ala and Cys), and another subgroup preferred large hydrophobic residues (Ile and Leu), while the protease of HIV-1 preferred an Asn residue. The specificity distinctions among the proteases correlated well with the phylogenetic tree of retroviruses prepared solely based on the protease sequences. Molecular models for all of the proteases studied were built, and they were used to interpret the results. While size complementarities appear to be the main specificity-determining features of the S2 subsite of retroviral proteases, electrostatic contributions may play a role only in the case of HIV proteases. In most cases the P2 residues of naturally occurring type 1 cleavage site sequences of the studied proteases agreed well with the observed P2 preferences.
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Masler, Edward P. "Protease inhibition by Heterodera glycines cyst content: evidence for effects on the Meloidogyne incognita proteasome." Nematology 17, no. 1 (2015): 91–102. http://dx.doi.org/10.1163/15685411-00002854.
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Proteases from Heterodera glycines and Meloidogyne incognita juveniles were inhibited by heat-stable content from H. glycines cysts (hHglCE), and by a polyphenol (EGCG) similar to a compound previously identified in Globodera cysts. General protease activities detected using the nematode peptide KSAYMRFa were inhibited by EGCG (IC50 1.19 mM, H. glycines; 0.34 mM, M. incognita) but not by hHglCE. However, hHglCE and EGCG each inhibited proteasome-associated chymotrypsin-like (CT-L) activity. EGCG IC50 values were 0.47 mM (H. glycines) and 0.15 mM (M. incognita). hHglCE IC50 values were 0.16 and 0.005 mM hHglCEeq μl−1 for H. glycines and M. incognita, respectively. Across all substrate-inhibitor combinations, M. incognita proteases were inhibited more robustly than those from H. glycines, particularly by hHglCE. In addition to CT-L protease, post-glutamate peptide hydrolysing (PGPH) and trypsin-like (T-L) proteasome proteases were detected in M. incognita, and each of these was also strongly inhibited by hHglCE. hHglCE inhibited CT-L, PGPH and T-L proteases within catalytic subunits from yeast (Saccharomyces cerevisiae) and human proteasomes. Proteasome inhibitors MG-132 and aclacinomycin A each inhibited M. incognita CT-L and PGPH activities by more than 80% at 20-100 μM, and hHglCE inhibited the same proteases by 70-80% at 0.04 hHglCEeq μl−1. hHglCE completely inhibited M. incognita T-L activity, but CT-L activity in native content from H. glycines cysts was not inhibited. Evidence that H. glycines cysts contain inhibitors of all proteases associated with the proteasome establishes the cyst as an important new target to explore for potential nematode control compounds. In addition, characterisation of protease activities from a core cellular metabolic component using M. incognita is novel for plant-parasitic nematodes.
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Lee, Jung-Yub, Su-Min Song, Eun-Kyung Moon, Yu-Ran Lee, Bijay Kumar Jha, Dinzouna-Boutamba Sylvatrie Danne, Hee-Jae Cha, et al. "Cysteine Protease Inhibitor (AcStefin) Is Required for Complete Cyst Formation of Acanthamoeba." Eukaryotic Cell 12, no. 4 (February 8, 2013): 567–74. http://dx.doi.org/10.1128/ec.00308-12.
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ABSTRACTThe encystation ofAcanthamoebaleads to the formation of resilient cysts from vegetative trophozoites. This process is essential for parasite survival under unfavorable conditions, such as those associated with starvation, low temperatures, and biocides. Furthermore, cysteine proteases have been implicated in the massive turnover of intracellular components required for encystation. Thus, strict modulation of the activities of cysteine proteases is required to protectAcanthamoebafrom intracellular damage. However, mechanisms underlying the control of protease activity during encystation have not been established inAcanthamoeba. In the present study, we identified and characterizedAcanthamoebacysteine protease inhibitor (AcStefin), which was found to be highly expressed during encystation and to be associated with lysosomes by fluorescence microscopy. Recombinant AcStefin inhibited various cysteine proteases, including human cathepsin B, human cathepsin L, and papain. Transfection with small interfering RNA against AcStefin increased cysteine protease activity during encystation and resulted in incomplete cyst formation, reduced excystation efficiency, and a significant reduction in cytoplasmic area. Taken together, these results indicate that AcStefin is involved in the modulation of cysteine proteases and that it plays an essential role during the encystation ofAcanthamoeba.
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Davis, David A., Cara A. Brown, Fonda M. Newcomb, Emily S. Boja, Henry M. Fales, Joshua Kaufman, Stephen J. Stahl, Paul Wingfield, and Robert Yarchoan. "Reversible Oxidative Modification as a Mechanism for Regulating Retroviral Protease Dimerization and Activation." Journal of Virology 77, no. 5 (March 1, 2003): 3319–25. http://dx.doi.org/10.1128/jvi.77.5.3319-3325.2003.
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ABSTRACT Human immunodeficiency virus protease activity can be regulated by reversible oxidation of a sulfur-containing amino acid at the dimer interface. We show here that oxidation of this amino acid in human immunodeficiency virus type 1 protease prevents dimer formation. Moreover, we show that human T-cell leukemia virus type 1 protease can be similarly regulated through reversible glutathionylation of its two conserved cysteine residues. Based on the known three-dimensional structures and multiple sequence alignments of retroviral proteases, it is predicted that the majority of retroviral proteases have sulfur-containing amino acids at the dimer interface. The regulation of protease activity by the modification of a sulfur-containing amino acid at the dimer interface may be a conserved mechanism among the majority of retroviruses.
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Batten, Margaret R., Bernard W. Senior, Mogens Kilian, and Jenny M. Woof. "Amino Acid Sequence Requirements in the Hinge of Human Immunoglobulin A1 (IgA1) for Cleavage by Streptococcal IgA1 Proteases." Infection and Immunity 71, no. 3 (March 2003): 1462–69. http://dx.doi.org/10.1128/iai.71.3.1462-1469.2003.
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ABSTRACT The amino acid sequence requirements in the hinge of human immunoglobulin A1 (IgA1) for cleavage by IgA1 proteases of different species of Streptococcus were investigated. Recombinant IgA1 antibodies were generated with point mutations at proline 227 and threonine 228, the residues lying on either side of the peptide bond at which all streptococcal IgA1 proteases cleave wild-type human IgA1. The amino acid substitutions produced no major effect upon the structure of the mutant IgA1 antibodies or their functional ability to bind to Fcα receptors. However, the substitutions had a substantial effect upon sensitivity to cleavage with some streptococcal IgA1 proteases, with, in some cases, a single point mutation rendering the antibody resistant to a particular IgA1 protease. This effect was least marked with the IgA1 protease from Streptococcus pneumoniae, which showed no absolute requirement for either proline or threonine at residues 227 to 228. By contrast, the IgA1 proteases of Streptococcus oralis, Streptococcus sanguis, and Streptococcus mitis had an absolute requirement for proline at 227 but not for threonine at 228, which could be replaced by valine. There was evidence in S. mitis that proteases from different strains may have different amino acid requirements for cleavage. Remarkably, some streptococcal proteases appeared able to cleave the hinge at a distant alternative site if substitution prevented efficient cleavage of the original site. Hence, this study has identified key residues required for the recognition of the IgA1 hinge as a substrate by streptococcal IgA1 proteases, and it marks a preliminary step towards development of specific enzyme inhibitors.
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Mallia-Milanes, Brendan, Antoine Dufour, Christopher Philp, Nestor Solis, Theo Klein, Marlies Fischer, Charlotte E. Bolton, Steven Shapiro, Christopher M. Overall, and Simon R. Johnson. "TAILS proteomics reveals dynamic changes in airway proteolysis controlling protease activity and innate immunity during COPD exacerbations." American Journal of Physiology-Lung Cellular and Molecular Physiology 315, no. 6 (December 1, 2018): L1003—L1014. http://dx.doi.org/10.1152/ajplung.00175.2018.
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Dysregulated protease activity is thought to cause parenchymal and airway damage in chronic obstructive pulmonary disease (COPD). Multiple proteases have been implicated in COPD, and identifying their substrates may reveal new disease mechanisms and treatments. However, as proteases interact with many substrates that may be protease inhibitors or proteases themselves, these webs of protease interactions make the wider consequences of therapeutically targeting proteases difficult to predict. We therefore used a systems approach to determine protease substrates and protease activity in COPD airways. Protease substrates were determined by proteomics using the terminal amine isotopic labeling of substrates (TAILS) methodology in paired sputum samples during stable COPD and exacerbations. Protease activity and specific protein degradation in airway samples were assessed using Western blotting, substrate assays, and ex vivo cleavage assays. Two hundred ninety-nine proteins were identified in human COPD sputum, 125 of which were proteolytically processed, including proteases, protease inhibitors, mucins, defensins, and complement and other innate immune proteins. During exacerbations, airway neutrophils and neutrophil proteases increased and more proteins were cleaved, particularly at multiple sites, consistent with degradation and inactivation. During exacerbations, different substrates were processed, including protease inhibitors, mucins, and complement proteins. Exacerbations were associated with increasing airway elastase activity and increased processing of specific elastase substrates, including secretory leukocyte protease inhibitor. Proteolysis regulates multiple processes including elastase activity and innate immune proteins in COPD airways and differs during stable disease and exacerbations. The complexity of protease, inhibitor, and substrate networks makes the effect of protease inhibitors hard to predict which should be used cautiously.
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Patra, Madhumita Dandopath. "Rational Lead Optimization Based on the Modeled Structure of Cysteine Protease of Leishmania donovani." Asian Journal of Organic & Medicinal Chemistry 4, no. 4 (2019): 256–66. http://dx.doi.org/10.14233/ajomc.2019.ajomc-p239.
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In present study, the molecular modeling techniques were applied to generate a refined model of a cysteine protease of Leishmania donovani using the crystal structure of a homologous protease and used for lead optimization. The structures of a series of complexes of the protease with the designed inhibitors were predicted using a novel docking technique comprising of repeated cycles of molecular dynamics and energy minimization. Calculation of the free energies of binding of the model with the designed inhibitors suggested that three compounds can form stable complexes with dissociation constants in the nanomolar range (0.038-1.41 nM). Search in the human genome revealed that a number of proteases of the cathepsin family had high homology with the parasite protease with amino acid identity around 45 %. The X-ray structures of all these were available in the protein data bank. The structures of the complexes of the selected inhibitors with a few homologous human proteases of known 3-D structures were also predicted using the same technique of optimization. The electrostatic potentials around the binding sites of the proteases were highly negative, which served as a clue for the introduction of positively charged groups in the designed inhibitors for higher affinity. The comparison of interaction energies and hydrogen bonding patterns among these complexes and similar complexes with homologous human proteases allowed us to short-listed three molecules as effective antileishmanial cysteine protease inhibitors.
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Böttcher, Eva, Tatyana Matrosovich, Michaela Beyerle, Hans-Dieter Klenk, Wolfgang Garten, and Mikhail Matrosovich. "Proteolytic Activation of Influenza Viruses by Serine Proteases TMPRSS2 and HAT from Human Airway Epithelium." Journal of Virology 80, no. 19 (October 1, 2006): 9896–98. http://dx.doi.org/10.1128/jvi.01118-06.
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ABSTRACT Host cell proteases that cleave the hemagglutinin (HA) of influenza viruses in the human respiratory tract are still not identified. Here we cloned two human type II transmembrane serine proteases with known airway localization, TMPRSS2 and HAT, into mammalian expression vector. Cotransfection of mammalian cells with plasmids encoding HA and either protease resulted in HA cleavage in situ. Transient expression of either protease in MDCK cells enabled multicycle replication of influenza viruses in these cells in the absence of exogenous trypsin. These data suggest that TMPRSS2 and HAT are candidates for proteolytic activation of influenza viruses in vivo.
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Kong, Y., Y. B. Chung, S. Y. Cho, S. H. Choi, and S. Y. Kang. "Characterization of three neutral proteases of Spirometra mansoni plerocercoid." Parasitology 108, no. 3 (April 1994): 359–68. http://dx.doi.org/10.1017/s0031182000076204.
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SUMMARYIn the pathogenesis of sparganosis, proteases have been considered to play important roles in tissue migration and parasite feeding. Several bands of proteolysis were observed when crude extracts of Spirometra mansoni plerocercoid (sparganum) were examined using gelatin substrate gel at neutral pH, of which two proteases of 198 and 104 kDa were purified by two chromatographic steps, and a 36 kDa protease was purified by gelatin-affinity and DEAE–anion exchange chromatography. All the purified proteases exhibited optimal activity at pH 7·5 and 0·1 M Tris–HCI. Proteolytic activities at 198 and 104 kDa were inhibited specifically by serine protease inhibitors, and 4-(amidinophenyl)methansulfonyl fluoride (APMSF, 0·5 m) and N-α–p–tosyl-L-lysine chloromethyl ketone (TLCK, 1 mM), which strongly suggested that these two proteases were trypsin-like proteases. The activity of the 36 kDa protease was inhibited by N-tosyl-l-phenylalanine chloromethyl ketone (TPCK, I mM) and chymostatin (0·1 mM), and was potentiated in 10 mM Ca2+ which showed that the protease had a chymotrypsin-like property. All the proteases were Schiff(PAS) positive. Proteases of 198 and 104 kDa degraded collagen completely within 24 h. The 36 kDa enzyme cleaved human recombinant interferon-γ (rIFNγ) and bovine myelin basic protein. In addition, all the purified proteins elicited strong antibody responses in the infected patients.
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Meléndez-Martínez, David, Luis Fernando Plenge-Tellechea, Ana Gatica-Colima, Martha Sandra Cruz-Pérez, José Manuel Aguilar-Yáñez, and Cuauhtémoc Licona-Cassani. "Functional Mining of the Crotalus Spp. Venom Protease Repertoire Reveals Potential for Chronic Wound Therapeutics." Molecules 25, no. 15 (July 28, 2020): 3401. http://dx.doi.org/10.3390/molecules25153401.
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Chronic wounds are a major health problem that cause millions of dollars in expenses every year. Among all the treatments used, active wound treatments such as enzymatic treatments represent a cheaper and specific option with a fast growth category in the market. In particular, bacterial and plant proteases have been employed due to their homology to human proteases, which drive the normal wound healing process. However, the use of these proteases has demonstrated results with low reproducibility. Therefore, alternative sources of proteases such as snake venom have been proposed. Here, we performed a functional mining of proteases from rattlesnakes (Crotalus ornatus, C. molossus nigrescens, C. scutulatus, and C. atrox) due to their high protease predominance and similarity to native proteases. To characterize Crotalus spp. Proteases, we performed different protease assays to measure and confirm the presence of metalloproteases and serine proteases, such as the universal protease assay and zymography, using several substrates such as gelatin, casein, hemoglobin, L-TAME, fibrinogen, and fibrin. We found that all our venom extracts degraded casein, gelatin, L-TAME, fibrinogen, and fibrin, but not hemoglobin. Crotalus ornatus and C. m. nigrescens extracts were the most proteolytic venoms among the samples. Particularly, C. ornatus predominantly possessed low molecular weight proteases (P-I metalloproteases). Our results demonstrated the presence of metalloproteases capable of degrading gelatin (a collagen derivative) and fibrin clots, whereas serine proteases were capable of degrading fibrinogen-generating fibrin clots, mimicking thrombin activity. Moreover, we demonstrated that Crotalus spp. are a valuable source of proteases that can aid chronic wound-healing treatments.
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Chang, Kyeong-Ok, Yunjeong Kim, Scott Lovell, Athri Rathnayake, and William Groutas. "Antiviral Drug Discovery: Norovirus Proteases and Development of Inhibitors." Viruses 11, no. 2 (February 25, 2019): 197. http://dx.doi.org/10.3390/v11020197.
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Proteases are a major enzyme group playing important roles in a wide variety of biological processes in life forms ranging from viruses to mammalians. The aberrant activity of proteases can lead to various diseases; consequently, host proteases have been the focus of intense investigation as potential therapeutic targets. A wide range of viruses encode proteases which play an essential role in viral replication and, therefore, constitute attractive targets for the development of antiviral therapeutics. There are numerous examples of successful drug development targeting cellular and viral proteases, including antivirals against human immunodeficiency virus and hepatitis C virus. Most FDA-approved antiviral agents are peptidomimetics and macrocyclic compounds that interact with the active site of a targeted protease. Norovirus proteases are cysteine proteases that contain a chymotrypsin-like fold in their 3D structures. This review focuses on our group’s efforts related to the development of norovirus protease inhibitors as potential anti-norovirus therapeutics. These protease inhibitors are rationally designed transition-state inhibitors encompassing dipeptidyl, tripeptidyl and macrocyclic compounds. Highly effective inhibitors validated in X-ray co-crystallization, enzyme and cell-based assays, as well as an animal model, were generated by launching an optimization campaign utilizing the initial hit compounds. A prodrug approach was also explored to improve the pharmacokinetics (PK) of the identified inhibitors.
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Takenouchi-Ohkubo, Nobuko, Lotte M. Mortensen, Kim R. Drasbek, Mogens Kilian, and Knud Poulsen. "Horizontal transfer of the immunoglobulin A1 protease gene (iga) from Streptococcus to Gemella haemolysans." Microbiology 152, no. 7 (July 1, 2006): 2171–80. http://dx.doi.org/10.1099/mic.0.28801-0.
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Bacterial IgA1 proteases share the ability to cleave human IgA1 at the hinge region. Nature has developed this trait along at least five independent evolutionary lineages. To obtain further insight into the phylogeny and function of IgA1 proteases, the nucleotide sequence of the iga gene that encodes the IgA1 protease was determined from two Streptococcus mitis strains and one Gemella haemolysans strain. Heterologous expression in Escherichia coli confirmed that the genes encode human IgA1-cleaving activity. IgA1 proteases from Streptococcus and G. haemolysans shared structural features, including a motif typical for zinc-dependent metalloproteases of clan MA(E) family M26 and an N-terminal signal sequence followed by an LPXTG cell-wall-anchor motif and two putative membrane-spanning domains. In addition, they all harboured a repeat region preceding the active site of the protease. In the streptococcal IgA1 proteases, a G5 domain, which has been suggested to bind N-acetylglucosamine, was identified. Conservation of these structures in otherwise diverse proteases suggests that they are essential to the biological function of the enzyme. The phylogenetic distribution of homologous iga genes and conservation of gene order in the iga gene region in different Streptococcus species, combined with the sequence homologies, strongly suggest that the iga gene is more ancient in Streptococcus than in G. haemolysans, and therefore that the IgA1 protease gene was transferred from Streptococcus to G. haemolysans.
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Glenthoj, Andreas, Katrin Nickles, Jack B. Cowland, and Niels Borregaard. "Processing of Neutrophil α-Defensins Does Not Rely on Serine Proteases in Vivo." Blood 124, no. 21 (December 6, 2014): 1400. http://dx.doi.org/10.1182/blood.v124.21.1400.1400.
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Abstract The α-defensins human neutrophil peptides (HNPs) are the predominant antimicrobial peptides of neutrophil granules. They are synthesized in promyelocytes and myelocytes as proHNPs, but only processed and stored as mature HNPs in promyelocytes. Despite decades of search, the mechanisms underlying the posttranslational processing of neutrophil defensins remain unidentified. Thus, neither the enzyme processing proHNPs nor the localization of processing has been identified. It has long been hypothesized that proHNPs are processed by the serine proteases highly expressed in promyelocytes: Neutrophil elastase (NE), cathepsin G (CG), and proteinase 3 (PR3), all of which have shown in vitrocapability to process recombinant proHNP into HNP. We investigated, whether serine proteases are in fact responsible for proHNP processing in human bone marrow cells and in human and murine myeloid cell lines. Subcellular fractionation of the human promyelocytic cell line PLB-985 demonstrated proHNP processing to commence in fractions containing endoplasmic reticulum. Processing of 35S-proHNP was insensitive to serine protease inhibitors. Simultaneous knockdown of NE, CG, and PR3 did not decrease proHNP processing by primary human neutrophil precursors. Furthermore, introduction of NE, CG, and PR3 into murine promyelocytic cells did not increase processing capability. Finally, two patients suffering from Papillon–Lefèvre syndrome, who lack the ability to activate neutrophil serine proteases, demonstrated normal levels of fully processed HNP in peripheral neutrophils. Contradicting earlier assumptions, our study found serine proteases dispensable for processing of proHNPs in vivo. This calls for study of other protease classes in the search for the proHNP processing protease(s). Disclosures No relevant conflicts of interest to declare.
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Park, Jung-Ho, Yoshihiro Yamaguchi, and Masayori Inouye. "Intramolecular Regulation of the Sequence-Specific mRNA Interferase Activity of MazF Fused to a MazE Fragment with a Linker Cleavable by Specific Proteases." Applied and Environmental Microbiology 78, no. 11 (March 23, 2012): 3794–99. http://dx.doi.org/10.1128/aem.00364-12.
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ABSTRACTThe genomes of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) consist of single-stranded RNA encoding polyproteins, which are processed to individual functional proteins by virus-encoded specific proteases. These proteases have been used as targets for drug development. Here, instead of targeting these proteases to inhibit viral infection, we utilized the protease activity to activate a toxic protein to prevent viral infection. We engineered the MazE-MazF antitoxin-toxin system ofEscherichia colito fuse a C-terminal 41-residue fragment of antitoxin MazE to the N-terminal end of toxin MazF with a linker having a specific protease cleavage site for either HIV PR (HIV-1 protease), NS3 protease (HCV protease), or factor Xa. These fusion proteins formed a stable dimer (instead of the MazF2-MazE2-MazF2heterohexamer in nature) to inactivate the ACA (sequence)-specific mRNA interferase activity of MazF. When the fusion proteins were incubated with the corresponding proteases, the MazE fragment was cleaved from the fusion proteins, releasing active MazF, which then acted as an ACA-specific mRNA interferase cleaving single-stranded MS2 phage RNA. The intramolecular regulation of MazF toxicity by proteases as demonstrated may provide a novel approach for preventive and therapeutic treatments of infection by HIV-1, HCV, and other single-stranded RNA viruses.
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Long, Shinong, Elaine Phan, and Michel C. Vellard. "The Expression of Soluble and Active RecombinantHaemophilus influenzaeIgA1 Protease inE. coli." Journal of Biomedicine and Biotechnology 2010 (2010): 1–9. http://dx.doi.org/10.1155/2010/253983.
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Immunoglobulin A1 (IgA1) proteases fromHaemophilus influenzaeare extracellular proteases that specifically cleave the hinge region of human IgA1, the predominant class of immunoglobulin present on mucosal membranes. The IgA1 proteases may have the potential to cleave IgA1 complexes in the kidney and be a therapeutic agent for IgA1 nephropathy (IgAN), a disease characterized by deposition of the IgA1 antibody in the glomerulus. We have screened for the expression of recombinantH. influenzaeIgA1 protease by combining various expression plasmids, IgA1 protease constructs, andE. colistrains under multiple conditions. Using the method we have developed, approximately 20–40 mg/L of soluble and activeH. influenzaeIgA1 protease can be produced fromE. colistrain C41(DE3), a significant increase in yield compared to the yield upon expression inH. influenzaeor other related bacteria.
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Wang, Q. May, Robert B. Johnson, Louis N. Jungheim, Jeffrey D. Cohen, and Elcira C. Villarreal. "Dual Inhibition of Human Rhinovirus 2A and 3C Proteases by Homophthalimides." Antimicrobial Agents and Chemotherapy 42, no. 4 (April 1, 1998): 916–20. http://dx.doi.org/10.1128/aac.42.4.916.
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ABSTRACT The 2A and 3C proteases encoded by human rhinoviruses (HRVs) are attractive targets for antiviral drug development due to their important roles in viral replication. Homophthalimides were originally identified as inhibitors of rhinovirus 3C protease through our screening effort. Previous studies have indicated that the antiviral activity of certain homophthalimides exceeded their in vitro inhibitory activity against the viral 3C protease, suggesting that an additional mechanism might be involved. Reported here is the identification of homophthalimides as potent inhibitors for another rhinovirus protease, designated 2A. Several homophthalimides exhibit time-dependent inhibition of the 2A protease in the low-micromolar range, and enzyme-inhibitor complexes were identified by mass spectrometry. Compound LY343814, one of the most potent inhibitors against HRV14 2A protease, had an antiviral 50% inhibitory concentration of 4.2 μM in the cell-based assay. Our data reveal that homophthalimides are not only 3C but also 2A protease inhibitors in vitro, implying that the antiviral activity associated with these compounds might result from inactivation of both 2A and 3C proteases in vivo. Since the processing of the viral polyprotein is hierarchical, dual inhibition of the two enzymes may result in cooperative inhibition of viral replication. On the basis of the current understanding of their enzyme inhibitory mechanism, homophthalimides, as a group of novel nonpeptidic antirhinovirus agents, merit further structure-action relationship studies.
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King, Cecile, Siobhan Brennan, Phillip J. Thompson, and Geoffrey A. Stewart. "Dust Mite Proteolytic Allergens Induce Cytokine Release from Cultured Airway Epithelium." Journal of Immunology 161, no. 7 (October 1, 1998): 3645–51. http://dx.doi.org/10.4049/jimmunol.161.7.3645.
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Abstract Endogenous proteolytic enzymes have been shown to be potential sources of airway inflammation inducing proinflammatory cytokine release from respiratory epithelial cells; however, whether any of the exogenous proteases from important allergen sources such as the house dust mite present in our environment behave in a similar fashion is unclear. To this end, we have investigated whether the mite cysteine and serine proteolytic allergens, Der p 1 and Der p 9, respectively, induced cytokine production from primary human bronchial epithelial cells and from the epithelial cell line BEAS-2B. Cells were exposed to mite proteases, and cells or supernatants were assayed for cytokine release, cytokine mRNA expression, and modulation of intracellular calcium ion concentration. Both proteases induced concentration- and time-dependent increases in the release of granulocyte-macrophage (GM)-CSF, IL-6, and IL-8 as well as an increase in the expression of IL-6 mRNA. Cytokine release and mRNA expression were first observed at 8 h and 2 h after protease exposure, respectively. The minimum concentration of each protease that was required to stimulate GM-CSF, IL-6, and IL-8 release was ∼10 ng/ml. Cytokine release was initiated by 1 to 2 h of protease exposure, although maximum concentrations were detected only after a 24-h incubation. IL-6, but not IL-8 and GM-CSF, was shown to be degraded by both proteases at concentrations of >2 μg/ml. The proteases also stimulated changes in the intracellular calcium ion concentration. All mite protease-induced phenomena were inhibited using appropriate protease inhibitors. These results suggest that the proteolytic activity of an allergen may stimulate the release of proinflammatory cytokines from human bronchial epithelium.
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Kryukov, V. S., S. V. Zinoviev, and R. V. Nekrasov. "Proteases in the diet of monogastric animals." Agrarian science 344, no. 1 (March 13, 2021): 30–38. http://dx.doi.org/10.32634/0869-8155-2021-344-1-30-38.
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There are many proteases, and about 2% of the human genome is involved in the regulation of their formation. The share of proteases involved in digestion accounts for only a small part. Despite this, the mechanisms of action of digestive proteases are less studied than carbohydrases and lipases. The incorporation of exogenous proteases into young animal feeds is often accompanied by improved utilization of protein and other nutrients. Exogenous proteases degrade inhibitors of the endogenous protease and lectins in feed. Alkaline proteases are of interest due to their broader substrate specificity and activity throughout the entire gastrointestinal tract. This group includes keratinases, which digest proteins inaccessible for cleavage by proteases and peptidases of animals. Keratinases digest agglutinins, glycinin and b-conglycinin and connective tissue proteins, which are resistant to the action of gastrointestinal enzymes and a number of exogenous proteases. The alleged reasons for the inconsistent results when using feed proteases are described. Their mediated positive effects not associated with proteolysis are indicated. It is advisable to use proteases with keratinolytic activity as fodder proteases.
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Abbinante-Nissen, J. M., L. G. Simpson, and G. D. Leikauf. "Neutrophil elastase increases secretory leukocyte protease inhibitor transcript levels in airway epithelial cells." American Journal of Physiology-Lung Cellular and Molecular Physiology 265, no. 3 (September 1, 1993): L286—L292. http://dx.doi.org/10.1152/ajplung.1993.265.3.l286.
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Airway inflammation is often associated with the infiltration of activated neutrophils and subsequent protease release. Although aiding in the digestion and phagocytosis of foreign proteins and microorganisms, neutrophil proteases can indiscriminately damage healthy lung tissue. In the conducting airway, proteases, particularly neutrophil elastase, are counter-balanced by several antiproteases, including secretory leukocyte protease inhibitor (SLPI). SLPI can be produced locally by a number of cells including the airway epithelial cell. To examine the effects of neutrophil granule components on SLPI transcript levels, airway epithelial cells were treated (up to 96 h) with elastase, other proteases, or enzymes isolated from human sputum. We found that neutrophil elastase increased SLPI transcript levels in primary and transformed human airway epithelial cells in a time- and dose-dependent manner. Other neutrophil products, such as cathepsin G, myeloperoxidase, and lysozyme, had little or no effect on SLPI transcript levels. However, two nonneutrophil proteases, trypsin and pancreatic elastase, also increased SLPI transcript levels at higher doses than that required of neutrophil elastase. Two inflammatory cytokines, tumor necrosis factor-alpha and interleukin-8, produced little or no effect on SLPI transcript levels. This study demonstrates one way in which SLPI is regulated, via a protease that it inhibits, neutrophil elastase.
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Álvarez, Enrique, Luis Menéndez-Arias, and Luis Carrasco. "The Eukaryotic Translation Initiation Factor 4GI Is Cleaved by Different Retroviral Proteases." Journal of Virology 77, no. 23 (December 1, 2003): 12392–400. http://dx.doi.org/10.1128/jvi.77.23.12392-12400.2003.
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ABSTRACT The initiation factor eIF4G plays a central role in the regulation of translation. In picornaviruses, as well as in human immunodeficiency virus type 1 (HIV-1), cleavage of eIF4G by the viral protease leads to inhibition of protein synthesis directed by capped cellular mRNAs. In the present work, cleavage of both eIF4GI and eIF4GII has been analyzed by employing the proteases encoded within the genomes of several members of the family Retroviridae, e.g., Moloney murine leukemia virus (MoMLV), mouse mammary tumor virus, human T-cell leukemia virus type 1, HIV-2, and simian immunodeficiency virus. All of the retroviral proteases examined were able to cleave the initiation factor eIF4GI both in intact cells and in cell-free systems, albeit with different efficiencies. The eIF4GI hydrolysis patterns obtained with HIV-1 and HIV-2 proteases were very similar to each other but rather different from those obtained with MoMLV protease. Both eIF4GI and eIF4GII were cleaved very efficiently by the MoMLV protease. However, eIF4GII was a poor substrate for HIV proteases. Proteolytic cleavage of eIF4G led to a profound inhibition of cap-dependent translation, while protein synthesis driven by mRNAs containing internal ribosome entry site elements remained unaffected or was even stimulated in transfected cells.
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Bertram, Stephanie, Ilona Glowacka, Paulina Blazejewska, Elizabeth Soilleux, Paul Allen, Simon Danisch, Imke Steffen, et al. "TMPRSS2 and TMPRSS4 Facilitate Trypsin-Independent Spread of Influenza Virus in Caco-2 Cells." Journal of Virology 84, no. 19 (July 14, 2010): 10016–25. http://dx.doi.org/10.1128/jvi.00239-10.
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ABSTRACT Proteolysis of influenza virus hemagglutinin by host cell proteases is essential for viral infectivity, but the proteases responsible are not well defined. Recently, we showed that engineered expression of the type II transmembrane serine proteases (TTSPs) TMPRSS2 and TMPRSS4 allows hemagglutinin (HA) cleavage. Here we analyzed whether TMPRSS2 and TMPRSS4 are expressed in influenza virus target cells and support viral spread in the absence of exogenously added protease (trypsin). We found that transient expression of TMPRSS2 and TMPRSS4 resulted in HA cleavage and trypsin-independent viral spread. Endogenous expression of TMPRSS2 and TMPRSS4 in cell lines correlated with the ability to support the spread of influenza virus in the absence of trypsin, indicating that these proteases might activate influenza virus in naturally permissive cells. Indeed, RNA interference (RNAi)-mediated knockdown of both TMPRSS2 and TMPRSS4 in Caco-2 cells, which released fully infectious virus without trypsin treatment, markedly reduced the spread of influenza virus, demonstrating that these proteases were responsible for efficient proteolytic activation of HA in this cell line. Finally, TMPRSS2 was found to be coexpressed with the major receptor determinant of human influenza viruses, 2,6-linked sialic acids, in human alveolar epithelium, indicating that viral target cells in the human respiratory tract express TMPRSS2. Collectively, our results point toward an important role for TMPRSS2 and possibly TMPRSS4 in influenza virus replication and highlight the former protease as a potential therapeutic target.
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OSSOVSKAYA, VALERIA S., and NIGEL W. BUNNETT. "Protease-Activated Receptors: Contribution to Physiology and Disease." Physiological Reviews 84, no. 2 (April 2004): 579–621. http://dx.doi.org/10.1152/physrev.00028.2003.
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Ossovskaya, Valeria S., and Nigel W. Bunnett. Protease-Activated Receptors: Contribution to Physiology and Disease. Physiol Rev 84: 579–621, 2004; 10.1152/physrev.00028.2003.—Proteases acting at the surface of cells generate and destroy receptor agonists and activate and inactivate receptors, thereby making a vitally important contribution to signal transduction. Certain serine proteases that derive from the circulation (e.g., coagulation factors), inflammatory cells (e.g., mast cell and neutrophil proteases), and from multiple other sources (e.g., epithelial cells, neurons, bacteria, fungi) can cleave protease-activated receptors (PARs), a family of four G protein-coupled receptors. Cleavage within the extracellular amino terminus exposes a tethered ligand domain, which binds to and activates the receptors to initiate multiple signaling cascades. Despite this irreversible mechanism of activation, signaling by PARs is efficiently terminated by receptor desensitization (receptor phosphorylation and uncoupling from G proteins) and downregulation (receptor degradation by cell-surface and lysosomal proteases). Protease signaling in tissues depends on the generation and release of proteases, availability of cofactors, presence of protease inhibitors, and activation and inactivation of PARs. Many proteases that activate PARs are produced during tissue damage, and PARs make important contributions to tissue responses to injury, including hemostasis, repair, cell survival, inflammation, and pain. Drugs that mimic or interfere with these processes are attractive therapies: selective agonists of PARs may facilitatehealing, repair, and protection, whereas protease inhibitors and PAR antagonists can impede exacerbated inflammation and pain. Major future challenges will be to understand the role of proteases and PARs in physiological control mechanisms and human diseases and to develop selective agonists and antagonists that can be used to probe function and treat disease.
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Atiakshin, Dmitri, Igor Buchwalow, Peter Horny, and Markus Tiemann. "Protease profile of normal and neoplastic mast cells in the human bone marrow with special emphasis on systemic mastocytosis." Histochemistry and Cell Biology 155, no. 5 (January 25, 2021): 561–80. http://dx.doi.org/10.1007/s00418-021-01964-3.
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AbstractMast cells (MC) are immune cells that produce a variety of mediators, such as proteases, that are important in the body’s immune responses. MC proteases have pronounced multifunctionality and in many respects determine the biological characteristics of the organ-specific MC population. Although, increased numbers of MC are one of the objective mastocytosis signs, a detailed assessment of the proteases biogenesis and excretion mechanisms in the bone marrow (BM) has not yet been carried out. Here, we performed an analysis of the expression of proteases in patients with various forms of systemic mastocytosis. We presented data on intracellular protease co-localization in human BM MCs and discussed their implication in secretory pathways of MCs in the development of the disease. Systemic mastocytosis, depending on the course, is featured by the formation of definite profiles of specific proteases in various forms of atypical mast cells. Intragranular accumulation of tryptase, chymase and carboxypeptidases in the hypochromic phenotype of atypical mast cells is characterized. Characterization of MC proteases expression during mastocytosis can be used to refine the MC classification, help in a prognosis, and increase the effectiveness of targeted therapy.
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Parsons, H. K., S. Vitovski, and J. R. Sayers. "Immunoglobulin A1 proteases: a structure–function update." Biochemical Society Transactions 32, no. 6 (October 26, 2004): 1130–32. http://dx.doi.org/10.1042/bst0321130.
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IgA1 (immunoglobulin A1) antibodies are the first line of defence against microbial pathogens such as Neisseria meningitidis and Haemophilus influenzae. However, these bacteria secrete a site-specific protease that is capable of cleaving human IgA1 and interacting with other host components. The IgA proteases are released by the type V secretion pathway, which involves translocation through two membranes and an autolytic, post-translational processing step. Results reported recently throw light on the type V secretion pathway and on the roles of the multifunctional IgA protease. The IgA1 protease-recognition sequence is present within the IgA1 hinge region as well as in the variable sequence connecting the IgA1 protease to its translocator domain. Recent results suggest that neisserial IgA1 proteases are capable of cleaving substrates lacking the classical recognition sequence. This review will cover recent advances in the IgA protease field.
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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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Bond, Judith S. "Proteases: History, discovery, and roles in health and disease." Journal of Biological Chemistry 294, no. 5 (February 1, 2019): 1643–51. http://dx.doi.org/10.1074/jbc.tm118.004156.
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The Journal of Biological Chemistry (JBC) has been a major vehicle for disseminating and recording the discovery and characterization of proteolytic enzymes. The pace of discovery in the protease field accelerated during the 1971–2010 period that Dr. Herb Tabor served as the JBC's editor-in-chief. When he began his tenure, the fine structure and kinetics of only a few proteases were known; now thousands of proteases have been characterized, and over 600 genes for proteases have been identified in the human genome. In this review, besides reflecting on Dr. Tabor's invaluable contributions to the JBC and the American Society for Biochemistry and Molecular Biology (ASBMB), I endeavor to provide an overview of the extensive history of protease research, highlighting a few discoveries and roles of proteases in vivo. In addition, metalloproteinases, particularly meprins of the astacin family, will be discussed with regard to structural characteristics, regulation, mechanisms of action, and roles in health and disease. Proteases and protein degradation play crucial roles in living systems, and I briefly address future directions in this highly diverse and thriving research area.
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Euzebio Alves, Vanessa Tubero, Henrique Aparecido Bueno da Silva, Bruno Nunes de França, Rosangela Santos Eichler, Luciana Saraiva, Maria Helena Catelli de Carvalho, and Marinella Holzhausen. "Periodontal Treatment Downregulates Protease-Activated Receptor 2 in Human Gingival Crevicular Fluid Cells." Infection and Immunity 81, no. 12 (September 16, 2013): 4399–407. http://dx.doi.org/10.1128/iai.01107-13.
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ABSTRACTProtease-activated receptor 2 (PAR2) is implicated in the pathogenesis of chronic inflammatory diseases, including periodontitis; it can be activated by gingipain and produced byPorphyromonas gingivalisand by neutrophil protease 3 (P3). PAR2 activation plays a relevant role in inflammatory processes by inducing the release of important inflammatory mediators associated with periodontal breakdown. The effects of periodontal treatment on PAR2 expression and its association with levels of proinflammatory mediators and activating proteases were investigated in chronic periodontitis patients. Positive staining for PAR2 was observed in gingival crevicular fluid cells and was reflective of tissue destruction. Overexpression of PAR2 was positively associated with inflammatory clinical parameters and with the levels of interleukin-6 (IL-6), IL-8, tumor necrosis factor alpha, matrix metalloprotease 2 (MMP-2), MMP-8, hepatocyte growth factor, and vascular endothelial growth factor. Elevated levels of gingipain and P3 and decreased levels of dentilisin and the protease inhibitors secretory leukocyte protease inhibitor and elafin were also associated with PAR2 overexpression. Healthy periodontal sites from individuals with chronic periodontitis showed diminished expression of PAR2 mRNA and the PAR2 protein (P< 0.05). Furthermore, periodontal treatment resulted in decreased PAR2 expression and correlated with decreased expression of inflammatory mediators and activating proteases. We concluded that periodontal treatment resulted in decreased levels of proteases and that proinflammatory mediators are associated with decreased PAR2 expression, suggesting that PAR2 expression is influenced by the presence of periodontal infection and is not a constitutive characteristic favoring periodontal inflammation.
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Alemka, Abofu, Harald Nothaft, Jing Zheng, and Christine M. Szymanski. "N-Glycosylation of Campylobacter jejuni Surface Proteins Promotes Bacterial Fitness." Infection and Immunity 81, no. 5 (March 4, 2013): 1674–82. http://dx.doi.org/10.1128/iai.01370-12.
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ABSTRACTCampylobacter jejuniis the etiologic agent of human bacterial gastroenteritis worldwide. In contrast, despite heavy colonization,C. jejunimaintains a commensal mode of existence in chickens. The consumption of contaminated chicken products is thought to be the principal mode ofC. jejunitransmission to the human population.C. jejuniharbors a system for N-linked protein glycosylation that has been well characterized and modifies more than 60 periplasmic and membrane-bound proteins. However, the precise role of this modification in the biology ofC. jejuniremains unexplored. We hypothesized that the N-glycans protectC. jejunisurface proteins from the action of gut proteases. TheC. jejuni pglBmutant, deficient in the expression of the oligosaccharyltransferase, exhibited reduced growth in medium supplemented with chicken cecal contents (CCC) compared with that of wild-type (WT) cells. Inactivation of the cecal proteases by heat treatment or with protease inhibitors completely restored bacterial viability and partially rescued bacterial growth. Physiological concentrations of trypsin, but not chymotrypsin, also reducedC. jejuni pglBmutant CFU. Live or dead staining indicated that CCC preferentially influencedC. jejunigrowth as opposed to bacterial viability. We identified multiple chicken cecal proteases by mass fingerprinting. The use of protease inhibitors that target specific classes indicated that both metalloproteases and serine proteases were involved in the attenuated growth of the oligosaccharyltransferase mutant. In conclusion, protein N-linked glycosylation of surface proteins may enhanceC. jejunifitness by protecting bacterial proteins from cleavage due to gut proteases.
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NA, Byoung-Kuk, Bhaskar R. SHENAI, Puran S. SIJWALI, Youngchool CHOE, Kailash C. PANDEY, Ajay SINGH, Charles S. CRAIK, and Philip J. ROSENTHAL. "Identification and biochemical characterization of vivapains, cysteine proteases of the malaria parasite Plasmodium vivax." Biochemical Journal 378, no. 2 (March 1, 2004): 529–38. http://dx.doi.org/10.1042/bj20031487.
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Cysteine proteases play important roles in the life cycles of malaria parasites. Cysteine protease inhibitors block haemoglobin hydrolysis and development in Plasmodium falciparum, suggesting that the cysteine proteases of this major human pathogen, termed falcipains, are appropriate therapeutic targets. To expand our understanding of plasmodial proteases to Plasmodium vivax, the other prevalent human malaria parasite, we identified and cloned genes encoding the P. vivax cysteine proteases, vivapain-2 and vivapain-3, and functionally expressed the proteases in Escherichia coli. The vivapain-2 and vivapain-3 genes predicted papain-family cysteine proteases, which shared a number of unusual features with falcipain-2 and falcipain-3, including large prodomains and short N-terminal extensions on the catalytic domain. Recombinant vivapain-2 and vivapain-3 shared properties with the falcipains, including acidic pH optima, requirements for reducing conditions for activity and hydrolysis of substrates with positively charged residues at P1 and Leu at P2. Both enzymes hydrolysed native haemoglobin at acidic pH and the erythrocyte cytoskeletal protein 4.1 at neutral pH, suggesting similar biological roles to the falcipains. Considering inhibitor profiles, the vivapains were inhibited by fluoromethylketone and vinyl sulphone inhibitors that also inhibited falcipains and have demonstrated potent antimalarial activity.
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Van Damme, Petra, Joel Vandekerckhove, and Kris Gevaert. "Disentanglement of protease substrate repertoires." Biological Chemistry 389, no. 4 (April 1, 2008): 371–81. http://dx.doi.org/10.1515/bc.2008.043.
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Abstract Identification of protease substrates and detailed characterization of processed sites are essential for understanding the biological function of proteases. Because of inherent complexity reasons, this however remains a formidable analytical challenge, illustrated by the fact that the majority of the more than 500 human proteases are uncharacterized to date. Recently, in addition to conventional genetic and biochemical approaches, diverse quantitative peptide-centric proteomics approaches, some of which selectively recover N-terminal peptides, have emerged. These latter proteomic technologies in particular allow the identification of natural protease substrates and delineation of cleavage sites in a complex, natural background of thousands of different proteins. We here review current biochemical, genetic and proteomic methods for global analysis of substrates of proteases and discuss selected applications.
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Fu, Zhirong, Srinivas Akula, Anna-Karin Olsson, Jukka Kervinen, and Lars Hellman. "Mast Cells and Basophils in the Defense against Ectoparasites: Efficient Degradation of Parasite Anticoagulants by the Connective Tissue Mast Cell Chymases." International Journal of Molecular Sciences 22, no. 23 (November 23, 2021): 12627. http://dx.doi.org/10.3390/ijms222312627.
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Ticks, lice, flees, mosquitos, leeches and vampire bats need to prevent the host’s blood coagulation during their feeding process. This is primarily achieved by injecting potent anticoagulant proteins. Basophils frequently accumulate at the site of tick feeding. However, this occurs only after the second encounter with the parasite involving an adaptive immune response and IgE. To study the potential role of basophils and mast cells in the defense against ticks and other ectoparasites, we produced anticoagulant proteins from three blood-feeding animals; tick, mosquito, and leech. We tested these anticoagulant proteins for their sensitivity to inactivation by a panel of hematopoietic serine proteases. The majority of the connective tissue mast cell proteases tested, originating from humans, dogs, rats, hamsters, and opossums, efficiently cleaved these anticoagulant proteins. Interestingly, the mucosal mast cell proteases that contain closely similar cleavage specificity, had little effect on these anticoagulant proteins. Ticks have been shown to produce serpins, serine protease inhibitors, upon a blood meal that efficiently inhibit the human mast cell chymase and cathepsin G, indicating that ticks have developed a strategy to inactivate these proteases. We show here that one of these tick serpins (IRS-2) shows broad activity against the majority of the mast cell chymotryptic enzymes and the neutrophil proteases from human to opossum. However, it had no effect on the mast cell tryptases or the basophil specific protease mMCP-8. The production of anticoagulants, proteases and anti-proteases by the parasite and the host presents a fascinating example of an arms race between the blood-feeding animals and the mammalian immune system with an apparent and potent role of the connective tissue mast cell chymases in the host defense.
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Sharon, Haim, Shelly Hagag, and Nir Osherov. "Transcription Factor PrtT Controls Expression of Multiple Secreted Proteases in the Human Pathogenic Mold Aspergillus fumigatus." Infection and Immunity 77, no. 9 (June 29, 2009): 4051–60. http://dx.doi.org/10.1128/iai.00426-09.
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ABSTRACT The role of secreted proteases in the virulence of the pathogenic fungus Aspergillus fumigatus remains controversial. Recently, the Aspergillus niger transcription factor PrtT was shown to control the expression of multiple secreted proteases. In this work, the gene which encodes the PrtT homolog in A. fumigatus was cloned and its function analyzed using a deletion mutant strain. Deletion of A. fumigatus prtT resulted in the loss of secreted protease activity. The expression of six secreted proteases (ALP, MEP, Dpp4, CpdS, AFUA_2G17330, and AFUA_7G06220) was markedly reduced. Culture filtrates from the prtT deletion strain exhibited reduced killing of lung epithelial cells and lysis of erythrocytes. However, the prtT deletion strain did not exhibit altered virulence in lung-infected mice. These results suggest that PrtT is not a significant virulence factor in A. fumigatus.
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Beaufort, Nathalie, Paulina Seweryn, Sophie de Bentzmann, Aihua Tang, Josef Kellermann, Nicolai Grebenchtchikov, Manfred Schmitt, Christian P. Sommerhoff, Dominique Pidard, and Viktor Magdolen. "Activation of human pro-urokinase by unrelated proteases secreted by Pseudomonas aeruginosa." Biochemical Journal 428, no. 3 (May 27, 2010): 473–82. http://dx.doi.org/10.1042/bj20091806.
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Pathogenic bacteria, including Pseudomonas aeruginosa, interact with and engage the host plasminogen (Plg) activation system, which encompasses the urokinase (uPA)-type Plg activator, and is involved in extracellular proteolysis, including matrilysis and fibrinolysis. We hypothesized that secreted bacterial proteases might contribute to the activation of this major extracellular proteolytic system, thereby participating in bacterial dissemination. We report that LasB, a thermolysin-like metalloprotease secreted by Ps. aeruginosa, converts the human uPA zymogen into its active form (kcat=4.9 s−1, Km=8.9 μM). Accordingly, whereas the extracellular secretome from the LasB-expressing pseudomonal strain PAO1 efficiently activates pro-uPA, the secretome from the isogenic LasB-deficient strain PDO240 is markedly less potent in pro-uPA activation. Still, both secretomes induce some metalloprotease-independent activation of the human zymogen. The latter involves a serine protease, which we identified via both recombinant protein expression in Escherichia coli and purification from pseudomonal cultures as protease IV (PIV; kcat=0.73 s−1, Km=6.2 μM). In contrast, neither secretomes nor the pure proteases activate Plg. Along with this, LasB converts Plg into mini-Plg and angiostatin, whereas, as reported previously, it processes the uPA receptor, inactivates the plasminogen activator inhibitor 1, and activates pro-matrix metalloproteinase 2. PIV does not target these factors at all. To conclude, LasB and PIV, although belonging to different protease families and displaying quite different substrate specificities, both activate the urokinase-type precursor of the Plg activation cascade. Direct pro-uPA activation, as also reported for other bacterial proteases, might be a frequent phenomenon that contributes to bacterial virulence.
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Dai, Rujuan, Deena Khan, and S. Ansar Ahmed. "Estrogen regulates the expression and activity of serine proteases in mouse splenocytes (87.11)." Journal of Immunology 184, no. 1_Supplement (April 1, 2010): 87.11. http://dx.doi.org/10.4049/jimmunol.184.supp.87.11.
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Abstract In addition to digesting proteins, serine proteases also play critical roles in regulation of inflammatory responses, innate and adaptive immunity via proteolytic cleavage of protease-activated receptors (PARs) and/or signaling molecules such as NF-κB. Dysregulation of serine proteases activity may result in the development of variety of human diseases. We and others have shown that estrogen, an important immune modulator, promoted the production of proinflammatory molecules in different types of cell including murine splenocytes and peritoneal macrophages. Our recent report has demonstrated that serine proteases mediated proteolysis play a critical role in regulation of STAT1 and NF-κB activity in splenocytes from estrogen-treated mice. Inhibition of serine protease activity decreased the production of inflammatory molecules IFNγ and nitric oxide in splenocytes from estrogen-treated mice. In this study, we found that estrogen treatment increased the serine proteases activity in splenocytes. Further, we confirmed that neutrophils-derived elastase mRNA expression level is increased, and serine protease inhibitor SerpinB7 is decreased in estrogen-treated splenocytes when compared to placebo control. Together, our data suggest a novel mechanism underlying estrogen-mediated promotion of inflammatory responses. These studies may form the basis for developing new alternative therapies for estrogen-influenced autoimmune diseases by intervening the activity of serine proteases.
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Martínez, Miguel-Angel, Marta Cabana, Mariona Parera, Arantxa Gutierrez, José A. Esté, and Bonaventura Clotet. "A Bacteriophage Lambda-Based Genetic Screen for Characterization of the Activity and Phenotype of the Human Immunodeficiency Virus Type 1 Protease." Antimicrobial Agents and Chemotherapy 44, no. 5 (May 1, 2000): 1132–39. http://dx.doi.org/10.1128/aac.44.5.1132-1139.2000.
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ABSTRACT Human immunodeficiency virus type 1 (HIV-1) resistance to antiretroviral drugs is the main cause of patient treatment failure. Despite the problems associated with interpretation of HIV-1 resistance testing, resistance monitoring should help in the rational design of initial or rescue antiretroviral therapies. It has previously been shown that the activity of the HIV-1 protease can be monitored by using a bacteriophage lambda-based genetic assay. This genetic screening system is based on the bacteriophage lambda regulatory circuit in which the viral repressor cI is specifically cleaved to initiate the lysogenic to lytic switch. We have adapted this simple lambda-based genetic assay for the analysis of the activities and phenotypes of different HIV-1 proteases. Lambda phages that encode HIV-1 proteases either from laboratory strains (strain HXB2) or from clinical samples are inhibited in a dose-dependent manner by the HIV-1 protease inhibitors indinavir, ritonavir, saquinavir, and nelfinavir. Distinct susceptibilities to different drugs were also detected among phages that encode HIV-1 proteases carrying different resistance mutations, further demonstrating the specificity of this assay. Differences in proteolytic processing activity can also be directly monitored with this genetic screen system since two phage populations compete in culture with each other until one phage outgrows the other. In summary, we present here a simple, safe, and rapid genetic screening system that may be used to predict the activities and phenotypes of HIV-1 proteases in the course of viral infection and antiretroviral therapy. This assay responds appropriately to well-known HIV-1 protease inhibitors and can be used to search for new protease inhibitors.
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Shamsi, Tooba Naz, and Sadaf Fatima. "Protease Inhibitors as Ad-hoc Antibiotics." Open Pharmaceutical Sciences Journal 3, no. 1 (June 29, 2016): 131–37. http://dx.doi.org/10.2174/1874844901603010131.
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Background:Proteases are important enzymes that can degrade proteins and are found in animals, plants, bacteria, fungi and viruses. The action of proteases can be controlled by Protease Inhibitors (PIs), chemical or proteinaceous in nature that can block the active site of protease. Since the step catalyzed by proteases may play important role in life cycle of microbes, hindering the action of proteases by PIs may act as therapeutic intervention for microbial infection.Material and Methods:A thorough study was performed and wide range of literature was surveyed to confirm our results of PIs showing antibacterial activity.Results:PIs have shown to be effective drugs against bacterial pathogens, pathogenic viruses- Human Immunodeficiency Virus (HIV), Herpes virus, Hepatitis Virus. PIs have recently been investigated for controlling protozoan parasites. Clinical value of proteases and their inhibitors has been studied inHelicobacter pyloriwhich is the etiologic agent of gastritis.Conclusion:This review is intended to highlight the role of PIs in the Battle against Microbial Pathogens.