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1
Watanabe, Katsuji, and Koichi Hayano. "Source of soil protease in paddy fields." Canadian Journal of Microbiology 39, no. 11 (November 1, 1993): 1035–40. http://dx.doi.org/10.1139/m93-157.
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Properties of soil proteases and proteases from Bacillus spp. obtained from water-logged paddy fields treated with organic manure or chemical fertilizer or not treated with fertilizer were compared to elucidate the sources of soil proteases. The major extractable soil proteases were metal chelator sensitive neutral proteases that were active in hydrolyzing benzyloxycarbonyl-L-phenylalanyl-L-leucine and benzyloxycarbonyl-L-phenylalanyl-L-tyrosyl-L-leucine. In this respect they resembled extracellular proteases from Bacillus subtilis (six isolates), Bacillus cereus (four isolates), and Bacillus mycoides (three isolates) isolated from the same fields. The major extractable soil protease from the manured field was a serine neutral protease that was active in hydrolyzing casein. It resembled an extracellular protease from B. subtilis (eight isolates) isolated from the same field. Extractable soil proteases accounted for 18–96% of the total soil protease in the aforementioned soil. We concluded that a major source of soil protease in water-logged paddy fields is proteolytic Bacillus spp.Key words: soil protease, metal chelator sensitive neutral protease, serine neutral protease, proteolytic Bacillus spp.
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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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Kostallas, George, and Patrik Samuelson. "Novel Fluorescence-Assisted Whole-Cell Assay for Engineering and Characterization of Proteases and Their Substrates." Applied and Environmental Microbiology 76, no. 22 (September 17, 2010): 7500–7508. http://dx.doi.org/10.1128/aem.01558-10.
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ABSTRACT We have developed a sensitive and highly efficient whole-cell methodology for quantitative analysis and screening of protease activity in vivo. The method is based on the ability of a genetically encoded protease to rescue a coexpressed short-lived fluorescent substrate reporter from cytoplasmic degradation and thereby confer increased whole-cell fluorescence in proportion to the protease's apparent activity in the Escherichia coli cytoplasm. We demonstrated that this system can reveal differences in the efficiency with which tobacco etch virus (TEV) protease processes different substrate peptides. In addition, when analyzing E. coli cells expressing TEV protease variants that differed in terms of their in vivo solubility, cells containing the most-soluble protease variant exhibited the highest fluorescence intensity. Furthermore, flow cytometry screening allowed for enrichment and subsequent identification of an optimal substrate peptide and protease variant from a large excess of cells expressing suboptimal variants (1:100,000). Two rounds of cell sorting resulted in a 69,000-fold enrichment and a 22,000-fold enrichment of the superior substrate peptide and protease variant, respectively. Our approach presents a new promising path forward for high-throughput substrate profiling of proteases, engineering of novel protease variants with desired properties (e.g., altered substrate specificity and improved solubility and activity), and identification of protease inhibitors.
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Paulin, John P., and Francisco C. Franco. "Modified bis‐tetrahydrofuran inhibitors toward improved binding to HIV‐1 proteases." Vietnam Journal of Chemistry 59, no. 5 (October 2021): 563–79. http://dx.doi.org/10.1002/vjch.202000179.
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AbstractHIV treatment includes inhibiting HIV‐1 protease which is responsible for viral maturation. However, HIV‐1 protease responds to drug treatment by mutation making the protease‐resistant to inhibitors. In this study, binding interactions between bis‐tetrahydrofuran‐derived (bis‐THF) inhibitors and HIV‐1 protease were described by molecular docking. We characterized the binding energies and all the amino acids present during the binding of the bis‐THF derivatives to the wild type HIV‐1 protease and several mutant HIV‐1 proteases. We found that the modifications to the structure of darunavir helped improve its binding to the wild‐type protease. Also, these structures were found to interact with the mutant HIV‐1 proteases better than darunavir. Results showed that compound 4 had the highest binding energy to the wild‐type HIV‐1 protease and the V654/84 mutant, while compound 5 was found to interact greatly with cyclic urea‐based inhibitor‐resistant proteases and the multi‐protease inhibitor‐resistant HIV‐1 protease. The results may help explain how structural modifications to bis‐tetrahydrofuran inhibitors affect their response to wild‐type and resistant HIV‐1 proteases. Furthermore, this study is the first demonstration of the differences in the amino acids interacting with protease inhibitors for wild‐type and mutated HIV‐1 proteases and may help in the design of bis‐THF derivatives as HIV‐1 protease inhibitors.
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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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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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Schuhmann, Holger, Ulrike Mogg, and Iwona Adamska. "A new principle of oligomerization of plant DEG7 protease based on interactions of degenerated protease domains." Biochemical Journal 435, no. 1 (March 15, 2011): 167–74. http://dx.doi.org/10.1042/bj20101613.
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Deg/HtrA proteases are a large group of ATP-independent serine endoproteases found in almost every organism. Their usual domain arrangement comprises a trypsin-type protease domain and one or more PDZ domains. All Deg/HtrA proteases form homo-oligomers with trimers as the basic unit, where the active protease domain mediates the interaction between individual monomers. Among the members of the Deg/HtrA protease family, the plant protease DEG7 is unique since it contains two protease domains (one active and one degenerated) and four PDZ domains. In the present study, we investigated the oligomerization behaviour of this unusual protease using yeast two-hybrid analysis in vivo and with recombinant protein in vitro. We show that DEG7 forms trimeric complexes, but in contrast with other known Deg/HtrA proteases, it shows a new principle of oligomerization, where trimerization is based on the interactions between degenerated protease domains. We propose that, during evolution, a duplicated active protease domain degenerated and specialized in protein–protein interaction and complex formation.
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Mann, Krin, and Hélène Sanfaçon. "Expanding Repertoire of Plant Positive-Strand RNA Virus Proteases." Viruses 11, no. 1 (January 15, 2019): 66. http://dx.doi.org/10.3390/v11010066.
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Many plant viruses express their proteins through a polyprotein strategy, requiring the acquisition of protease domains to regulate the release of functional mature proteins and/or intermediate polyproteins. Positive-strand RNA viruses constitute the vast majority of plant viruses and they are diverse in their genomic organization and protein expression strategies. Until recently, proteases encoded by positive-strand RNA viruses were described as belonging to two categories: (1) chymotrypsin-like cysteine and serine proteases and (2) papain-like cysteine protease. However, the functional characterization of plant virus cysteine and serine proteases has highlighted their diversity in terms of biological activities, cleavage site specificities, regulatory mechanisms, and three-dimensional structures. The recent discovery of a plant picorna-like virus glutamic protease with possible structural similarities with fungal and bacterial glutamic proteases also revealed new unexpected sources of protease domains. We discuss the variety of plant positive-strand RNA virus protease domains. We also highlight possible evolution scenarios of these viral proteases, including evidence for the exchange of protease domains amongst unrelated viruses.
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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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Greenfield, Lucy M., Paul W. Hill, Eric Paterson, Elizabeth M. Baggs, and Davey L. Jones. "Do plants use root-derived proteases to promote the uptake of soil organic nitrogen?" Plant and Soil 456, no. 1-2 (September 23, 2020): 355–67. http://dx.doi.org/10.1007/s11104-020-04719-6.
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Abstract Aims The capacity of plant roots to directly acquire organic nitrogen (N) in the form of oligopeptides and amino acids from soil is well established. However, plants have poor access to protein, the central reservoir of soil organic N. Our question is: do plants actively secrete proteases to enhance the breakdown of soil protein or are they functionally reliant on soil microorganisms to undertake this role? Methods Growing maize and wheat under sterile hydroponic conditions with and without inorganic N, we measured protease activity on the root surface (root-bound proteases) or exogenously in the solution (free proteases). We compared root protease activities to the rhizosphere microbial community to estimate the ecological significance of root-derived proteases. Results We found little evidence for the secretion of free proteases, with almost all protease activity associated with the root surface. Root protease activity was not stimulated under N deficiency. Our findings suggest that cereal roots contribute one-fifth of rhizosphere protease activity. Conclusions Our results indicate that plant N uptake is only functionally significant when soil protein is in direct contact with root surfaces. The lack of protease upregulation under N deficiency suggests that root protease activity is unrelated to enhanced soil N capture.
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Ferrall-Fairbanks, Meghan C., Chris A. Kieslich, and Manu O. Platt. "Reassessing enzyme kinetics: Considering protease-as-substrate interactions in proteolytic networks." Proceedings of the National Academy of Sciences 117, no. 6 (January 24, 2020): 3307–18. http://dx.doi.org/10.1073/pnas.1912207117.
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Enzymes are catalysts in biochemical reactions that, by definition, increase rates of reactions without being altered or destroyed. However, when that enzyme is a protease, a subclass of enzymes that hydrolyze other proteins, and that protease is in a multiprotease system, protease-as-substrate dynamics must be included, challenging assumptions of enzyme inertness, shifting kinetic predictions of that system. Protease-on-protease inactivating hydrolysis can alter predicted protease concentrations used to determine pharmaceutical dosing strategies. Cysteine cathepsins are proteases capable of cathepsin cannibalism, where one cathepsin hydrolyzes another with substrate present, and misunderstanding of these dynamics may cause miscalculations of multiple proteases working in one proteolytic network of interactions occurring in a defined compartment. Once rates for individual protease-on-protease binding and catalysis are determined, proteolytic network dynamics can be explored using computational models of cooperative/competitive degradation by multiple proteases in one system, while simultaneously incorporating substrate cleavage. During parameter optimization, it was revealed that additional distraction reactions, where inactivated proteases become competitive inhibitors to remaining, active proteases, occurred, introducing another network reaction node. Taken together, improved predictions of substrate degradation in a multiple protease network were achieved after including reaction terms of autodigestion, inactivation, cannibalism, and distraction, altering kinetic considerations from other enzymatic systems, since enzyme can be lost to proteolytic degradation. We compiled and encoded these dynamics into an online platform (https://plattlab.shinyapps.io/catKLS/) for individual users to test hypotheses of specific perturbations to multiple cathepsins, substrates, and inhibitors, and predict shifts in proteolytic network reactions and system dynamics.
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Hooper, Nigel M. "Proteases: a primer." Essays in Biochemistry 38 (October 1, 2002): 1–8. http://dx.doi.org/10.1042/bse0380001.
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A protease can be defined as an enzyme that hydrolyses peptide bonds. Proteases can be divided into endopeptidases, which cleave internal peptide bonds in substrates, and exopeptidases, which cleave the terminal peptide bonds. Exopeptidases can be further subdivided into aminopeptidases and carboxypeptidases. The Schechter and Berger nomenclature provides a model for describing the interactions between the peptide substrate and the active site of a protease. Proteases can also be classified as aspartic proteases, cysteine proteases, metalloproteases, serine proteases and threonine proteases, depending on the nature of the active site. Different inhibitors can be used experimentally to distinguish between these classes of protease. The MEROPs database groups proteases into families on the basis of similarities in sequence and structure. Protease activity can be regulated in vivo by endogenous inhibitors, by the activation of zymogens and by altering the rate of their synthesis and degradation.
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Alias, Maslinda, Hakim Che Harun Mohammad, Ashraf Razali Nurul, Jasnizat Saidin, Nazaitulshila Rasit, Munirah Mohd Zaideen Izyan, and Sofiah Hamzah. "Production and characterisation of thermostable alkaline protease from Bacillus subtilis isolated from LA hot spring, Terengganu." Research Journal of Biotechnology 16, no. 7 (June 25, 2021): 84–91. http://dx.doi.org/10.25303/167rjbt8421.
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This research aims to produce thermostable alkaline protease from Bacillus subtilis isolated from La Hot Spring, Terengganu, Malaysia. The study was also conducted to determine the optimum conditions for protease production and stability by considering several parameters including pH, temperature and salt concentration. All seven bacteria were screened on skim milk agar overnight at 37 °C. Three strains with the highest proteolytic activity were identified in protease specific medium. The thermostable alkaline protease had an optimum temperature of 60 °C which achieved 85.73, 82.90 and 83.05 U/mL of protease activity for the three strains respectively. Furthermore, the strains exhibited significant activity of more than 90% from their original activity. Meanwhile, the optimum pH for protease production was pH 9 with the protease activity of 76.76, 79.71 and 88.39 U/mL for TB4, TB6 and TB9 strains, respectively. Proteases were found stable at pH 9 where the loss did not exceed 30% of its original activity. Collectively, all of the data emphasised that proteases from B. subtilis were alkaline thermostable proteases in accordance with a recent report. The finding highlights the viability of the proteases for biotechnological and industrial applications.
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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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Su, Hongfei, Zhenlun Xiao, Kefu Yu, Qinyu Huang, Guanghua Wang, Yinghui Wang, Jiayuan Liang, et al. "Diversity of cultivable protease-producing bacteria and their extracellular proteases associated to scleractinian corals." PeerJ 8 (May 6, 2020): e9055. http://dx.doi.org/10.7717/peerj.9055.
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Protease-producing bacteria play a vital role in degrading organic nitrogen in marine environments. However, the diversity of the bacteria and extracellular proteases has seldom been addressed, especially in communities of coral reefs. In this study, 136 extracellular protease-producing bacterial strains were isolated from seven genera of scleractinian corals from Luhuitou fringing reef, and their protease types were characterized. The massive coral had more cultivable protease-producing bacteria than branching or foliose corals. The abundance of cultivable protease-producing bacteria reached 106 CFU g−1 of coral. Phylogenetic analysis of 16S rRNA gene sequences revealed that the isolates were assigned to 24 genera, from which 20 corresponded to the phyla Firmicutes and Proteobacteria. Bacillus and Fictibacillus were retrieved from all coral samples. Moreover, Vibrio and Pseudovibrio were most prevalent in massive or foliose coral Platygyra and Montipora. In contrast, 11 genera were each identified in only one isolate. Nearly all the extracellular proteases from the bacteria were serine proteases or metalloproteases; 45.83% of isolates also released cysteine or aspartic proteases. These proteases had different hydrolytic ability against different substrates. This study represents a novel insight on the diversity of cultivable protease-producing bacteria and their extracellular proteases in scleractinian corals.
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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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FAMPA, P., C. V. LISBOA, A. M. JANSEN, A. L. S. SANTOS, and M. I. RAMIREZ. "Protease expression analysis in recently field-isolated strains of Trypanosoma cruzi: a heterogeneous profile of cysteine protease activities between TC I and TC II major phylogenetic groups." Parasitology 135, no. 9 (July 14, 2008): 1093–100. http://dx.doi.org/10.1017/s0031182008004587.
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SUMMARYProtease expression among TCI and TCII field isolates was analysed. Gelatin-containing gels revealed hydrolysis bands with molecular masses ranging from 45 to 66 kDa. The general protease expression profile showed that TCII isolates presented higher heterogeneity compared to TCI. By utilizing protease inhibitors, we showed that all active proteases at acid pH are cysteine-proteases and all proteases active at alkaline pH are metalloproteases. However, the expression of cruzipain, the T. cruzi major cysteine-protease, did not reproduce a heterogeneous TCII cysteine zymogram profile. Dendogram analyses based on presence/absence matrices of proteases and cruzipain bands showed a TCI separation from the TCII group with 50–60% similarity. We suggest that the observed cysteine protease diversification contributes to differential host infection between TCI and II genotypes.
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Uba, Garba, Muntari Bala, Muhammad Mushidi Abdullah, Muhammad Nor Farhan Saat, Baskaran Gunasekaran, and Mohd Yunus Shukor. "The Application of Crude Ginger Protease as an Inhibitive Assay for Heavy Metals." Journal of Environmental Microbiology and Toxicology 7, no. 1 (July 31, 2019): 48–53. http://dx.doi.org/10.54987/jemat.v7i1.472.
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In this work, a novel source of protease for the bioassay of heavy metals has been developed using proteases extracted from ginger. The result shows that the optimum protease activity was reached at 1 mg/mL protein concentration of the ginger protease. The optimum casein concentration toward crude ginger protease activity was 1.75 mg/mL. The most suitable pH for protease from crude ginger protease was within the range from pH 5.0 to 7.0. The proteases exhibited high protease activity in a broad range of temperature from 20 to 60 oC. The optimum incubation time for the enzyme occurred at minute 30. Among the six heavy metals tested, only three heavy metals inhibited proteolytic activity of ginger crude with an inhibition more than 30% at 1 mg/L. The calculated LC50 for mercury, copper and silver were, 0.182, (95%, confidence interval (C.I.) 0.134 to 0.283), 0.071, (95% C.I. of 0.056 to 0.096), 0.054, (95% C.I. of 0.039 to 0.085), respectively. Data on the sensitivity of various proteases to heavy metals shows that the crude ginger protease is comparable in sensitivity for mercury to the achromopeptidase, bromelain, papain assays. The crude ginger protease assay for copper is comparable in sensitivity to the papain assay and appears to be more sensitive than the rest of the assays. For silver, the ginger protease was the most sensitive while other assay methods are either unable or be able to detect higher concentration of silver. The crude proteases extracted from ginger showed a good potential for the development of a rapid, sensitive, and economic inhibitive assay for the biomonitoring of mercury, copper, and silver in the environment.
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Fu, Runzhong, Wannita Klinngam, Martin Heur, Maria C. Edman, and Sarah F. Hamm-Alvarez. "Tear Proteases and Protease Inhibitors." Eye & Contact Lens: Science & Clinical Practice 46 (March 2020): S70—S83. http://dx.doi.org/10.1097/icl.0000000000000641.
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Vuorinen, Emmiliisa, Salla Valtonen, Nazia Hassan, Randa Mahran, Huda Habib, Morteza Malakoutikhah, Kari Kopra, and Harri Härmä. "Protease Substrate-Independent Universal Assay for Monitoring Digestion of Native Unmodified Proteins." International Journal of Molecular Sciences 22, no. 12 (June 14, 2021): 6362. http://dx.doi.org/10.3390/ijms22126362.
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Proteases are a group of enzymes with a catalytic function to hydrolyze peptide bonds of proteins. Proteases regulate the activity, signaling mechanism, fate, and localization of many proteins, and their dysregulation is associated with various pathological conditions. Proteases have been identified as biomarkers and potential therapeutic targets for multiple diseases, such as acquired immunodeficiency syndrome, cardiovascular diseases, osteoporosis, type 2 diabetes, and cancer, where they are essential to disease progression. Thus, protease inhibitors and inhibitor-like molecules are interesting drug candidates. To study proteases and their substrates and inhibitors, simple, rapid, and sensitive protease activity assays are needed. Existing fluorescence-based assays enable protease monitoring in a high-throughput compatible microtiter plate format, but the methods often rely on either molecular labeling or synthetic protease targets that only mimic the hydrolysis site of the true target proteins. Here, we present a homogenous, label-free, and time-resolved luminescence utilizing the protein-probe method to assay proteases with native and denatured substrates at nanomolar sensitivity. The developed protein-probe method is not restricted to any single protein or protein target class, enabling digestion and substrate fragmentation studies with the natural unmodified substrate proteins. The versatility of the assay for studying protease targets was shown by monitoring the digestion of a substrate panel with different proteases. These results indicate that the protein-probe method not only monitors the protease activity and inhibition, but also studies the substrate specificity of individual proteases.
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Das, Kamonashis, Sourav Chakraborty, Mahmudul Hasan, and Abdullah Maruf Rahman Shovo. "In silico analysis to elect superior bacterial alkaline protease for detergent and leather industries." JOURNAL OF ADVANCES IN BIOTECHNOLOGY 5, no. 3 (December 30, 2015): 685–98. http://dx.doi.org/10.24297/jbt.v5i3.1482.
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Alkaline protease contributes 40% of the total worldwide enzyme sales. Alkaline protease that is stable at very high temperature and pH is massively desirable for detergent industry and leather industry specially in tanning process. So the present study aims to elect superior bacterial alkaline protease (high temperature and pH stable) as compared to the alkaline proteases of currently industrially used bacteria (Bacillus subtilis and Bacillus cereus). A total of 50 protein sequences of alkaline proteases of different bacteria were analyzed through in silico characterization. ProtParam result revealed that isoelectric point and aliphatic index of alkaline protease of Bacillus megaterium were 8.83 and 93.35 respectively. In case of alkaline protease of B. megaterium, these two properties were significant in comparison to alkaline proteases of industrially used bacteria and other considered bacteria. A common motif of 28 amino acid residues i.e., IQSTYPGEDYEYMSGTSMATPHVAGVAA was found using MEME software in 46 protein sequences. It can be concluded that alkaline protease of Bacillus megaterium may be superior to alkaline proteases of industrially used bacteria and other considered bacteria. In addition, obtained common motif indicates its probable role in catalytic function and structure of alkaline proteases.Â
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El-Shanshoury, Abd El-Raheem R., Mostafa A. El-Sayed, Reda H. Sammour, and Wageeh A. El-Shouny. "Purification and partial characterization of two extracellular alkaline proteases from Streptomyces corchorusii ST36." Canadian Journal of Microbiology 41, no. 1 (January 1, 1995): 99–104. http://dx.doi.org/10.1139/m95-013.
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Two distinct alkaline proteases were detected in the culture medium of Streptomyces corchorusii ST36 isolated from an Egyptian soil sample. These enzymes were purified by precipitation with chilled ethanol and gel filtration on carboxymethyl Sepharose and on Sephadex G-120. The enzymes were purified 6 and 6.5 fold to homogeneity. The purified enzymes had final specific activities on substrate of 3.6 and 3.9 U/mg. Protease 1 had a molecular mass of 31 kDa and protease 2 was composed of two subunits, of molecular masses 18.6 and 17.4 kDa. The optimal pH and temperature for catalytic activity of protease 1 were pH 11 and 70 °C, respectively, and for protease 2 they were pH 10 and 70 °C, respectively. Protease 1 was more thermostable than protease 2. Both enzymes showed substrate specificity to casein, serum albumin, and ovalbumin. Calcium, copper, and cobalt stimulated protease 2 but did not significantly affect protease 1. Mercury was the strongest inhibitor for protease 2. The proteolytic activities of both proteases were inhibited by 10 mM phenanthroline and 50 mM ethylenediaminetetraacetic acid (EDTA). The inhibitory effect of EDTA on both enzymes was reversed by the addition of 50 mM of copper, calcium, or cobalt. Both enzymes were more stable at −20 °C under alkaline conditions than under neutral or acidic conditions.Key words: Streptomyces corchorusii, alkaline proteases, partial characterization.
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Kuklina, M. M., and V. V. Kuklin. "PROTEASE ACTIVITY IN THE SMALL INTESTINE OF THE NORTHERN FULMAR <i>FULMARUS GLACIALIS</i> BY INFECTION OF <i>TETRABOTHRIUS MINOR</i> (CESTODA: TETRABOTHRIIDAE)." Журнал эволюционной биохимии и физиологии 59, no. 5 (September 1, 2023): 361–69. http://dx.doi.org/10.31857/s0044452923050054.
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The effect of infection of Tetrabothrius minor (Cestoda: Tetrabothriidae) on the protease activity of the mucous membrane of the small intestine of the Northern Fulmar Fulmarus glacialis was studied. Aspects of changes in the activity of proteases and protease subclasses (metalloproteases, serine proteases and cysteine proteases) by infection of T. minor, and the ability of T. minor to inactivate proteases from the intestinal mucosa and commercial trypsin were evaluated. It has been established that in the localization of T. minor (proximal and medial sections of the small intestine) decreased protease activity due to a decrease in the activity of serine proteases and metalloproteases. The dependence of the decrease of protease activity in the mucous membrane of the small intestine of the host on the parameters of infection with cestodes was found – the higher the infection intensity of T. minor, the lower the activity of proteases, including metalloproteases and serine proteases. The ability of T. minor homogenates to inhibit the activity of proteases from the mucosa of Northern Fulmar and the activity of commercial trypsin of different concentrations was noted.
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Liu, Ying, Jun Ying An, Xu Qiong Hu, and Chun Hou Xu. "Isolation and Identification of Thermophilic Protease Bacterium 0701 and its Protease Characterization." Advanced Materials Research 781-784 (September 2013): 1032–36. http://dx.doi.org/10.4028/www.scientific.net/amr.781-784.1032.
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Soil was collected from Huguangyan of Zhanjiang city and regarded as separate material, It was screened thermophilic protease-producing bacteria though dilution flat, transparent circle and Folin method. Strain 0701 isolated was identificated by its morphological features, physiological and biochemical characteristics and 16s rRNA. This experiment also researched on the genetic stability of enzyme producing, and different temperature, pH influence on proteases activity. 25 high temperature-tolerance protease producing strains were isolated, which accounted for 28.1 % of all the isolated strains. Among them, strain 0701 proteases activity was 247 U/mL and it was far higher than that of proteases for other strains and the most optimum reaction temperature and pH of the proteases were 70oC and pH 7.0, respectively, and retained high enzyme activity within 50-80 oC, pH6.5-9.0. The strain 0703 still had the ability to produce protease after consecutive 4 generation, and was identified as Bacillus subtilis by morphological, physiological-biochemical characteristics and 16s rRNA.
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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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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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Achmad, Ayu Ashari, M. Saifur Rohman, and Irfan D. Prijambada. "Biochemical Properties of Crude Extracellular Proteases from Chromohalobacter salexigens BKL5 and Micrococcus luteus 11A." Indonesian Journal of Biotechnology 21, no. 1 (July 22, 2017): 56. http://dx.doi.org/10.22146/ijbiotech.26705.
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In this work, we have reported an enzymatic activity and biochemical properties of extracellular proteases from Chromohalobacter salexigens BKL5 and Micrococcus luteus 11A. C. salexigens BKL5 and M. luteus 11A were previously isolated from Bledug Kuwu mud volcano and dietary industry wastewater treatment, respectively. Both bacterial strains were able to produce extracellular proteases, when grown on minimal agar medium supplemented with 1% of skim milk. Proteolytic indexes of C. salexigens BKL5 and M. luteus 11A were 2.5±0.14 and 2.9±0.42, respectively. Both extracellular proteases exhibited optimum enzymatic activity at pH 7, with specific activity of C. salexigens BKL5 was 13.3% higher than that of M. luteus 11A. Optimum temperature for enzymatic activity of both proteases was 45°C. Metal cofactor preferences assay showed that extracellular protease from C. salexigens BKL5 preferred Zn2+, meanwhile extracellular protease from M. luteus 11A mainly preferred Ca2+ ion. Metal cofactor preferences assay also suggested that crude extracellular protease from C. salexigens BKL5 was categorized as metalloprotease, meanwhile crude extracellular protease of M. luteus 11A was common neutral protease. The enzymatic stability assay against various salt concentrations showed that crude extracellular protease from C. salexigens BKL5 was more stable than that of M. luteus 11A.
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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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Hook, V. Y. H., and S. R. Hwang. "Novel Secretory Vesicle Serpins, Endopin 1 and Endopin 2: Endogenous Protease Inhibitors with Distinct Target Protease Specificities." Biological Chemistry 383, no. 7-8 (August 27, 2002): 1067–74. http://dx.doi.org/10.1515/bc.2002.115.
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Abstract Secretory vesicles of neuroendocrine cells possess multiple proteases for proteolytic processing of proteins into biologically active peptide components, such as peptide hormones and neurotransmitters. The importance of proteases within secretory vesicles predicts the presence of endogenous protease inhibitors in this subcellular compartment. Notably, serpins represent a diverse class of endogenous protease inhibitors that possess selective target protease specificities, defined by the reactive site loop domains (RSL). In the search for endogenous serpins in model secretory vesicles of neuroendocrine chromaffin cells, the presence of serpins related to α1-antichymotrypsin (ACT) was detected by Western blots with antiACT. Molecular cloning revealed the primary structures of two unique serpins, endopin 1 and endopin 2, that possess homology to ACT. Of particular interest was the observation that distinct RSL domains of these new serpins predicted that endopin 1 would inhibit trypsinlike serine proteases cleaving at basic residues, and endopin 2 would inhibit both elastase and papain that represent serine and cysteine proteases, respectively. Endopin 1 showed selective inhibition of trypsin, but did not inhibit chymotrypsin, elastase, or subtilisin. Endopin 2 demonstrated crossclass inhibition of the cysteine protease papain and the serine protease elastase. Endopin 2 did not inhibit chymotrypsin, trypsin, plasmin, thrombin, furin, or cathepsin B. Endopin 1 and endopin 2 each formed SDSstable complexes with target proteases, a characteristic property of serpins. In neuroendocrine chromaffin cells from adrenal medulla, endopin 1 and endopin 2 were both localized to secretory vesicles. Moreover, the inhibitory activity of endopin 2 was optimized under reducing conditions, which required reduced Cys-374; this property is consistent with the presence of endogenous reducing agents in secretory vesicles in vivo. These new findings demonstrate the presence of unique secretory vesicle serpins, endopin 1 and endopin 2, which possess distinct target protease selectivities. Endopin 1 inhibits trypsinlike proteases; endopin 2 possesses crossclass inhibition for inhibition of papainlike cysteine proteases and elastaselike serine proteases. It will be of interest in future studies to define the endogenous protease targets of these two novel secretory vesicle serpins.
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Gondzik, Veronika, Wolf Michael Weber, and Mouhamed S. Awayda. "Coupling of epithelial Na+ and Cl− channels by direct and indirect activation by serine proteases." American Journal of Physiology-Cell Physiology 303, no. 9 (November 1, 2012): C936—C946. http://dx.doi.org/10.1152/ajpcell.00395.2011.
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The mammalian collecting duct (CD) is continuously exposed to urinary proteases. The CD expresses an epithelial Na+ channel (ENaC) that is activated after cleavage by serine proteases. ENaC also exists at the plasma membrane in the uncleaved form, rendering activation by extracellular proteases an important mechanism for regulating Na+ transport. Many exogenous and a small number of endogenous extracellular serine proteases have been shown to activate the channel. Recently, kallikrein 1 (KLK1) was shown to increase γENaC cleavage in the native CD indicating a possible direct role of this endogenous protease in Na+ homeostasis. To explore this process, we examined the coordinated effect of this protease on Na+ and Cl− transport in a polarized renal epithelial cell line (Madin-Darby canine kidney). We also examined the role of native urinary proteases in this process. Short-circuit current ( Isc) was used to measure transport of these ions. The Isc exhibited an ENaC-dependent Na+ component that was amiloride blockable and a cystic fibrosis transmembrane conductance regulator (CFTR)-dependent Cl− component that was blocked by inhibitor 172. Apical application of trypsin, an exogenous S1 serine protease, activated IENaC but was without effects on ICFTR. Subtilisin an exogenous S8 protease that mimics endogenous furin-type proteases activated both currents. A similar activation was also observed with KLK1 and native rat urinary proteases. Activation with urinary proteases occurred within minutes and at protease concentrations similar to those in the CD indicating physiological significance of this process. ENaC activation was irreversible and mediated by enhanced cleavage of γENaC. The activation of CFTR was indirect and likely dependent on activation of an endogenous apical membrane protease receptor. Collectively, these data demonstrate coordinated stimulation of separate Na+ and Cl− transport pathways in renal epithelia by extracellular luminal proteases. They also indicate that baseline urinary proteolytic activity is sufficient to modify Na+ and Cl− transport in these epithelia.
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Zhu, Yiwen, Jinzhi Song, Xu Zhang, Mengchu Gao, Biyu Peng, and Chunxiao Zhang. "Effect of Electrostatic Interaction between Collagen and Enzymes on Permeation of Protease into the Pelt during Leather Bating Process." Journal of the American Leather Chemists Association 118, no. 10 (October 2, 2023): 428–38. http://dx.doi.org/10.34314/jalca.v118i10.8231.
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The enzymatic delimed pelts bating process using proteases is critical to improving the overall performance of the leather. Bating effectiveness is determined not only by the properties but also by the permeation behavior of the proteases. Imperfect methods to control protease permeation often results in uneven distribution of enzyme proteins in the pelts, leading to excessive enzymolysis of the surface layer and inadequate opening-up of the inner layer. In this study, the relative size of proteases and delimed pelts were analyzed, the permeation behavior of fluorescein-labeled proteases in the pelt was observed using a confocal laser scanning microscope (CLSM), and the effect of electrostatic interaction between protease and collagen proteins on the permeation of protease into the pelt was investigated. The results showed that, after dehairing, liming and deliming operations, the enzyme can easily permeate into the pelts due to the formation of large cavities and interfibrillar gaps. The permeation of protease within the delimed pelt is significantly influenced by the affinity (electrostatic interactions) between the collagen and protease proteins. The isoelectric point (pI) of the protease protein, the collagen and the pH of the solution directly influence the electrostatic properties and interactions. When the enzyme and collagen are similarly charged (electrostatic repulsion), the enzyme can easily permeate into the pelts; when the enzyme and collagen are oppositely charged (electrostatic attraction), the permeation of the enzyme into the inner layer is difficult, resulting in the accumulation of protease on the grain and excessive hydrolysis of the grain layer. Therefore, the established permeation regulation mechanism of protease based on electrostatic interactions between enzyme and collagen could serve as an important basis for the selection of protease and the regulation of the enzymatic bating process.
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Li, Qingxin, and Congbao Kang. "Structure and Dynamics of Zika Virus Protease and Its Insights into Inhibitor Design." Biomedicines 9, no. 8 (August 19, 2021): 1044. http://dx.doi.org/10.3390/biomedicines9081044.
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Zika virus (ZIKV)—a member of the Flaviviridae family—is an important human pathogen. Its genome encodes a polyprotein that can be further processed into structural and non-structural proteins. ZIKV protease is an important target for antiviral development due to its role in cleaving the polyprotein to release functional viral proteins. The viral protease is a two-component protein complex formed by NS2B and NS3. Structural studies using different approaches demonstrate that conformational changes exist in the protease. The structures and dynamics of this protease in the absence and presence of inhibitors were explored to provide insights into the inhibitor design. The dynamic nature of residues binding to the enzyme cleavage site might be important for the function of the protease. Due to the charges at the protease cleavage site, it is challenging to develop small-molecule compounds acting as substrate competitors. Developing small-molecule compounds to inhibit protease activity through an allosteric mechanism is a feasible strategy because conformational changes are observed in the protease. Herein, structures and dynamics of ZIKV protease are summarized. The conformational changes of ZIKV protease and other proteases in the same family are discussed. The progress in developing allosteric inhibitors is also described. Understanding the structures and dynamics of the proteases are important for designing potent inhibitors.
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Lebuan, Urbanus Yustus, Roga Florida Kembaren, Merry Meryam Martgrita, and Cut Rizlani Kholibrina. "Thrombolytic protease characterization from leaves and fruit flesh of the jernang rattan plant (Daemonorops draco)." Indonesian Journal of Biotechnology 28, no. 4 (December 30, 2023): 248. http://dx.doi.org/10.22146/ijbiotech.82390.
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Thrombolytic agents are used for thrombolytic therapy to dissolve blood clots that form in a blood vessel. All currently used thrombolytic agents have unfavorable shortcomings, such as gastrointestinal bleeding, allergic reactions, and thrombolytic agent resistance, treatment for some of which can be quite expensive. As a result, the search for thrombolytic agents derived from plants is currently taking place. Some plants have been discovered to contain protease enzymes with thrombolytic activity; pharmaceuticals derived from plants are believed to be safer. Jernang rattan (Daemonorops draco) is a plant of the Arecaceae family and is known to produce resin. Jernang rattan resin is also known to have antioxidant, antiseptic, antitumor, antimicrobial, and cytotoxic activity, but very limited information on proteolytic activity of the protease from this plant. This research aims to isolate proteases from the leaves and fruit flesh of the rattan jernang plant (D. draco) and to investigate the proteolytic activity of the isolated proteases. The protease was isolated from the leaves and the fruit flesh, and then partially purified by ammonium sulfate precipitation. The radial caseinolytic assay showed that protease in a 60% ammonium sulfate fraction gave a clear zone, with diameters of 1.4 cm and 1.8 cm for the protease isolated from leaves and fruit flesh, respectively. A Folin‐Ciocalteau assay showed that the enzymes isolated were able to hydrolyze casein and release L‐tyrosine, with activity of 0.158 U/mL and 0.174 U/mL for the protease from the leaves and fruit flesh, respectively. A fibrinogenolytic assay showed that the protease from the fruit flesh hydrolyzed the A‐α, B‐β and the γ chain of human fibrinogen, while the protease from the leaves hydrolyzed the A‐α and γ chain. Both proteases were inhibited by 56% by phenylmethylsulfonyl fluoride (PMSF), indicating that the enzymes are serine proteases. Based on the assay results obtained, it can be concluded that proteases isolated from the leaves and fruit flesh have potential as thrombolytic proteases.
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Weeda, Sarah M., G. N. Mohan Kumar, and N. Richard Knowles. "Correlative changes in proteases and protease inhibitors during mobilisation of protein from potato (Solanum tuberosum) seed tubers." Functional Plant Biology 37, no. 1 (2010): 32. http://dx.doi.org/10.1071/fp09188.
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Potato tubers (Solanum tuberosum L.) contain protease inhibitors that function in plant defence and as storage proteins. A multi-domain cysteine protease inhibitor, potato multicystatin (PMC), has also been implicated in regulating protein accumulation in developing tubers by inhibiting proteases. Unlike developing tubers, sprouting tubers mobilise protein reserves to support growth of developing plants and, therefore, show an increase in protease activity. Using single-eye containing cores (seedcores) from seed tubers, we characterised the relative changes in patatin, PMC, proteases and serine (Ser) protease inhibitors, as a prerequisite to further research on their potential roles in protein mobilisation from tubers during plant establishment. Approximately 63% of seedcore dry matter was mobilised over a 29-day period of plant establishment (1.7 mg seedcore dry matter mobilised for every mg increase in plant dry matter). The gelatinolytic protease isoforms induced in seedcores during plant establishment differed from those characterised previously in developing tubers. Total protease activity increased progressively in seedcores and reached a maximum 23 days after planting. Conversely, seedcore soluble protein content declined, with patatin accounting for the greatest decrease in the soluble protein fraction during plant establishment. PMC also decreased 44% and Ser (trypsin) protease inhibitors decreased to levels barely detectable in seedcores over the 29-day growth interval. Moreover, the temporal changes in PMC, protease activity and patatin content were highly correlated. As PMC decreased from 6 to 4 ng core–1, protease activity increased 9-fold, patatin decreased 2.6-fold and total soluble protein decreased by 58%. These results suggest that catabolism of protease inhibitors may facilitate protein mobilisation from seed tubers. Further work to define unequivocally the role of protease inhibitors in modulating the activity of proteases during protein mobilisation from tubers is warranted.
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Strachecka, A. J., M. M. Gryzińska, M. Krauze, and K. Grzywnowicz. "Profile of the body surface proteolytic systém in Apis mellifera quee." Czech Journal of Animal Science 56, No. 1 (January 20, 2011): 15–22. http://dx.doi.org/10.17221/150/2009-cjas.
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The proteolytic system on the body surface of the honey bee has been insufficiently researched. In this study the body surface proteolytic activity was examined in queens at various developmental stages (eggs, larvae, pupae and imagines) in different seasons (spring, summer, autumn, winter). Extracts of the body surface material with water and detergent were used for an in vitro analysis of the proteolytic activity and protease inhibitor level assaying, as well as for an electrophoretic separation of the extracts in polyacrylamide gels. The following methods were used: protein content testing by the Lowry method (modified by Schacterle-Pollack), protease activity testing by the Anson method and protease inhibitor activity testing by the Lee and Lin method. Our studies revealed a high protease activity in an acidic environment (pH = 2.4; the material rinsed with detergent), as well as in neutral (pH = 7) and alkaline (pH = 11.2) environments (the material rinsed with water in both cases). The highest protein concentration values were observed in the imagines from summer. The lowest activities of the proteases and protease inhibitors were determined in the eggs from summer. The highest activities of the acidic, neutral and alkaline proteases were observed in the pupae from spring. The highest number of protease activity bands in PAGE zymography was obtained for the neutral and alkaline activities in the queens for all the seasons. In the queens all the catalytic protease types were present: asparagine and cysteine proteases at pH = 2.4; cysteine proteases and metalloproteases at pH = 7 and serine proteases at pH = 11.2. These results were crucial for the analysis of immunity mechanisms on the body surface of the honey bee.
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Pettit, Steven C., Sergei Gulnik, Lori Everitt, and Andrew H. Kaplan. "The Dimer Interfaces of Protease and Extra-Protease Domains Influence the Activation of Protease and the Specificity of GagPol Cleavage." Journal of Virology 77, no. 1 (January 1, 2003): 366–74. http://dx.doi.org/10.1128/jvi.77.1.366-374.2003.
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ABSTRACT Activation of the human immunodeficiency virus type 1 (HIV-1) protease is an essential step in viral replication. As is the case for all retroviral proteases, enzyme activation requires the formation of protease homodimers. However, little is known about the mechanisms by which retroviral proteases become active within their precursors. Using an in vitro expression system, we have examined the determinants of activation efficiency and the order of cleavage site processing for the protease of HIV-1 within the full-length GagPol precursor. Following activation, initial cleavage occurs between the viral p2 and nucleocapsid proteins. This is followed by cleavage of a novel site located in the transframe domain. Mutational analysis of the dimer interface of the protease produced differential effects on activation and specificity. A subset of mutations produced enhanced cleavage at the amino terminus of the protease, suggesting that, in the wild-type precursor, cleavages that liberate the protease are a relatively late event. Replacement of the proline residue at position 1 of the protease dimer interface resulted in altered cleavage of distal sites and suggests that this residue functions as a cis-directed specificity determinant. In summary, our studies indicate that interactions within the protease dimer interface help determine the order of precursor cleavage and contribute to the formation of extended-protease intermediates. Assembly domains within GagPol outside the protease domain also influence enzyme activation.
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Chourasia, P., B. Patel, M. M. Prakash, and S. Gaherwal. "Screening and Optimization of Extracellular Alkaline Protease Production from Bacillus Spp." Environment Conservation Journal 13, no. 3 (December 20, 2012): 49–52. http://dx.doi.org/10.36953/ecj.2012.130309.
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Protease enzyme catalyzes the hydrolysis of protein. Among the various proteases, bacterial proteases are most significant when compared with animal and fungal proteases. In the present study a protease producing bacteria were isolated from soil collected from Govt. Holkar Science College, Indore campus and identified as Bacillus spp. They were grown within a temperature range between 25°C & 45 °C and pH range of 6.0 to 11.0. The optimum condition for protease production obtained was 35 °C at pH 9. The best carbon and organic nitrogen sources for this bacterial strain were fructose and yeast extract, respectively, while the most effective inorganic nitrogen sources was urea. It is envisaged that the isolate can be a potential source of alkaline protease for use as additive in industrial applications like detergent industry.
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Serrano-Luna, José de Jesús, Isaac Cervantes-Sandoval, Jesús Calderón, Fernando Navarro-García, Victor Tsutsumi, and Mineko Shibayama. "Protease activities of Acanthamoeba polyphaga and Acanthamoeba castellanii." Canadian Journal of Microbiology 52, no. 1 (January 1, 2006): 16–23. http://dx.doi.org/10.1139/w05-114.
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Acanthamoeba spp. are free-living amoebae that cause amoebic granulomatous encephalitis, skin lesions, and ocular amoebic keratitis in humans. Several authors have suggested that proteases could play a role in the pathogenesis of these diseases. In the present work, we performed a partial biochemical characterization of proteases in crude extracts of Acanthamoeba spp. and in conditioned medium using 7.5% SDS–PAGE copolymerized with 0.1% m/v gelatin as substrate. We distinguished a total of 17 bands with proteolytic activity distributed in two species of Acanthamoeba. The bands ranged from 30 to 188 kDa in A. castellanii and from 34 to 144 kDa in A. polyphaga. Additionally, we showed that the pattern of protease activity differed in the two species of Acanthamoeba when pH was altered. By using protease inhibitors, we found that the proteolytic activities belonged mostly to the serine protease family and secondly to cysteine proteases and that the proteolytic activities from A. castellanii were higher than those in A. polyphaga. Furthermore, aprotinin was found to in hibit crude extract protease activity on Madin–Darby canine kidney (MDCK) monolayers. These data suggest that protease patterns could be more complex than previously reported.Key words: Acanthamoeba spp., amoebic keratitis, serine proteases, cysteine proteases, cytopathic effect.
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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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Tyagi, R. D., V. Sikati Foko, S. Barnabe, A. S. Vidyarthi, J. R. Valéro, and R. Y. Surampalli. "Simultaneous production of biopesticide and alkaline proteases by Bacillus thuringiensis using sewage sludge as a raw material." Water Science and Technology 46, no. 10 (November 1, 2002): 247–54. http://dx.doi.org/10.2166/wst.2002.0344.
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The simultaneous production of Bacillus thuringiensis (Bt) based biopesticide and proteases was studied using synthetic medium and wastewater sludge as a raw material. The studies were conducted in shake flask and computer controlled 15-L capacity fermentors. Measuring viable cell and spore counts, entomotoxicity and protease activity monitored the progress of the biopesticide production process. A higher viable cell count and spore count was observed in synthetic Soya medium, however, higher entomotoxicity and protease activity were observed in wastewater sludge medium. Thus, the wastewater sludge is a better raw material than commercial Soya medium for the biopesticides and enzyme production. The maximum entomotoxicity and protease activity observed in the fermentor was 9,332 IU/μL and 4.58 IU/mL, respectively. The proteases produced by Bt were also characterised. Two types of proteases were detected; neutral proteases with pH optimum 7.0 and alkaline proteases with pH optimum 10-11. Further, two types of alkaline proteases were detected; one having a pH and temperature optimum at 10 and 50°C while the other at 11 and 70°C. The protease thermal stability was found to increase in the presence of CaCl2, indicating the proteases were metalloproteases.
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Sakoda, Yoshihiro, Natalie Ross-Smith, Toru Inoue, and Graham J. Belsham. "An Attenuating Mutation in the 2A Protease of Swine Vesicular Disease Virus, a Picornavirus, Regulates Cap- and Internal Ribosome Entry Site-Dependent Protein Synthesis." Journal of Virology 75, no. 22 (November 15, 2001): 10643–50. http://dx.doi.org/10.1128/jvi.75.22.10643-10650.2001.
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ABSTRACT Virulent and avirulent strains of swine vesicular disease virus (SVDV), a picornavirus, have been characterized previously. The major determinants for attenuation have been mapped to specific residues in the 1D-2A-coding region. The properties of the 2A proteases from the virulent and avirulent strains of SVDV have now been examined. Both proteases efficiently cleaved the 1D/2A junction in vitro and in vivo. However, the 2A protease of the avirulent strain of SVDV was much less effective than the virulent-virus 2A protease at inducing cleavage of translation initiation factor eIF4GI within transfected cells. Hence the virulent-virus 2A protease is much more effective at inhibiting cap-dependent protein synthesis. Furthermore, the virulent-virus 2A protease strongly stimulated the internal ribosome entry sites (IRESs) from coxsackievirus B4 and from SVDV, while the avirulent-virus 2A protease was significantly less active in these assays. Thus, the different properties of the 2A proteases from the virulent and avirulent strains of SVDV in regulating protein synthesis initiation reflect the distinct pathogenic properties of the viruses from which they are derived. A single amino acid substitution, adjacent to His21 of the catalytic triad, is sufficient to confer the characteristics of the virulent-strain 2A protease on the avirulent-strain protease. It is concluded that the efficiency of picornavirus protein synthesis, controlled directly by the IRES or indirectly by the 2A protease, can determine virus virulence.
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Kryvalap, Yury, and Jan Czyzyk. "The Role of Proteases and Serpin Protease Inhibitors in β-Cell Biology and Diabetes." Biomolecules 12, no. 1 (January 2, 2022): 67. http://dx.doi.org/10.3390/biom12010067.
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Regulation of the equilibrium between proteases and their inhibitors is fundamental to health maintenance. Consequently, developing a means of targeting protease activity to promote tissue regeneration and inhibit inflammation may offer a new strategy in therapy development for diabetes and other diseases. Specifically, recent efforts have focused on serine protease inhibitors, known as serpins, as potential therapeutic targets. The serpin protein family comprises a broad range of protease inhibitors, which are categorized into 16 clades that are all extracellular, with the exception of Clade B, which controls mostly intracellular proteases, including both serine- and papain-like cysteine proteases. This review discusses the most salient, and sometimes opposing, views that either inhibition or augmentation of protease activity can bring about positive outcomes in pancreatic islet biology and inflammation. These potential discrepancies can be reconciled at the molecular level as specific proteases and serpins regulate distinct signaling pathways, thereby playing equally distinct roles in health and disease development.
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Semenov, Andrey, Jed E. Olson, and Philip J. Rosenthal. "Antimalarial Synergy of Cysteine and Aspartic Protease Inhibitors." Antimicrobial Agents and Chemotherapy 42, no. 9 (September 1, 1998): 2254–58. http://dx.doi.org/10.1128/aac.42.9.2254.
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ABSTRACT It has been proposed that the Plasmodium falciparumcysteine protease falcipain and aspartic proteases plasmepsin I and plasmepsin II act cooperatively to hydrolyze hemoglobin as a source of amino acids for erythrocytic parasites. Inhibitors of each of these proteases have potent antimalarial effects. We have now evaluated the antimalarial effects of combinations of cysteine and aspartic protease inhibitors. When incubated with cultured P. falciparumparasites, cysteine and aspartic protease inhibitors exhibited synergistic effects in blocking parasite metabolism and development. The inhibitors also demonstrated apparent synergistic inhibition of plasmodial hemoglobin degradation both in culture and in a murine malaria model. When evaluated for the treatment of murine malaria, a combination of cysteine and aspartic protease inhibitors was much more effective than higher concentrations of either compound used alone. These results support a model whereby plasmodial cysteine and aspartic proteases participate in the degradation of hemoglobin, and they suggest that combination antimalarial therapy with inhibitors of the two classes of proteases is worthy of further study.
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Mintoo, Mubashir, Amritangshu Chakravarty, and Ronak Tilvawala. "N-Terminomics Strategies for Protease Substrates Profiling." Molecules 26, no. 15 (August 3, 2021): 4699. http://dx.doi.org/10.3390/molecules26154699.
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Proteases play a central role in various biochemical pathways catalyzing and regulating key biological events. Proteases catalyze an irreversible post-translational modification called proteolysis by hydrolyzing peptide bonds in proteins. Given the destructive potential of proteolysis, protease activity is tightly regulated. Dysregulation of protease activity has been reported in numerous disease conditions, including cancers, neurodegenerative diseases, inflammatory conditions, cardiovascular diseases, and viral infections. The proteolytic profile of a cell, tissue, or organ is governed by protease activation, activity, and substrate specificity. Thus, identifying protease substrates and proteolytic events under physiological conditions can provide crucial information about how the change in protease regulation can alter the cellular proteolytic landscape. In recent years, mass spectrometry-based techniques called N-terminomics have become instrumental in identifying protease substrates from complex biological mixtures. N-terminomics employs the labeling and enrichment of native and neo-N-termini peptides, generated upon proteolysis followed by mass spectrometry analysis allowing protease substrate profiling directly from biological samples. In this review, we provide a brief overview of N-terminomics techniques, focusing on their strengths, weaknesses, limitations, and providing specific examples where they were successfully employed to identify protease substrates in vivo and under physiological conditions. In addition, we explore the current trends in the protease field and the potential for future developments.
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Howng, Bruce, Michael B. Winter, Carol LePage, Irina Popova, Michael Krimm, and Olga Vasiljeva. "Novel Ex Vivo Zymography Approach for Assessment of Protease Activity in Tissues with Activatable Antibodies." Pharmaceutics 13, no. 9 (September 2, 2021): 1390. http://dx.doi.org/10.3390/pharmaceutics13091390.
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Proteases are involved in the control of numerous physiological processes, and their dysregulation has been identified in a wide range of pathologies, including cancer. Protease activity is normally tightly regulated post-translationally and therefore cannot be accurately estimated based on mRNA or protein expression alone. While several types of zymography approaches to estimate protease activity exist, there remains a need for a robust and reliable technique to measure protease activity in biological tissues. We present a novel quantitative ex vivo zymography (QZ) technology based on Probody® therapeutics (Pb-Tx), a novel class of protease-activated cancer therapeutics that contain a substrate linker cleavable by tumor-associated proteases. This approach enables the measurement and comparison of protease activity in biological tissues via the detection of Pb-Tx activation. By exploiting substrate specificity and selectivity, cataloguing and differentiating protease activities is possible, with further refinement achieved using protease-specific inhibitors. Using the QZ assay and human tumor xenografts, patient tumor tissues, and patient plasma, we characterized protease activity in preclinical and clinical samples. The QZ assay offers the potential to increase our understanding of protease activity in tissues and inform diagnostic and therapeutic development for diseases, such as cancer, that are characterized by dysregulated proteolysis.
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Chan, Edward D., Paul T. King, Xiyuan Bai, Allen M. Schoffstall, Robert A. Sandhaus, and Ashley M. Buckle. "The Inhibition of Serine Proteases by Serpins Is Augmented by Negatively Charged Heparin: A Concise Review of Some Clinically Relevant Interactions." International Journal of Molecular Sciences 25, no. 3 (February 2, 2024): 1804. http://dx.doi.org/10.3390/ijms25031804.
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Serine proteases are members of a large family of hydrolytic enzymes in which a particular serine residue in the active site performs an essential role as a nucleophile, which is required for their proteolytic cleavage function. The array of functions performed by serine proteases is vast and includes, among others, the following: (i) the ability to fight infections; (ii) the activation of blood coagulation or blood clot lysis systems; (iii) the activation of digestive enzymes; and (iv) reproduction. Serine protease activity is highly regulated by multiple families of protease inhibitors, known collectively as the SERine Protease INhibitor (SERPIN). The serpins use a conformational change mechanism to inhibit proteases in an irreversible way. The unusual conformational change required for serpin function provides an elegant opportunity for allosteric regulation by the binding of cofactors, of which the most well-studied is heparin. The goal of this review is to discuss some of the clinically relevant serine protease–serpin interactions that may be enhanced by heparin or other negatively charged polysaccharides. The paired serine protease–serpin in the framework of heparin that we review includes the following: thrombin–antithrombin III, plasmin–anti-plasmin, C1 esterase/kallikrein–C1 esterase inhibitor, and furin/TMPRSS2 (serine protease Transmembrane Protease 2)–alpha-1-antitrypsin, with the latter in the context of COVID-19 and prostate cancer.
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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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Braaksma, Machtelt, Age K. Smilde, Mariët J. van der Werf, and Peter J. Punt. "The effect of environmental conditions on extracellular protease activity in controlled fermentations of Aspergillus niger." Microbiology 155, no. 10 (October 1, 2009): 3430–39. http://dx.doi.org/10.1099/mic.0.031062-0.
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Proteolytic degradation by host proteases is one of the key issues in the application of filamentous fungi for non-fungal protein production. In this study the influence of several environmental factors on the production of extracellular proteases of Aspergillus niger was investigated systematically in controlled batch cultures. Of all factors investigated in a series of initial screening experiments, culture pH and nitrogen concentration in particular strongly affected extracellular protease activities. For instance, at a culture pH of 4, protease activity was higher than at pH 5, and protease activity increased with increasing concentrations of ammonium as nitrogen source. Interestingly, an interdependence was observed for several of the factors studied. These possible interaction effects were investigated further using a full factorial experimental design. Amongst others, the results showed a clear interaction effect between nitrogen source and nitrogen concentration. Based on the observed interactions, the selection of environmental factors to reduce protease activity is not straightforward, as unexpected antagonistic or synergistic effects occur. Furthermore, not only were the effects of the process parameters on maximum protease activity investigated, but five other protease-related phenotypes were studied as well, such as maximum specific protease activity and maximum protease productivity. There were significant differences in the effect of the environmental parameters on the various protease-related phenotypes. For instance, pH significantly affected final levels of protease activity, but not protease productivity. The results obtained in this study are important for the optimization of A. niger for protein production.
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Gunasekaran, Baskaran, M. A. Syed, Shamala Salvamani, Subathra Sinniah, Parveen Devi Pattiram, Mohd Khalizan Sabullah, and Mohd Yunus Shukor. "Purification of protease from Coriandrum sativum using ion-exchange chromatography and gel filtration method." Bulletin of Environmental Science and Sustainable Management (e-ISSN 2716-5353) 2, no. 2 (December 3, 2014): 53–57. http://dx.doi.org/10.54987/bessm.v2i2.197.
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In the past decades, the interest towards plant proteases has increased significantly. Plant proteases are widely used in environment field, food and medicine industries. Proteases such as bromelain, papain and ficin are used in various areas such as in biosensors for detection of heavy metals, meat tenderization, brewing, cancer treatment, milk-clotting, viral disorders and digestion. In this study, protease from coriander leaf (Coriandrum sativum) was evaluated for protease activity using a Bradford-protease-casein assay system. This enzyme was purified through anion exchanger using DEAE-Cellulose column and gel filtration using Agilent ZORBAX column. Its molecular weight was around 55 kDa. The specific activity of the purified protein is 45.0 units/mg protein, total activity is 2745.0 units, yield 33.2% and fold purification is 2.8. Further investigation on gene sequence should be performed in order to identify the type of protease involved.
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Squires, E. James, N. F. Haard, and L. A. W. Feltham. "Gastric proteases of the Greenland cod Gadus ogac. II. Structural properties." Biochemistry and Cell Biology 64, no. 3 (March 1, 1986): 215–22. http://dx.doi.org/10.1139/o86-031.
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Three gastric proteases were isolated from the stomach mucosa of the Greenland cod (Gadus ogac). The cod proteases were all less stable to heating and protease 1 retained less activity at 5 °C when the pH was greater than 5 in comparison with porcine pepsin. The activities of cod proteases 1 and 2, with hemoglobin as the substrate, were doubled in the presence of 25 mM NaCl, while cod protease 3 and porcine pepsin were not stimulated by the salt. The cod proteases did not cross-react with antibodies raised against porcine pepsin. However, some cross-reactivity was noted with antibodies raised against proteases from psychrotrophic pseudomonads. The molecular weights of all the cod proteases were in the range of 36 000–38 000. The amino acid compositions of the cod proteases as compared by the Metzger difference index differed from the mammalian gastric proteases by about the same extent that pepsin, gastricsin, and chymosin differ from each other. Of the cod enzymes, protease 1 differed from mammalian gastric proteases, while cod proteases 3 was more like chymosin with respect to amino acid composition. Cod protease 1 had the lowest hydrophobicity index and chymosin had the highest. The hydrophobicity indices of cod proteases 2 and 3 were intermediate between that of porcine pepsin and bovine chymosin. It is suggested that the Greenland cod proteases represent less differentiated forms of gastric proteases than the mammalian pepsins, gastricsins, and chymosins.