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1
Mamo, Jermen, and Fassil Assefa. "The Role of Microbial Aspartic Protease Enzyme in Food and Beverage Industries." Journal of Food Quality 2018 (July 3, 2018): 1–15. http://dx.doi.org/10.1155/2018/7957269.
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Proteases represent one of the three largest groups of industrial enzymes and account for about 60% of the total global enzymes sale. According to the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology, proteases are classified in enzymes of class 3, the hydrolases, and the subclass 3.4, the peptide hydrolases or peptidase. Proteases are generally grouped into two main classes based on their site of action, that is, exopeptidases and endopeptidases. Protease has also been grouped into four classes based on their catalytic action: aspartic, cysteine, metallo, and serine proteases. However, lately, three new systems have been defined: the threonine-based proteasome system, the glutamate-glutamine system of eqolisin, and the serine-glutamate-aspartate system of sedolisin. Aspartic proteases (EC 3.4.23) are peptidases that display various activities and specificities. It has two aspartic acid residues (Asp32 and Asp215) within their active site which are useful for their catalytic activity. Most of the aspartic proteases display best enzyme activity at low pH (pH 3 to 4) and have isoelectric points in the pH range of 3 to 4.5. They are inhibited by pepstatin. The failure of the plant and animal proteases to meet the present global enzyme demand has directed to an increasing interest in microbial proteases. Microbial proteases are preferred over plant protease because they have most of the characteristics required for their biotechnological applications. Aspartic proteases are found in molds and yeasts but rarely in bacteria. Aspartic protease enzymes from microbial sources are mainly categorized into two groups: (i) the pepsin-like enzymes produced byAspergillus,Penicillium,Rhizopus, andNeurosporaand (ii) the rennin-like enzymes produced byEndothiaandMucorspp., such asMucor miehei,M. pusillus, andEndothia parasitica. Aspartic proteases of microbial origin have a wide range of application in food and beverage industries. These include as milk-clotting enzyme for cheese manufacturing, degradation of protein turbidity complex in fruit juices and alcoholic liquors, and modifying wheat gluten in bread by proteolysis.
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Salvesen, Guy S., Gillian Murphy, and Hideaki Nagase. "The trap hypothesis: α2 and protease inhibition." Biochemist 28, no. 3 (June 1, 2006): 46–48. http://dx.doi.org/10.1042/bio02803046.
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In the 1970s, the Strangeways Laboratory in Cambridge consisted of a small number of groups collectively focused on the mechanisms of pathological connective-tissue damage. One of these groups, headed by Alan Barrett, was breaking ground on the destruction of the protein components of the matrix and was therefore heavily involved in identifying and categorizing newly emerging types of tissue-degrading enzymes. These enzymes, which Alan Barrett urges scientists to call peptidases, are also commonly called proteases or proteinases*. In the early 1970s, there were about 100 described human peptidases, a reasonable sampling of the 500–600 now known in humans in the post-genomic age. Approximately 2% of the human genome encodes peptidases, and roughly 1% encodes proteins with the ability to inhibit these enzymes. As the peptidases developed different catalytic mechanisms to solve the problem of cleaving the notoriously stable peptide bond, so the families of protease inhibitors acquired distinct strategies to regulate peptidase action. The strategies are usually directed towards blocking the peptidase active site directly or, less commonly, by allosteric mechanisms. But perhaps the most bizarre mechanism is that performed by members of the protein clan exemplified by the human protein 2-macroglobulin (α2).
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Wolke, Carmen, Alexander Teumer, Karlhans Endlich, Nicole Endlich, Rainer Rettig, Sylvia Stracke, Beate Fiene, et al. "Serum protease activity in chronic kidney disease patients: The GANI_MED renal cohort." Experimental Biology and Medicine 242, no. 5 (December 30, 2016): 554–63. http://dx.doi.org/10.1177/1535370216684040.
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Serum or plasma proteases have been associated with various diseases including cancer, inflammation, or reno-cardiovascular diseases. We aimed to investigate whether the enzymatic activities of serum proteases are associated with the estimated glomerular filtration rate (eGFR) in patients with different stages of chronic kidney disease (CKD). Our study population comprised 268 participants of the “Greifswald Approach to Individualized Medicine” (GANI_MED) cohort. Enzymatic activity of aminopeptidase A, aminopeptidase B, alanyl (membrane) aminopeptidase, insulin-regulated aminopeptidase, puromycin-sensitive aminopeptidase, leucine aminopeptidase 3, prolyl-endopeptidase (PEP), dipeptidyl peptidase 4 (DPP4), angiotensin I-converting enzyme, and angiotensin I-converting enzyme 2 (ACE2) proteases was measured in serum. Linear regression of the respective protease was performed on kidney function adjusted for age and sex. Kidney function was modeled either by the continuous Modification of Diet in Renal Disease (MDRD)-based eGFR or dichotomized by eGFR < 15 mL/min/1.73 m2 or <45 mL/min/1.73 m2, respectively. Results with a false discovery rate below 0.05 were deemed statistically significant. Among the 10 proteases investigated, only the activities of ACE2 and DPP4 were correlated with eGFR. Patients with lowest eGFR exhibited highest DPP4 and ACE2 activities. DPP4 and PEP were correlated with age, but all other serum protease activities showed no associations with age or sex. Our data indicate that ACE2 and DPP4 enzymatic activity are associated with the eGFR in patients with CKD. This finding distinguishes ACE2 and DPP4 from other serum peptidases analyzed and clearly indicates that further analyses are warranted to identify the precise role of these serum ectopeptidases in the pathogenesis of CKD and to fully elucidate underlying molecular mechanisms. Impact statement • Renal and cardiac diseases are very common and often occur concomitantly, resulting in increased morbidity and mortality. Understanding of molecular mechanisms linking both diseases is limited, available fragmentary data point to a role of the renin–angiotensin system (RAS) and, in particular, Ras-related peptidases. • Here, a comprehensive analysis of serum peptidase activities in patients with different stages of chronic kidney disease (CKD) is presented, with special emphasis given to RAS peptidases • The serum activities of the peptidases angiotensin I-converting enzyme 2 and dipeptidyl peptidase 4 were identified as closely associated with kidney function, specifically with the estimated glomerular filtration rate. The findings are discussed in the context of available data suggesting protective roles for both enzymes in reno-cardiac diseases. • The data add to our understanding of pathomechanisms underlying development and progression of CKD and indicate that both enzymes might represent potential pharmacological targets for the preservation of renal function.
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SHAN, Lu, Thomas MARTI, Ludvig M. SOLLID, Gary M. GRAY, and Chaitan KHOSLA. "Comparative biochemical analysis of three bacterial prolyl endopeptidases: implications for coeliac sprue." Biochemical Journal 383, no. 2 (October 8, 2004): 311–18. http://dx.doi.org/10.1042/bj20040907.
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Prolyl endopeptidases have potential for treating coeliac sprue, a disease of the intestine caused by proteolytically resistant peptides from proline-rich prolamins of wheat, barley and rye. We compared the properties of three similar bacterial prolyl endopeptidases, including the known enzymes from Flavobacterium meningosepticum (FM) and Sphingomonas capsulate (SC) and a novel enzyme from Myxococcus xanthus (MX). These enzymes were interrogated with reference chromogenic substrates, as well as two related gluten peptides (PQPQLPYPQPQLP and LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF), believed to play a key role in coeliac sprue pathogenesis. In vitro and in vivo studies were conducted to evaluate the activity, specificity and acid/protease stability of the enzymes. All peptidases were relatively resistant to acid, pancreatic proteases and membrane peptidases of the small intestinal mucosa. Although their activities against reference substrates were similar, the enzymes exhibited substantial differences with respect to chain length and subsite specificity. SC hydrolysed PQPQLPYPQPQLP well, but had negligible activity against LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF. In contrast, the FM and MX peptidases cleaved both substrates, although the FM enzyme acted more rapidly on LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF than MX. Whereas the FM enzyme showed a preference for Pro–Gln bonds, SC cleaved both Pro–Gln and Pro–Tyr bonds with comparable efficiency, and MX had a modest preference for Pro–(Tyr/Phe) sites over Pro–Gln sites. While a more comprehensive understanding of sequence and chain-length specificity may be needed to assess the relative utility of alternative prolyl endopeptidases for treating coeliac sprue, our present work has illustrated the diverse nature of this class of enzymes from the standpoint of proteolysing complex substrates such as gluten.
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Marokházi, Judit, Katalin Lengyel, Szilvia Pekár, Gabriella Felföldi, András Patthy, László Gráf, András Fodor, and István Venekei. "Comparison of Proteolytic Activities Produced by Entomopathogenic Photorhabdus Bacteria: Strain- and Phase-Dependent Heterogeneity in Composition and Activity of Four Enzymes." Applied and Environmental Microbiology 70, no. 12 (December 2004): 7311–20. http://dx.doi.org/10.1128/aem.70.12.7311-7320.2004.
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ABSTRACT Twenty strains (including eight phase variant pairs) of nematode-symbiotic and insect-pathogenic Photorhabdus bacteria were examined for the production of proteolytic enzymes by using a combination of several methods, including gelatin liquefaction, zymography coupled to native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and activity measurement with two chromogen substrate types. Four protease activities (∼74, ∼55, ∼54, and ∼37 kDa) could be separated. The N-terminal sequences of three of the proteases were determined, and a comparison with sequences in databases allowed identification of these proteases as HEXXH metallopeptidases. Thus, the 74-kDa protease (described formerly as Php-B [J. Marokházi, G. Kóczán, F. Hudecz, L. Gráf, A. Fodor, and I. Venekei, Biochem. J. 379:633-640, 2004) is an ortholog of OpdA, a member the thimet oligopeptidase family, and the 55-kDa protease is an ortholog of PrtA, a HEXXH+H peptidase in clan MB (metzincins), while the 37-kDa protease (Php-C) belongs to the HEXXH+E peptidases in clan MA. The 54-kDa protease (Php-D) is a nonmetalloenzyme. PrtA and Php-C were zymographically detected, and they occurred in several smaller forms as well. OpdA could not be detected by zymography. PrtA, Php-C, and Php-D were secreted proteases; OpdA, in contrast, was an intracellular enzyme. OpdA activity was found in every strain tested, while Php-D was detected only in the Brecon/1 strain. There was significant strain variation in the secretion of PrtA and Php-C activities, but reduced activity or a lack of activity was not specific to secondary-phase variants. The presence of PrtA, OpdA, and Php-C activities could be detected in the hemolymph of Galleria melonella larvae 20 to 40 h postinfection. These proteases appear not to be directly involved in the pathogenicity of Photorhabdus, since strains or phase variants lacking any of these proteases do not show reduced virulence when they are injected into G. melonella larvae.
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Kryukov, V. S., S. V. Zinoviev, and R. V. Nekrasov. "Proteases in the diet of monogastric animals." Agrarian science 344, no. 1 (March 13, 2021): 30–38. http://dx.doi.org/10.32634/0869-8155-2021-344-1-30-38.
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There are many proteases, and about 2% of the human genome is involved in the regulation of their formation. The share of proteases involved in digestion accounts for only a small part. Despite this, the mechanisms of action of digestive proteases are less studied than carbohydrases and lipases. The incorporation of exogenous proteases into young animal feeds is often accompanied by improved utilization of protein and other nutrients. Exogenous proteases degrade inhibitors of the endogenous protease and lectins in feed. Alkaline proteases are of interest due to their broader substrate specificity and activity throughout the entire gastrointestinal tract. This group includes keratinases, which digest proteins inaccessible for cleavage by proteases and peptidases of animals. Keratinases digest agglutinins, glycinin and b-conglycinin and connective tissue proteins, which are resistant to the action of gastrointestinal enzymes and a number of exogenous proteases. The alleged reasons for the inconsistent results when using feed proteases are described. Their mediated positive effects not associated with proteolysis are indicated. It is advisable to use proteases with keratinolytic activity as fodder proteases.
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Massaoud, Mustafa K., Judit Marokh�zi, Andr�s Fodor, and Istv�n Venekei. "Proteolytic Enzyme Production by Strains of the Insect Pathogen Xenorhabdus and Characterization of an Early-Log-Phase-Secreted Protease as a Potential Virulence Factor." Applied and Environmental Microbiology 76, no. 20 (August 27, 2010): 6901–9. http://dx.doi.org/10.1128/aem.01567-10.
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ABSTRACT As a comparison to a similar study on Photorhabdus strains, 15 Xenorhabdus bacterial strains and secondary phenotypic variants of two strains were screened for proteolytic activity by five detection methods. Although the number and intensity of proteolytic activities were different, every strain was positive for proteolytic activity by several tests. Zymography following native PAGE detected two groups of activities with different substrate affinities and a higher and lower electrophoretic mobility that were distinguished as activity 1 and 2, respectively. Zymography following SDS-PAGE resolved three activities, which were provisionally named proteases A, B, and C. Only protease B, an ∼55-kDa enzyme, was produced by every strain. This enzyme exhibited higher affinity to the gelatin substrate than to the casein substrate. Of the chromogenic substrates used, three were hydrolyzed: furylacryloyl-Ala-Leu-Val-Tyr (Fua-ALVY), Fua-LGPA (LGPA is Leu-Gly-Pro-Ala) (a substrate for collagen peptidases), and succinyl-Ala-Ala-Pro-Phe-thiobenzyl (Succ-AAPF-SBzl). All but the Fua-LGPA-ase activity seemed to be from secreted enzymes. According to their substrate preference profiles and inhibitor sensitivities, at least six such proteolytic enzymes could be distinguished in the culture medium of Xenorhabdus strains. The proteolytic enzyme that was secreted the earliest, protease B and the Succ-AAPF-SBzl-hydrolyzing enzyme, appeared from the early logarithmic phase of growth. Protease B could also be detected in the hemolymph of Xenorhabdus-infected Galleria mellonella larvae from 15 h postinfection. The purified protease B hydrolyzed in vitro seven proteins in the hemolymph of Manduca sexta that were also cleaved by PrtA peptidase from Photorhabdus. The N-terminal sequence of protease B showed similarity to a 55-kDa serralysin type metalloprotease in Xenorhabdus nematophila, which had been identified as an orthologue of Photorhabdus PrtA peptidase.
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Suido, H., T. Eguchi, T. Tanaka, and M. Nakamura. "Identification of Periodontopathic Bacteria Based Upon their Peptidase Activities." Advances in Dental Research 2, no. 2 (November 1988): 304–9. http://dx.doi.org/10.1177/08959374880020021701.
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Black-pigmented Bacteroides (BPB) and spirochetes are associated with some forms of periodontal diseases. The enzymes produced by these bacteria may participate in the destruction of gingival and periodontal tissues. Certain proteases and peptidases are unique to Bacteroides gingivalis and Treponema denticola. Our purpose was to study the peptidases of periodontopathogens and to evaluate the use of unique peptidases for detection and identification of these bacteria. Bacteria used were BPB, Treponema, Fusobacterium, Capnocytophaga, Actinobacillus (Haemophilus), and Eikenella species. Twenty-five substrates, including mono-, di-, and tri-peptides of β-naphthylamide (β-NA) were employed for examination of peptidase activity. Clinically isolated BPB were obtained from 16 adult periodontitis patients. One hundred and ninety-three BPB strains were identified by conventional identification methods, and the peptidase activity was determined with N-Carbobenzoxy-glycyl-glycyl-L-arginine-β-naphthylamide (N-CBz-Gly-Gly-Arg-β-NA) used as a substrate. Among tested periodontopathic bacteria, only B. gingivalis and T. denticola could strongly hydrolyze some substrates such as N-CBz-Gly-Gly-Arg-β-NA and N-Benzoyl-L-valyl-glycyl-L-arginine-4-methoxy-(3-naphthylamide (Bz-Val-Gly-Arg-β-NA). In subgingival plaque samples, all patients showed BPB, and eight out of 16 patients possessed B. gingivalis by culture. One hundred and ten strains out of 193 BPB isolated were identified as B. gingivalis. Ninety-nine percent of these B. gingivalis strains identified showed N-CBz-Gly-Gly-Arg-β-NAhydrolyzing activity on a newly developed colorimetric plate assay. However, none of the other strains showed this activity in cultures of subgingival plaque which did not allow growth of spirochetes. Enzymes, such as N-CBz-Gly-Gly-Arg-peptidase and Bz-Val-Gly-Arg-peptidase, specific for B. gingivalis and T. denticola seem to be useful for rapid detection and identification of these bacteria.
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Sriranganadane, Dev, Utz Reichard, Karine Salamin, Marina Fratti, Olivier Jousson, Patrice Waridel, Manfredo Quadroni, Jean-Marc Neuhaus, and Michel Monod. "Secreted glutamic protease rescues aspartic protease Pep deficiency in Aspergillus fumigatus during growth in acidic protein medium." Microbiology 157, no. 5 (May 1, 2011): 1541–50. http://dx.doi.org/10.1099/mic.0.048603-0.
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In an acidic protein medium Aspergillus fumigatus secretes an aspartic endoprotease (Pep) as well as tripeptidyl-peptidases, a prolyl-peptidase and carboxypeptidases. In addition, LC-MS/MS revealed a novel glutamic protease, AfuGprA, homologous to Aspergillus niger aspergillopepsin II. The importance of AfuGprA in protein digestion was evaluated by deletion of its encoding gene in A. fumigatus wild-type D141 and in a pepΔ mutant. Either A. fumigatus Pep or AfuGprA was shown to be necessary for fungal growth in protein medium at low pH. Exoproteolytic activity is therefore not sufficient for complete protein hydrolysis and fungal growth in a medium containing proteins as the sole nitrogen source. Pep and AfuGprA constitute a pair of endoproteases active at low pH, in analogy to A. fumigatus alkaline protease (Alp) and metalloprotease I (Mep), where at least one of these enzymes is necessary for fungal growth in protein medium at neutral pH. Heterologous expression of AfuGprA in Pichia pastoris showed that the enzyme is synthesized as a preproprotein and that the propeptide is removed through an autoproteolytic reaction at low pH to generate the mature protease. In contrast to A. niger aspergillopepsin II, AfuGprA is a single-chain protein and is structurally more similar to G1 proteases characterized in other non-Aspergillus fungi.
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Heywood, Astra, and Iain L. Lamont. "Cell envelope proteases and peptidases of Pseudomonas aeruginosa: multiple roles, multiple mechanisms." FEMS Microbiology Reviews 44, no. 6 (August 17, 2020): 857–73. http://dx.doi.org/10.1093/femsre/fuaa036.
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ABSTRACT Pseudomonas aeruginosa is a Gram-negative bacterium that is commonly isolated from damp environments. It is also a major opportunistic pathogen, causing a wide range of problematic infections. The cell envelope of P. aeruginosa, comprising the cytoplasmic membrane, periplasmic space, peptidoglycan layer and outer membrane, is critical to the bacteria's ability to adapt and thrive in a wide range of environments. Over 40 proteases and peptidases are located in the P. aeruginosa cell envelope. These enzymes play many crucial roles. They are required for protein secretion out of the cytoplasm to the periplasm, outer membrane, cell surface or the environment; for protein quality control and removal of misfolded proteins; for controlling gene expression, allowing adaptation to environmental changes; for modification and remodelling of peptidoglycan; and for metabolism of small molecules. The key roles of cell envelope proteases in ensuring normal cell functioning have prompted the development of inhibitors targeting some of these enzymes as potential new anti-Pseudomonas therapies. In this review, we summarise the current state of knowledge across the breadth of P. aeruginosa cell envelope proteases and peptidases, with an emphasis on recent findings, and highlight likely future directions in their study.
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Valencia, Ricardo, Valentina González, Agustina Undabarrena, Leonardo Zamora-Leiva, Juan A. Ugalde, and Beatriz Cámara. "An Integrative Bioinformatic Analysis for Keratinase Detection in Marine-Derived Streptomyces." Marine Drugs 19, no. 6 (May 21, 2021): 286. http://dx.doi.org/10.3390/md19060286.
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Keratinases present promising biotechnological applications, due to their ability to degrade keratin. Streptomyces appears as one of the main sources of these enzymes, but complete genome sequences of keratinolytic bacteria are still limited. This article reports the complete genomes of three marine-derived streptomycetes that show different levels of feather keratin degradation, with high (strain G11C), low (strain CHD11), and no (strain Vc74B-19) keratinolytic activity. A multi-step bioinformatics approach is described to explore genes encoding putative keratinases in these genomes. Despite their differential keratinolytic activity, multiplatform annotation reveals similar quantities of ORFs encoding putative proteases in strains G11C, CHD11, and Vc74B-19. Comparative genomics classified these putative proteases into 140 orthologous groups and 17 unassigned orthogroup peptidases belonging to strain G11C. Similar network analysis reveals three network communities of putative peptidases related to known keratinases of the peptidase families S01, S08, and M04. When combined with the prediction of cellular localization and phylogenetic reconstruction, seven putative keratinases from the highly keratinolytic strain Streptomyces sp. G11C are identified. To our knowledge, this is the first multi-step bioinformatics analysis that complements comparative genomics with phylogeny and cellular localization prediction, for the prediction of genes encoding putative keratinases in streptomycetes.
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MAROKHÁZI, Judit, György KÓCZÁN, Ferenc HUDECZ, László GRÁF, András FODOR, and István VENEKEI. "Enzymic characterization with progress curve analysis of a collagen peptidase from an enthomopathogenic bacterium, Photorhabdus luminescens." Biochemical Journal 379, no. 3 (May 1, 2004): 633–40. http://dx.doi.org/10.1042/bj20031116.
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A proteolytic enzyme, Php-B (Photorhabdus protease B), was purified from the entomopathogenic bacterium, Photorhabdus luminescens. The enzyme is intracellular, and its molecular mass is 74 kDa. Tested on various peptide and oligopeptide substrates, Php-B hydrolysed only oligopeptides, with significant activity against bradykinin and a 2-furylacryloyl-blocked peptide, Fua-LGPA (2-furylacryloyl-Leu-Gly-Pro-Ala; kcat=3.6×102 s−1, Km=5.8×10−5 M−1, pH optimum approx. 7.0). The pKa1 and the pKa2 values of the enzyme activity (6.1 and 7.9 respectively), as well as experiments with enzyme inhibitors and bivalent metal ions, suggest that the activity of Php-B is dependent on histidine and cysteine residues, but not on serine residues, and that it is a metalloprotease, which most probably uses Zn2+ as a catalytic ion. The enzyme's ability to cleave oligopeptides that contain a sequence similar to collagen repeat (-Pro-Xaa-Gly-), bradykinin and Fua-LGPA (a synthetic substrate for bacterial collagenases and collagen peptidases), but not native collagens (types I and IV) or denatured collagen (gelatin), indicates that Php-B is probably a collagen peptidase, the first enzyme of this type to be identified in an insect pathogen, that might have a role in the nutrition of P. luminescens by degrading small collagen fragments. For the determination of enzyme kinetic constants, we fitted a numerically integrated Michaelis–Menten model to the experimental progress curves. Since this approach has not been used before in the characterization of proteases that are specific for the P1´–P4´ substrate sites (e.g. collagenolytic enzymes), we present a comparison of this method with more conventional ones. The results confirm the reliability of the numerical integration method in the kinetic analysis of collagen-peptide-hydrolysing enzymes.
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Zilda, Dewi Seswita, Yusro Nuri Fawzya, and Agustinus Robert Uria. "Identification of Protease-Producing Bacteria Isolated from Banyuwedang, Bali, and Characterization of its Protease." Squalen Bulletin of Marine and Fisheries Postharvest and Biotechnology 13, no. 3 (December 30, 2018): 101. http://dx.doi.org/10.15578/squalen.v13i3.367.
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Proteases or peptidases is known as a largest group of hydrolytic enzymes and have been applied in various industries such as food, pharmacy, leather, detergent and waste treatment. Although they are also produced by plants and animals, microbes remain the main source of proteases in the world market which mostly derived from Bacillus sp. Aims of this research were to identify isolate BII-1 and study its protease. Analysis of 16Sr RNA sequencing showed the identity of BII-1 as Bacillus subtilis (99% similarity with the same species in GenBank). It was found that protease from BII-1 exhibited optimal temperature and pH of 50 oC and 8-9, respectively. It was activated by Li2+, Na2+, Mg2+ and K+. The degenerated primer for protease gene was designed, and a partial protease gene was amplified from BII-1. The sequencing result showed that this amplified gene shared 100 and 99% similarity with those from Geobacillus thermophiles and Bacillus subtilis in the GenBank, respectively.Keywords: protease, bacteria, Bacillus subtilis, Geobacillus thermophylus
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De Oliveira Martinez, Juan Pinheiro, Guiqin Cai, Matthias Nachtschatt, Laura Navone, Zhanying Zhang, Karen Robins, and Robert Speight. "Challenges and Opportunities in Identifying and Characterising Keratinases for Value-Added Peptide Production." Catalysts 10, no. 2 (February 3, 2020): 184. http://dx.doi.org/10.3390/catal10020184.
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Keratins are important structural proteins produced by mammals, birds and reptiles. Keratins usually act as a protective barrier or a mechanical support. Millions of tonnes of keratin wastes and low value co-products are generated every year in the poultry, meat processing, leather and wool industries. Keratinases are proteases able to breakdown keratin providing a unique opportunity of hydrolysing keratin materials like mammalian hair, wool and feathers under mild conditions. These mild conditions ameliorate the problem of unwanted amino acid modification that usually occurs with thermochemical alternatives. Keratinase hydrolysis addresses the waste problem by producing valuable peptide mixes. Identifying keratinases is an inherent problem associated with the search for new enzymes due to the challenge of predicting protease substrate specificity. Here, we present a comprehensive review of twenty sequenced peptidases with keratinolytic activity from the serine protease and metalloprotease families. The review compares their biochemical activities and highlights the difficulties associated with the interpretation of these data. Potential applications of keratinases and keratin hydrolysates generated with these enzymes are also discussed. The review concludes with a critical discussion of the need for standardized assays and increased number of sequenced keratinases, which would allow a meaningful comparison of the biochemical traits, phylogeny and keratinase sequences. This deeper understanding would facilitate the search of the vast peptidase family sequence space for novel keratinases with industrial potential.
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LEITING, Barbara, KellyAnn D. PRYOR, Joseph K. WU, Frank MARSILIO, Reshma A. PATEL, Charles S. CRAIK, Jonathan A. ELLMAN, Richard T. CUMMINGS, and Nancy A. THORNBERRY. "Catalytic properties and inhibition of proline-specific dipeptidyl peptidases II, IV and VII." Biochemical Journal 371, no. 2 (April 15, 2003): 525–32. http://dx.doi.org/10.1042/bj20021643.
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There is currently intense interest in the emerging group of proline-specific dipeptidases, and their roles in the regulation of biological processes. Dipeptidyl peptidase IV (DPP-IV) is involved in glucose metabolism by contributing to the regulation of glucagon family peptides and has emerged as a potential target for the treatment of metabolic diseases. Two other proline-specific dipeptidases, DPP-VII (also known as quiescent cell proline dipeptidase) and DPP-II, have unknown functions and have recently been suggested to be identical proteases based on a sequence comparison of human DPP-VII and rat DPP-II (78% identity) [Araki, Li, Yamamoto, Haneda, Nishi, Kikkawa and Ohkubo (2001) J. Biochem. 129, 279–288; Fukasawa, Fukasawa, Higaki, Shiina, Ohno, Ito, Otogoto and Ota (2001) Biochem. J. 353, 283–290]. To facilitate the identification of selective substrates and inhibitors for these enzymes, a complete biochemical profile of these enzymes was obtained. The pH profiles, substrate specificities as determined by positional scanning, Michaelis–Menten constants and inhibition profiles for DPP-VII and DPP-II were shown to be virtually identical, strongly supporting the hypothesis that they are the same protease. In addition, substrate specificities, catalytic constants and IC50 values were shown to be markedly different from those of DPP-IV. Selective DPP-IV and DPP-VII substrates were identified and they can be used to design selective inhibitors and probe further into the biology of these enzymes.
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Icimoto, Marcelo Yudi, Adrianne Marlise Mendes Brito, Marcos Paulo Cyrillo Ramos, Vitor Oliveira, and Iseli Lourenço Nantes-Cardoso. "Increased Stability of Oligopeptidases Immobilized on Gold Nanoparticles." Catalysts 10, no. 1 (January 4, 2020): 78. http://dx.doi.org/10.3390/catal10010078.
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The metallopeptidases thimet oligopeptidase (THOP, EC 3.4.24.25) and neurolysin (NEL, EC 3.4.24.26) are enzymes that belong to the zinc endopeptidase M13 family. Numerous studies suggest that these peptidases participate in the processing of bioactive peptides such as angiotensins and bradykinin. Efforts have been conducted to develop biotechnological tools to make possible the use of both proteases to regulate blood pressure in mice, mainly limited by the low plasmatic stability of the enzymes. In the present study, it was investigated the use of nanotechnology as an efficient strategy for to circumvent the low stability of the proteases. Recombinant THOP and NEL were immobilized in gold nanoparticles (GNPs) synthesized in situ using HEPES and the enzymes as reducing and stabilizing agents. The formation of rTHOP-GNP and rNEL-GNP was characterized by the surface plasmon resonance band, zeta potential and atomic force microscopy. The gain of structural stability and activity of rTHOP and rNEL immobilized on GNPs was demonstrated by assays using fluorogenic substrates. The enzymes were also efficiently immobilized on GNPs fabricated with sodium borohydride. The efficient immobilization of the oligopeptidases in gold nanoparticles with gain of stability may facilitate the use of the enzymes in therapies related to pressure regulation and stroke, and as a tool for studying the physiological and pathological roles of both proteases.
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Jedeszko, Christopher, and Bonnie F. Sloane. "Cysteine cathepsins in human cancer." Biological Chemistry 385, no. 11 (November 1, 2004): 1017–27. http://dx.doi.org/10.1515/bc.2004.132.
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Abstract Proteases play causal roles in the malignant progression of human tumors. This review centers on the roles in this process of cysteine cathepsins, i.e., peptidases belonging to the papain family (C1) of the CA clan of cysteine proteases. Cysteine cathepsins, most likely along with matrix metalloproteases (MMPs) and serine proteases, degrade the extracellular matrix, thereby facilitating growth and invasion into surrounding tissue and vasculature. Studies on tumor tissues and cell lines have shown changes in expression, activity and distribution of cysteine cathepsins in numerous human cancers. Molecular, immunologic and pharmacological strategies to modulate expression and activity of cysteine cathepsins have provided evidence for a causal role for these enzymes in tumor progression and invasion. Clinically, the levels, activities and localization of cysteine cathepsins and their endogenous inhibitors have been shown to be of diagnostic and prognostic value. Understanding the roles that cysteine proteases play in cancer could lead to the development of more efficacious therapies.
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Reinheckel, T., J. Deussing, W. Roth, and C. Peters. "Towards Specific Functions of Lysosomal Cysteine Peptidases: Phenotypes of Mice Deficient for Cathepsin B or Cathepsin L." Biological Chemistry 382, no. 5 (May 5, 2001): 735–41. http://dx.doi.org/10.1515/bc.2001.089.
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Abstract The lysosomal cysteine peptidases cathepsin B and cathepsin L are abundant and ubiquitously expressed members of the papain family, and both enzymes contribute to the terminal degradation of proteins in the lysosome. However, there is accumulating evidence for specific functions of lysosomal proteases in health and disease. The generation of knock out mouse strains that are deficient in lysosomal proteases provides a valuable tool for evaluation of existing hypotheses and gaining new insights into the in vivo functions of these proteases. In this minireview, we summarise and discuss the findings obtained by analysis of mice that are devoid of cathepsin B or cathepsin L. In brief, cathepsin L appears to be critically involved in epidermal homeostasis, regulation of the hair cycle, and MHC class IImediated antigen presentation in cortical epithelial cells of the thymus. Cathepsin B plays a major role in pathological trypsinogen activation in the early course of experimental pancreatitis and contributes significantly to TNFα induced hepatocyte apoptosis.
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Sheikhs, Nabiha Naeem, Qurat-ul-ain, and Saba Altaf. "Production of Extracellular Protease from Bacterial Co-cultures using Solid State Fermentation." BioScientific Review 2, no. 4 (December 14, 2020): 13–23. http://dx.doi.org/10.32350/bsr/2020/24/726.
Full textAbstract:
Proteases (also known as peptidases or proteinases) are hydrolytic enzymes that cleave proteins into amino acids. They comprise 60% of the total industrial usage of enzymes worldwide and can be obtained from many sources. The current study aims to isolate and screen protease-producing bacterial strains from the soil and to produce protease from the bacterial co-cultures using solid-state fermentation (SSF). Primary screening of the protease-producing bacterial strains was carried out on skim milk agar and they were sub-cultured and preserved on the nutrient agar for further testing. Thirty-two compatibility tests of twenty-seven bacterial isolates were performed and SSF was carried out. Afterward, absorbance was taken at 660 nm against tyrosine as standard. According to the results, the bacterial co-culture 19 showed the highest absorbance with an enzyme activity of 10.2 U/ml. The bacterial strains of the co-culture 19 were identified through morphological and biochemical tests. Bacterial strain 1 was observed as cocci and irregular, while bacterial strain 2 was bacillus and rod-shaped. Both strains were positive for gram staining, catalase test, casein hydrolysis test and methyl red test. As for endospore staining, bacterial strain 1 was spore forming while bacterial strain 2 was a non-spore former. It was concluded that the bacterial co-culture 19 can act as a potent co-culture for protease production. Compatibility test was carried out to enhance the production of protease by utilizing cheap and readily available agro-waste products, which benefit the industry by being cost effective and the environment by being eco-friendly.
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Sheikhs, Nabiha Naeem, Qurat-ul-ain, and Saba Altaf. "Production of Extracellular Protease from Bacterial Co-cultures using Solid State Fermentation." BioScientific Review 2, no. 4 (December 14, 2020): 13–23. http://dx.doi.org/10.32350/bsr.0204.02.
Full textAbstract:
Proteases (also known as peptidases or proteinases) are hydrolytic enzymes that cleave proteins into amino acids. They comprise 60% of the total industrial usage of enzymes worldwide and can be obtained from many sources. The current study aims to isolate and screen protease-producing bacterial strains from the soil and to produce protease from the bacterial co-cultures using solid-state fermentation (SSF). Primary screening of the protease-producing bacterial strains was carried out on skim milk agar and they were sub-cultured and preserved on the nutrient agar for further testing. Thirty-two compatibility tests of twenty-seven bacterial isolates were performed and SSF was carried out. Afterward, absorbance was taken at 660 nm against tyrosine as standard. According to the results, the bacterial co-culture 19 showed the highest absorbance with an enzyme activity of 10.2 U/ml. The bacterial strains of the co-culture 19 were identified through morphological and biochemical tests. Bacterial strain 1 was observed as cocci and irregular, while bacterial strain 2 was bacillus and rod-shaped. Both strains were positive for gram staining, catalase test, casein hydrolysis test and methyl red test. As for endospore staining, bacterial strain 1 was spore forming while bacterial strain 2 was a non-spore former. It was concluded that the bacterial co-culture 19 can act as a potent co-culture for protease production. Compatibility test was carried out to enhance the production of protease by utilizing cheap and readily available agro-waste products, which benefit the industry by being cost effective and the environment by being eco-friendly.
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Ferraris, R. P., W. W. Kwan, and J. Diamond. "Regulatory signals for intestinal amino acid transporters and peptidases." American Journal of Physiology-Gastrointestinal and Liver Physiology 255, no. 2 (August 1, 1988): G151—G157. http://dx.doi.org/10.1152/ajpgi.1988.255.2.g151.
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Dietary protein ultimately regulates many processes involved in protein digestion, but it is often unclear whether proteins themselves, peptides, or amino acids (AAs) are the proximate regulatory signal. Hence we compared several processes involved in protein digestion in mice adapted to one of three rations, identical except for containing 54% of either casein, a partial hydrolysate of casein, or a free AA mixture simulating a complete hydrolysate of casein. We measured brush-border uptakes of seven AAs that variously serve as substrates for four AA transporters, and brush-border and cytosolic activities of four peptidases. The three rations yielded essentially the same AA uptake rates. Peptidase activities tended to be lower on the AA ration than on the protein ration. In other studies, all three rations yielded the same rates of brush-border peptide uptake; protein is only modestly more effective than AAs at inducing synthesis of pancreatic proteases; and, depending on the animal species, protein is either much less or much more effective than AAs at stimulating release of cholecystokinin and hence of pancreatic enzymes. Thus the regulators of each process involved in protein digestion are not necessarily that process's substrate. We call attention to other cases in which the functional significance of regulatory signals remains to be understood.
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Kumari, Saravana, and Reshma R. "Effect of alkaline protease produced from fish waste as substrate by Bacillus clausii on destaining of blood stained fabric." Journal of Tropical Life Science 11, no. 1 (February 3, 2021): 59–66. http://dx.doi.org/10.11594/jtls.11.01.08.
Full textAbstract:
Alkaline protease or peptidases are the largest groups of enzymes in the biological industry with a variety of application in manufacturing units used in the process of substrate stabilization, dehairing, diagnosis, extraction, food production, destaining, etc., where the pH of the environmental conditions remain above neutral pH. Because of these wider applications of alkaline proteases in industries, their demand is increasing to compete with their chemical counterpart. An alkaline tolerant bacterial strain Bacillus clausii wasisolated from fish waste and used for mass production of alkaline protease using fish waste homogenate as media. Preliminary study on optimization of conditions for the mass production carried out. The optimum temperature for production ranges between 25°C and 35°C and pH determined as 9. The mass production of extracellular alkaline protease carried out using mobilized and immobilized cells of B. clausii at optimized condition using production media, the mixture of production media and fish waste homogenate and in nutrient broth. The recorded results showed that the maximum enzyme production obtained with immobilized cells in nutrient broth media and followed by fish waste homogenate media of 8900 U/ml and 8600 U/ml. Purification protease enzyme yielded 0.35 g/ml from the production media . Bloodstained cloth treated with immobilized enzyme removed the stain completely compared to treatment with non-immobilized enzyme and commercially used detergent. So, the current study suggests the usage of microbial alkaline protease in household detergents to replace the usage of synthetic detergents and save the environment from chemical pollutants.
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González, Valentina, María José Vargas-Straube, Walter O. Beys-da-Silva, Lucélia Santi, Pedro Valencia, Fabrizio Beltrametti, and Beatriz Cámara. "Enzyme Bioprospection of Marine-Derived Actinobacteria from the Chilean Coast and New Insight in the Mechanism of Keratin Degradation in Streptomyces sp. G11C." Marine Drugs 18, no. 11 (October 28, 2020): 537. http://dx.doi.org/10.3390/md18110537.
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Marine actinobacteria are viewed as a promising source of enzymes with potential technological applications. They contribute to the turnover of complex biopolymers, such as pectin, lignocellulose, chitin, and keratin, being able to secrete a wide variety of extracellular enzymes. Among these, keratinases are a valuable alternative for recycling keratin-rich waste, which is generated in large quantities by the poultry industry. In this work, we explored the biocatalytic potential of 75 marine-derived actinobacterial strains, focusing mainly on the search for keratinases. A major part of the strains secreted industrially important enzymes, such as proteases, lipases, cellulases, amylases, and keratinases. Among these, we identified two streptomycete strains that presented great potential for recycling keratin wastes—Streptomyces sp. CHA1 and Streptomyces sp. G11C. Substrate concentration, incubation temperature, and, to a lesser extent, inoculum size were found to be important parameters that influenced the production of keratinolytic enzymes in both strains. In addition, proteomic analysis of culture broths from Streptomyces sp. G11C on turkey feathers showed a high abundance and diversity of peptidases, belonging mainly to the serine and metallo-superfamilies. Two proteases from families S08 and M06 were highly expressed. These results contributed to elucidate the mechanism of keratin degradation mediated by streptomycetes.
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Sharikov, A. Y., E. N. Sokolova, M. V. Amelyakina, T. V. Yuraskina, V. V. Ivanov, and E. M. Serba. "Development of a concept for the production of wheat snacks with the elimination of gluten by the biocatalysis." Proceedings of the Voronezh State University of Engineering Technologies 82, no. 4 (January 20, 2021): 77–83. http://dx.doi.org/10.20914/2310-1202-2020-4-77-83.
Full textAbstract:
The increase in the number of cases of allergic reactions and celiac disease is an important problem. The solution to this problem is the search and development of relevant and effective ways to eliminate gluten. Specific amino acid sequences glutamine and proline determine the resistance to protease hydrolysis of the structural domains of gluten fractions. The analysis of the literature data showed that an alternative to the gluten-free diet is the use of biotechnological methods for modifying ingredients containing gluten. Such methods include the use of leavens on the base of lactic acid bacteria or enzyme preparations containing peptidases specific to gluten biocatalysis. In addition, the pretreatment of raw materials by extrusion cooking contributes to an increase in the degree of gluten hydrolysis. The effect of the thermoplastic extrusion and various enzyme systems containing proteases, amylolytic, cellulolytic and hemicellulolytic enzymes on the changes in the molecular weights of wheat protein fractions was studied. It was found that extrusion as a factor of protein modification significantly affects the proteolysis of wheat proteins using enzyme systems of different substrate specificity. The most effective hydrolysis was shown by the use of a complex enzyme preparation Amyloprotoorizin. including The effect was also noted after bioconversion of non-extruded wheat. An algorithm for the technology of wheat snacks based on the processes of extrusion and biocatalysis of proteins with specific proteases for the elimination of gluten is devepoped. The practical implementation of the technology will make it possible to obtain ready-to-eat snacks, which will be investigated for the preservation or elimination of antigenic properties during clinical trials.
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Michalska, Karolina, Andrew Steen, Gekleng Chhor, Katlyn Fayman, Michael Endres, Gyorgy Babnigg, Karen Lloyd, Robert Jedrzejczak, and Andrzej Joachimiak. "Structure and specificity of novel aminopeptidase from marine sediment Archaea." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C468. http://dx.doi.org/10.1107/s205327331409531x.
Full textAbstract:
The Earth's microbial diversity remained largely unexplored until recent developments in DNA sequencing. Novel methods enabled us to access genomic information of uncultured microbial organisms and create hypotheses about their metabolic capabilities. These predictions primarily rely on the sequence similarity between a novel protein and characterized proteins. Such an approach introduces a "culture" bias: the well-understood proteins come from a set of laboratory-grown bacteria, while novel microbial proteins are obtained from a variety of environments, including the most extreme. One such niche is ocean sediment – an unexplored ecosystem that plays important roles in geochemical cycles. Single-cell genomics targeting sedimentary populations identified four new archaeons encoding putative intra- and extra-cellular proteases [1]. This discovery suggests that heterotrophic marine Archaea evolved to degrade detrital proteins and might contribute to global carbon cycling. The novel proteases share some sequence similarity with well-known protein-degrading enzymes, but generally are distant homologs. Thus, functional screening is necessary to validate sequence-based predictions. One of the proteases shares sequence similarity with S15 peptidases, cocaine esterases and α-amino acid ester hydrolases (AEH). Phylogeny indicates that the gene is of bacterial origin. Enzymatic assays reveal α-aminopeptidase activity towards dipeptides with a preference for a small, L-configured hydrophobic residue at the N-terminus. The crystal structure shows a homotetrameric, self-compartmentalizing enzyme with four independent active sites localized inside the oligomeric assembly accessible from the internal channel. The active site contains a serine protease triad (Ser-His-Asp) and a cluster of negatively charged residues that bind the N-terminal NH3+ group of the substrate molecule. Therefore, the observed activity suggests that the enzyme (designated as AP TA1) may act on di- or tri-peptides produced during extracellular degradation and subsequently imported to the cell. As a close homolog of AEHs, it is also possible that AP TA1 might participate in the synthesis of yet-to-be-discovered secondary metabolites. Supported by NIH GM094585, DOE/BER DE-AC02-06CH11357 & C-DEBI 36202823 & 157595.
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John-White, Marietta, Geoff J. Dumsday, Priscilla Johanesen, Dena Lyras, Nyssa Drinkwater, and Sheena McGowan. "Crystal structure of a β-aminopeptidase from an AustralianBurkholderiasp." Acta Crystallographica Section F Structural Biology Communications 73, no. 7 (June 17, 2017): 386–92. http://dx.doi.org/10.1107/s2053230x17007737.
Full textAbstract:
β-Aminopeptidases are a unique group of enzymes that have the unusual capability to hydrolyze N-terminal β-amino acids from synthetic β-peptides. β-Peptides can form secondary structures mimicking α-peptide-like structures that are resistant to degradation by most known proteases and peptidases. These characteristics of β-peptides give them great potential as peptidomimetics. Here, the X-ray crystal structure of BcA5-BapA, a β-aminopeptidase from a Gram-negativeBurkholderiasp. that was isolated from activated sludge from a wastewater-treatment plant in Australia, is reported. The crystal structure of BcA5-BapA was determined to a resolution of 2.0 Å and showed a tetrameric assembly typical of the β-aminopeptidases. Each monomer consists of an α-subunit (residues 1–238) and a β-subunit (residues 239–367). Comparison of the structure of BcA5-BapA with those of other known β-aminopeptidases shows a highly conserved structure and suggests a similar proteolytic mechanism of action.
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Uddin, Md Jalal, Jirapat Dawan, Gibeom Jeon, Tao Yu, Xinlong He, and Juhee Ahn. "The Role of Bacterial Membrane Vesicles in the Dissemination of Antibiotic Resistance and as Promising Carriers for Therapeutic Agent Delivery." Microorganisms 8, no. 5 (May 5, 2020): 670. http://dx.doi.org/10.3390/microorganisms8050670.
Full textAbstract:
The rapid emergence and spread of antibiotic-resistant bacteria continues to be an issue difficult to deal with, especially in the clinical, animal husbandry, and food fields. The occurrence of multidrug-resistant bacteria renders treatment with antibiotics ineffective. Therefore, the development of new therapeutic methods is a worthwhile research endeavor in treating infections caused by antibiotic-resistant bacteria. Recently, bacterial membrane vesicles (BMVs) have been investigated as a possible approach to drug delivery and vaccine development. The BMVs are released by both pathogenic and non-pathogenic Gram-positive and Gram-negative bacteria, containing various components originating from the cytoplasm and the cell envelope. The BMVs are able to transform bacteria with genes that encode enzymes such as proteases, glycosidases, and peptidases, resulting in the enhanced antibiotic resistance in bacteria. The BMVs can increase the resistance of bacteria to antibiotics. However, the biogenesis and functions of BMVs are not fully understood in association with the bacterial pathogenesis. Therefore, this review aims to discuss BMV-associated antibiotic resistance and BMV-based therapeutic interventions.
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Delfini, Claudio, Chiara Cocito, and M. Bonino. "A review. Biochemical and molecular mechanisms in Saccharomyces cerevisiae that are involved in the formation of some volatile compounds in wines." OENO One 33, no. 4 (December 31, 1999): 195. http://dx.doi.org/10.20870/oeno-one.1999.33.4.1018.
Full textAbstract:
<p style="text-align: justify;">There are evidences that a grape must of a non aromatic vine, not having perfume and revealing by gaschromatographie only some classes of compounds common to the musts of all the vine varieties, can originate a pool of characterizing fragrant substances after contact with the yeast during fermentation. Therefore, despite the scarce scientific knowledge available on biochemical mechanisms involved in <em>Saccharomyces cerevisiae</em> in the formation of a wine aromatic pattern, it can be likely hypothesized that the yeast could be the biological motor of this aromatic transformation. The yeast can act on the compounds of the must with many periplasmic enzymes (estérases, glycosidases, lyases, lipases, proteases, peptidases, pectolytiques) and several are the scientific contributions underlining the existence of an interaction between the yeast and the vine variety in the formation of wine aromatic characteristics. Besides the individual contribution of substances sensorially active, the yeast would contribute to the transformation of unknown varietal aromatic precursors that are in the grape skins and/or musts. The biochemical, genetic and physiological aspects of this transformation still have to be understood. At the end, we have to answer some important questions such as the mutual role that grape and/or yeast enzymes have during and soon after crushing in the liberation of the varietal precursors and in the conversion of these in fragrant compounds.</p>
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Llorente-Bousquets, Adriana, Sandra Pérez-Munguía, and Amelia Farrés. "Novel extracellular proteolytic activity inPediococcus acidilacticiATCC 8042." Canadian Journal of Microbiology 54, no. 8 (August 2008): 694–99. http://dx.doi.org/10.1139/w08-055.
Full textAbstract:
Proteolytic systems are common in lactic acid bacteria, but there are few reports about proteases or peptidases in the genus Pediococcus . To evaluate the presence of these types of enzymes, Pediococcus acidilactici ATCC 8042 was cultured in MRS broth. Supernatants collected during the log phase showed proteolytic activity towards an elastin dispersion when assayed using a spectrophotometer. Zn2+showed a stimulatory effect, and the proteolytic activity reached its maximum when 200 mmol/L NaCl was included in the reaction buffer. On the other hand, activity was reduced when 5 mmol/L EDTA, 10 mmol/L phenylmethylsulfonyl fluoride, and 10 mmol/L 1,10-phenanthroline were used or when the sample was heat treated. Zymograms showed two different proteolytic bands when gelatin was used as a substrate (>200 and 107 kDa), but only the higher molecular mass band was detected when casein or elastin was used. The gelatinolytic activity was not detected with zymograms of the 107 kDa band, which was the one inactivated by heat treatment. The use of a renaturing SDS–PAGE gel with embedded Micrococcus lysodeikticus cells allowed for the detection of a band with peptidoglycan hydrolase activity migrating at about 110 kDa. This activity was lost when 10 mmol/L EDTA was added to the renaturing buffer. Therefore, Pediococcus showed at least three different extracellular enzymes that were produced during the logarithmic growth phase and acted on peptide substrates. Each showed different substrate specificity, ion requirements, and thermostability.
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Peng, Sheng-Bin, Li Wang, John Moomaw, Robert B. Peery, Pei-Ming Sun, Robert B. Johnson, Jin Lu, Patti Treadway, Paul L. Skatrud, and Q. May Wang. "Biochemical Characterization of Signal Peptidase I from Gram-Positive Streptococcus pneumoniae." Journal of Bacteriology 183, no. 2 (January 15, 2001): 621–27. http://dx.doi.org/10.1128/jb.183.2.621-627.2001.
Full textAbstract:
ABSTRACT Bacterial signal peptidase I is responsible for proteolytic processing of the precursors of secreted proteins. The enzymes from gram-negative and -positive bacteria are different in structure and specificity. In this study, we have cloned, expressed, and purified the signal peptidase I of gram-positive Streptococcus pneumoniae. The precursor of streptokinase, an extracellular protein produced in pathogenic streptococci, was identified as a substrate of S. pneumoniae signal peptidase I. Phospholipids were found to stimulate the enzymatic activity. Mutagenetic analysis demonstrated that residues serine 38 and lysine 76 of S. pneumoniae signal peptidase I are critical for enzyme activity and involved in the active site to form a serine-lysine catalytic dyad, which is similar to LexA-like proteases andEscherichia coli signal peptidase I. Similar to LexA-like proteases, S. pneumoniae signal peptidase I catalyzes an intermolecular self-cleavage in vitro, and an internal cleavage site has been identified between glycine 36 and histidine 37. Sequence analysis revealed that the signal peptidase I and LexA-like proteases show sequence homology around the active sites and some common properties around the self-cleavage sites. All these data suggest that signal peptidase I and LexA-like proteases are closely related and belong to a novel class of serine proteases.
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Koppen, Mirko, Florian Bonn, Sarah Ehses, and Thomas Langer. "Autocatalytic Processing of m-AAA Protease Subunits in Mitochondria." Molecular Biology of the Cell 20, no. 19 (October 2009): 4216–24. http://dx.doi.org/10.1091/mbc.e09-03-0218.
Full textAbstract:
m-AAA proteases are ATP-dependent proteolytic machines in the inner membrane of mitochondria which are crucial for the maintenance of mitochondrial activities. Conserved nuclear-encoded subunits, termed paraplegin, Afg3l1, and Afg3l2, form various isoenzymes differing in their subunit composition in mammalian mitochondria. Mutations in different m-AAA protease subunits are associated with distinct neuronal disorders in human. However, the biogenesis of m-AAA protease complexes or of individual subunits is only poorly understood. Here, we have examined the processing of nuclear-encoded m-AAA protease subunits upon import into mitochondria and demonstrate autocatalytic processing of Afg3l1 and Afg3l2. The mitochondrial processing peptidase MPP generates an intermediate form of Afg3l2 that is matured autocatalytically. Afg3l1 or Afg3l2 are also required for maturation of newly imported paraplegin subunits after their cleavage by MPP. Our results establish that mammalian m-AAA proteases can act as processing enzymes in vivo and reveal overlapping activities of Afg3l1 and Afg3l2. These findings might be of relevance for the pathogenesis of neurodegenerative disorders associated with mutations in different m-AAA protease subunits.
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Sannes, P. L., B. H. Schofield, and D. F. McDonald. "Histochemical localization of cathepsin B, dipeptidyl peptidase I, and dipeptidyl peptidase II in rat bone." Journal of Histochemistry & Cytochemistry 34, no. 8 (August 1986): 983–88. http://dx.doi.org/10.1177/34.8.3016074.
Full textAbstract:
The histochemical distribution of the thiol proteases cathepsin B and dipeptidyl peptidase I and the serine protease dipeptidyl peptidase II was examined in rat bone and joint using amino acid derivatives of 4-methoxy-2-naphthylamine (MNA). The liberated MNA was then visualized by simultaneous coupling with fast blue B. Cathepsin B was examined with CBZ-Arg-Arg-MNA, dipeptidyl peptidase I (DPP I) with Gly-Arg- or Pro-Arg-MNA, and dipeptidyl peptidase II (DPP II) with Lys-ALA- or Lys-Pro-MNA. Bright red reaction product indicative of proteolytic activity was observed in most cell types associated with bone and its surrounding connective tissues, including osteocytes, osteoblasts, chondrocytes, chondroblasts, fibroblasts, and macrophages. Surprisingly, protease activity in osteoclasts could not be established with certainty, and it was concluded that these enzymes are either absent, present in very low amounts, or secreted as soon as they are synthesized rather than stored within the cell. The cells of the resting zone of the growth plate were intensely reactive for DPP II but were only moderately reactive for cathepsin B and DPP I. The reverse was true of the proliferating and hypertrophic layers. The protease activity observed in bone, cartilage, tendon, ligament, and synovium would be expected to contribute significantly to normal protein metabolism as well as to pathological destruction in these tissues.
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Wilson, Claire H., Hui Emma Zhang, Mark D. Gorrell, and Catherine A. Abbott. "Dipeptidyl peptidase 9 substrates and their discovery: current progress and the application of mass spectrometry-based approaches." Biological Chemistry 397, no. 9 (September 1, 2016): 837–56. http://dx.doi.org/10.1515/hsz-2016-0174.
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Abstract The enzyme members of the dipeptidyl peptidase 4 (DPP4) gene family have the very unusual capacity to cleave the post-proline bond to release dipeptides from the N-terminus of peptide/protein substrates. DPP4 and related enzymes are current and potential therapeutic targets in the treatment of type II diabetes, inflammatory conditions and cancer. Despite this, the precise biological function of individual dipeptidyl peptidases (DPPs), other than DPP4, and knowledge of their in vivo substrates remains largely unknown. For many years, identification of physiological DPP substrates has been difficult due to limitations in the available tools. Now, with advances in mass spectrometry based approaches, we can discover DPP substrates on a system wide-scale. Application of these approaches has helped reveal some of the in vivo natural substrates of DPP8 and DPP9 and their unique biological roles. In this review, we provide a general overview of some tools and approaches available for protease substrate discovery and their applicability to the DPPs with a specific focus on DPP9 substrates. This review provides comment upon potential approaches for future substrate elucidation.
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Brüning, Mareke, Martina Lummer, Caterina Bentele, Marcel M. W. Smolenaars, Kees W. Rodenburg, and Hermann Ragg. "The Spn4 gene from Drosophila melanogaster is a multipurpose defence tool directed against proteases from three different peptidase families." Biochemical Journal 401, no. 1 (December 11, 2006): 325–31. http://dx.doi.org/10.1042/bj20060648.
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By alternative use of four RSL (reactive site loop) coding exon cassettes, the serpin (serine protease inhibitor) gene Spn4 from Drosophila melanogaster was proposed to enable the synthesis of multiple protease inhibitor isoforms, one of which has been shown to be a potent inhibitor of human furin. Here, we have investigated the inhibitory spectrum of all Spn4 RSL variants. The analyses indicate that the Spn4 gene encodes inhibitors that may inhibit serine proteases of the subtilase family (S8), the chymotrypsin family (S1), and the papain-like cysteine protease family (C1), most of them at high rates. Thus a cohort of different protease inhibitors is generated simply by grafting enzyme-adapted RSL sequences on to a single serpin scaffold, even though the target proteases contain different types and/or a varying order of catalytic residues and are descendents of different phylogenetic lineages. Since all of the Spn4 RSL isoforms are produced as intracellular residents and additionally as variants destined for export or associated with the secretory pathway, the Spn4 gene represents a versatile defence tool kit that may provide multiple antiproteolytic functions.
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Oriano, Martina, Francesco Amati, Andrea Gramegna, Anthony De Soyza, Marco Mantero, Oriol Sibila, Sanjay H. Chotirmall, et al. "Protease–Antiprotease Imbalance in Bronchiectasis." International Journal of Molecular Sciences 22, no. 11 (June 1, 2021): 5996. http://dx.doi.org/10.3390/ijms22115996.
Full textAbstract:
Airway inflammation plays a central role in bronchiectasis. Protease–antiprotease balance is crucial in bronchiectasis pathophysiology and increased presence of unopposed proteases activity may contribute to bronchiectasis onset and progression. Proteases’ over-reactivity and antiprotease deficiency may have a role in increasing inflammation in bronchiectasis airways and may lead to extracellular matrix degradation and tissue damage. Imbalances in serine proteases and matrix-metallo proteinases (MMPs) have been associated to bronchiectasis. Active neutrophil elastase has been associated with disease severity and poor long-term outcomes in this disease. Moreover, high levels of MMPs have been associated with radiological and disease severity. Finally, severe deficiency of α1-antitrypsin (AAT), as PiSZ and PiZZ (proteinase inhibitor SZ and ZZ) phenotype, have been associated with bronchiectasis development. Several treatments are under study to reduce protease activity in lungs. Molecules to inhibit neutrophil elastase activity have been developed in both oral or inhaled form, along with compounds inhibiting dipeptydil-peptidase 1, enzyme responsible for the activation of serine proteases. Finally, supplementation with AAT is in use for patients with severe deficiency. The identification of different targets of therapy within the protease–antiprotease balance contributes to a precision medicine approach in bronchiectasis and eventually interrupts and disrupts the vicious vortex which characterizes the disease.
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De Toni, C. H., M. F. Richter, J. R. Chagas, J. AP Henriques, and C. Termignoni. "Purification and characterization of an alkaline serine endopeptidase from a feather-degradingXanthomonas maltophiliastrain." Canadian Journal of Microbiology 48, no. 4 (April 1, 2002): 342–48. http://dx.doi.org/10.1139/w02-027.
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A keratinolytic Xanthomonas maltophilia strain (POA-1), cultured on feather meal broth, using keratin as its sole source of carbon and nitrogen, secretes several extracellular peptidases. The major serine peptidase was purified to homogeneity by a five-step procedure. Its purity was evaluated by capillary zone electrophoresis. This enzyme has a molecular mass of 36 kDa, an optimum pH of 9.0, and an optimum temperature of 60°C. The inhibitory profile using protease inhibitors shows that this enzyme is a serine endopeptidase. Besides keratin, the enzyme is active upon the substrates azokeratin, azocasein, and the following fluorogenic peptide substrates: Abz-Leu-Gly-Met-Ile-Ser-Leu-Met-Lys-Arg-Pro-Gln-EDDnp, Abz-Lys-Leu-Cys(SBzl)-Gly-Pro-Lys-Gln-EDDnp, and Abz-Lys-Pro-Cys(SBzl)-Phe-Ser-Lys-Gln-EDDnp.Key words: serine endopeptidase, Xanthomonas maltophilia, keratinase, alkaline endopeptidase.
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Hedger, Mark P., and Michael D. Culler. "Comparison of LHRH-peptidase and plasminogen activator activity in rat testis extracts." Reproduction, Fertility and Development 9, no. 7 (1997): 659. http://dx.doi.org/10.1071/r97062.
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Testicular LHRH-peptidase and testicular urokinase-type plasminogen activator are Sertoli cell-secreted proteases which display similar molecular properties. However, there is relatively little information regarding the substrate specificity and potential cross-reactivity of these enzymes. Testicular extracts were prepared from homogenates of whole rat testes and assessed by LHRH-peptidase assay, and by radial caseinolysis assays for plasminogen activator and plasmin-like activity. Following partial purification of the protease activities in testicular extracts by gel filtration and ion-exchange chromatography, it was conrmed that testicular LHRH-peptidase and plasminogen activator are clearly separable. There was no detectable plasmin-like activity in the testicular extracts; however, the extracts were found to contain an inhibitor, or inhibitors, of both plasminogen activator and plasmin activity. In addition to LHRH and Gly 6 -substituted LHRH analogues, the partially purified LHRH-peptidase degraded both angiotensins I and II, but not the gonadotrophin-releasing-hormone-associated peptide derived from the LHRH precursor molecule. These properties of the LHRH-peptidase provide further evidence that it is a testis-specific prolyl endopeptidase, involved in regulating and/or limiting peptide activity in the testis.
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Kudzhaev, A. M., A. G. Andrianova, E. S. Dubovtseva, O. V. Serova, and T. V. Rotanova. "Role of the Inserted α-Helical Domain in E. coli ATP-Dependent Lon Protease Function." Acta Naturae 9, no. 2 (June 15, 2017): 75–81. http://dx.doi.org/10.32607/20758251-2017-9-2-75-81.
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Multidomain ATP-dependent Lon protease of E. coli (Ec-Lon) is one of the key enzymes of the quality control system of the cellular proteome. A recombinant form of Ec-Lon with deletion of the inserted characteristic -helical HI(CC) domain (Lon-dHI(CC)) has been prepared and investigated to understand the role of this domain. A comparative study of the ATPase, proteolytic, and peptidase activities of the intact Lon protease and Lon-dHI(CC) has been carried out. The ability of the enzymes to undergo autolysis and their ability to bind DNA have been studied as well. It has been shown that the HI(CC) domain of Ec-Lon protease is required for the formation of a functionally active enzyme structure and for the implementation of protein-protein interactions.
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Swatek, Anita, and Magdalena Staszczak. "Effect of Ferulic Acid, a Phenolic Inducer of Fungal Laccase, on 26S Proteasome Activities In Vitro." International Journal of Molecular Sciences 21, no. 7 (April 2, 2020): 2463. http://dx.doi.org/10.3390/ijms21072463.
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The 26S proteasome is an ATP-dependent protease complex (2.5 MDa) that degrades most cellular proteins in Eukaryotes, typically those modified by a polyubiquitin chain. The proteasome-mediated proteolysis regulates a variety of critical cellular processes such as transcriptional control, cell cycle, oncogenesis, apoptosis, protein quality control, and stress response. Previous studies conducted in our laboratory have shown that 26S proteasomes are involved in the regulation of ligninolytic enzymes (such as laccase) in white-rot fungi in response to nutrient starvation, cadmium exposure, and ER stress. Laccases are useful biocatalysts for a wide range of biotechnological applications. The goal of the current study was to determine the effect of ferulic acid (4-hydroxy-3-methoxycinnamic acid), a phenolic compound known to induce some ligninolytic enzymes, on proteasomes isolated from mycelia of the wood-decomposing basidiomycete Trametes versicolor. The peptidase activities of 26S proteasomes were assayed by measuring the hydrolysis of fluorogenic peptide substrates specific for each active site: Suc-LLVY-AMC, Z-GGR-AMC and Z-LLE-AMC for chymotrypsin-like, trypsin-like, and caspase-like site, respectively. Ferulic acid affected all peptidase activities of the 26S fungal proteasomes in a concentration-dependent manner. A possible inhibitory effect of ferulic acid on peptidase activities of the 26S human proteasomes was tested as well. Moreover, the ability of ferulic acid to inhibit (at concentrations known to induce laccase activity in white-rot fungi) the rate of 26S proteasome-catalyzed degradation of a model full-length protein substrate (β-casein) was demonstrated by a fluorescamine assay and by a gel-electrophoretic analysis. Our findings provide new insights into the role of ferulic acid in lignin-degrading fungi. However, the detailed molecular mechanisms involved remain to be elucidated by future studies.
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Magnen, Mélia, Brigitta Margit Elsässer, Olga Zbodakova, Petr Kasparek, Fabien Gueugnon, Agnès Petit-Courty, Radislav Sedlacek, Peter Goettig, and Yves Courty. "Kallikrein-related peptidase 5 and seasonal influenza viruses, limitations of the experimental models for activating proteases." Biological Chemistry 399, no. 9 (September 25, 2018): 1053–64. http://dx.doi.org/10.1515/hsz-2017-0340.
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Abstract Every year, influenza A virus (IAV) affects and kills many people worldwide. The viral hemagglutinin (HA) is a critical actor in influenza virus infectivity which needs to be cleaved by host serine proteases to exert its activity. KLK5 has been identified as an activating protease in humans with a preference for the H3N2 IAV subtype. We investigated the origin of this preference using influenza A/Puerto Rico/8/34 (PR8, H1N1) and A/Scotland/20/74 (Scotland, H3N2) viruses. Pretreatment of noninfectious virions with human KLK5 increased infectivity of Scotland IAV in MDCK cells and triggered influenza pneumonia in mice. These effects were not observed with the PR8 IAV. Molecular modeling and in vitro enzymatic studies of peptide substrates and recombinant HAs revealed that the sequences around the cleavage site do not represent the sole determinant of the KLK5 preference for the H3N2 subtype. Using mouse Klk5 and Klk5-deficient mice, we demonstrated in vitro and in vivo that the mouse ortholog protease is not an IAV activating enzyme. This may be explained by unfavorable interactions between H3 HA and mKlk5. Our data highlight the limitations of some approaches used to identify IAV-activating proteases.
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Levesque, Jean-Pierre, Fulu Liu, Paul J. Simmons, Tomoko Betsuyaku, Robert M. Senior, Christine Pham, and Daniel C. Link. "Characterization of hematopoietic progenitor mobilization in protease-deficient mice." Blood 104, no. 1 (July 1, 2004): 65–72. http://dx.doi.org/10.1182/blood-2003-05-1589.
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Abstract Recent evidence suggests that protease release by neutrophils in the bone marrow may contribute to hematopoietic progenitor cell (HPC) mobilization. Matrix metalloproteinase-9 (MMP-9), neutrophil elastase (NE), and cathepsin G (CG) accumulate in the bone marrow during granulocyte colony-stimulating factor (G-CSF) treatment, where they are thought to degrade key substrates including vascular cell adhesion molecule-1 (VCAM-1) and CXCL12. To test this hypothesis, HPC mobilization was characterized in transgenic mice deficient in one or more hematopoietic proteases. Surprisingly, HPC mobilization by G-CSF was normal in MMP-9–deficient mice, NE × CG-deficient mice, or mice lacking dipeptidyl peptidase I, an enzyme required for the functional activation of many hematopoietic serine proteases. Moreover, combined inhibition of neutrophil serine proteases and metalloproteinases had no significant effect on HPC mobilization. VCAM-1 expression on bone marrow stromal cells decreased during G-CSF treatment of wild-type mice but not NE × CG-deficient mice, indicating that VCAM-1 cleavage is not required for efficient HPC mobilization. G-CSF induced a significant decrease in CXCL12α protein expression in the bone marrow of Ne × CG-deficient mice, indicating that these proteases are not required to down-regulate CXCL12 expression. Collectively, these data suggest a complex model in which both protease-dependent and -independent pathways may contribute to HPC mobilization.
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Gonçalves, Rayane Natshe, Suellen Duarte Gozzini Barbosa, and Raquel Elisa da Silva-López. "Proteases from Canavalia ensiformis: Active and Thermostable Enzymes with Potential of Application in Biotechnology." Biotechnology Research International 2016 (August 17, 2016): 1–11. http://dx.doi.org/10.1155/2016/3427098.
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Extracts of leaves, seeds, roots, and stem from a tropical legume, C. ensiformis, were prepared employing buffers and detergent in aqueous solution. Leaf extracts had the highest protein content and the most pronounced peptidase activity with optimal pH in the neutral to alkaline range. All extracts exhibited peaks of activity at various pH values, suggesting the presence of distinctive classes of proteases. N-α-Tosyl-L-arginine methyl ester hydrolysis was maximal at 30°C to 60°C and peptidase activity from all extracts presented very good thermal stability after 24 h incubation at 70°C. C. ensiformis proteases exhibited molecular masses of about 200–57, 40–37, and 20–15 kDa by SDS-PAGE analysis. These enzymes cleaved hemoglobin, bovine serum albumin, casein, and gelatin at different levels. Serine and metalloproteases are the major proteases in C. ensiformis extracts, modulated by divalent cations, stable at 1% of surfactant Triton X-100 and at different concentrations of the reducing agent β-mercaptoethanol. Thus, C. ensiformis expresses a particular set of proteases in distinctive organs with high activity and stability, making this legume an important source of proteases with biotechnological potential.
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Wiederanders, Bernd. "Structure-function relationships in class CA1 cysteine peptidase propeptides." Acta Biochimica Polonica 50, no. 3 (September 30, 2003): 691–713. http://dx.doi.org/10.18388/abp.2003_3661.
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Regulation of proteolytic enzyme activity is an essential requirement for cells and tissues because proteolysis at a wrong time and location may be lethal. Proteases are synthesized as inactive or less active precursor molecules in order to prevent such inappropriate proteolysis. They are activated by limited intra- or intermolecular proteolysis cleaving off an inhibitory peptide. These regulatory proenzyme regions have attracted much attention during the last decade, since it became obvious that they harbour much more information than just triggering activation. In this review we summarize the structural background of three functions of clan CA1 cysteine peptidase (papain family) proparts, namely the selectivity of their inhibitory potency, the participation in correct intracellular targeting and assistance in folding of the mature enzyme. Today, we know more than 500 cysteine peptidases of this family from the plant and animal kingdoms, e.g. papain and the lysosomal cathepsins L and B. As it will be shown, the propeptide functions are determined by certain structural motifs conserved over millions of years of evolution.
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Madhu, Swati N., Savitri Sharma, and Devarshi U. Gajjar. "Identification of Proteases: Carboxypeptidase and Aminopeptidase as Putative Virulence Factors of Fusarium solani Species Complex." Open Microbiology Journal 14, no. 1 (November 25, 2020): 266–77. http://dx.doi.org/10.2174/1874434602014010266.
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Background: Fusarium keratitis accounts for around 50% of mycotic keratitis cases. Major virulence factors produced by keratopathogenic fungi are proteases. Objective: The aim of the current study was to identify proteases contributing to corneal pathogenicity of Fusarium species. Methods: Culture filtrates from fourteen Fusarium solani species complex (FSSC) isolates and three F. delphinoides isolates were evaluated for protease activity and gelatine zymography. Mass spectroscopy was carried out using a partially purified enzyme and total extracellular extract. Protease gene expression in an in-vitro condition and an ex-vivo goat corneal infection model was measured using qRT-PCR. Specific activity was observed in a wide range and at a broad pH range; and isolates Cs1 (maximum) and Cc50 (minimum) were selected for the infection model. Results: Gene expression in in-vitro condition showed the highest fold change for proteases (C7YY94, C7Z7U2 and C7Z6W1) while in an ex-vivo infection highest fold change was seen for proteases (C7Z6W1, C7YQJ2 and C7Z7U2); in decreasing order, respectively. Expression of aminopeptidase (C7Z6W1) was 50-fold higher in the infected cornea in both isolates (Cs1 and Cc50); while expression of carboxypeptidase (C7YVF3) was 15-fold higher only in isolate Cs1. Corneal histology showed less penetration of Cc50 than Cs1 into the stroma. Mass spectrometry showed the presence of carboxypeptidase (C7YVF3) and tripeptidyl amino peptidase. Conclusion: It can be concluded that clinical isolates of FSSC produce varying amounts of proteases and differ in specific activity and gene expression in both conditions (in vitro and ex vivo). Carboxypeptidase and aminopeptidase contribute to the pathogenic potential of Fusarium solani species complex.
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Banbula, Agnieszka, Marcin Bugno, Jason Goldstein, Jane Yen, Daniel Nelson, James Travis, and Jan Potempa. "Emerging Family of Proline-Specific Peptidases ofPorphyromonas gingivalis: Purification and Characterization of Serine Dipeptidyl Peptidase, a Structural and Functional Homologue of Mammalian Prolyl Dipeptidyl Peptidase IV." Infection and Immunity 68, no. 3 (March 1, 2000): 1176–82. http://dx.doi.org/10.1128/iai.68.3.1176-1182.2000.
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ABSTRACT Porphyromonas gingivalis is an asaccharolytic and anaerobic bacterium that possesses a complex proteolytic system which is essential for its growth and evasion of host defense mechanisms. In this report, we show the purification and characterization of prolyl dipeptidyl peptidase IV (DPPIV) produced by this organism. The enzyme was purified to homogeneity, and its enzymatic activity and biochemical properties were investigated. P. gingivalis DPPIV, like its human counterpart, is able to cleave the N terminus of synthetic oligopeptides with sequences analogous to those of interleukins 1β and 2. Additionally, this protease hydrolyzes biologically active peptides including substance P, fibrin inhibitory peptide, and β-casomorphin. Southern blot analysis of genomic DNA isolated from several P. gingivalis strains reveal that a single copy of the DPPIV gene was present in all strains tested.
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Fukasawa, Kayoko M., Toshiyuki Hata, Yukio Ono, and Junzo Hirose. "Metal Preferences of Zinc-Binding Motif on Metalloproteases." Journal of Amino Acids 2011 (May 11, 2011): 1–7. http://dx.doi.org/10.4061/2011/574816.
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Almost all naturally occurring metalloproteases are monozinc enzymes. The zinc in any number of zinc metalloproteases has been substituted by some other divalent cation. Almost all Co(II)- or Mn(II)-substituted enzymes maintain the catalytic activity of their zinc counterparts. However, in the case of Cu(II) substitution of zinc proteases, a great number of enzymes are not active, for example, thermolysin, carboxypeptidase A, endopeptidase from Lactococcus lactis, or aminopeptidase B, while some do have catalytic activity, for example, astacin (37%) and DPP III (100%). Based on structural studies of various metal-substituted enzymes, for example, thermolysin, astacin, aminopeptidase B, dipeptidyl peptidase (DPP) III, and del-DPP III, the metal coordination geometries of both active and inactive Cu(II)-substituted enzymes are shown to be the same as those of the wild-type Zn(II) enzymes. Therefore, the enzyme activity of a copper-ion-substituted zinc metalloprotease may depend on the flexibility of catalytic domain.
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Su, Q., and A. Boschetti. "Substrate- and species-specific processing enzymes for chloroplast precursor proteins." Biochemical Journal 300, no. 3 (June 15, 1994): 787–92. http://dx.doi.org/10.1042/bj3000787.
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Using different precursors of chloroplast proteins and stromal extracts from both Chlamydomonas reinhardii and pea chloroplasts, we analysed the specificity of stroma-localized processing peptidases. By gel filtration of a stromal extract from isolated Chlamydomonas chloroplasts, fractions could be separated containing enzymic activities for processing the precursors of the small subunit of ribulose-1,5-bisphosphate carboxylase (pSS) and of the protein OEE1 from the photosynthetic water-splitting complex (pOEE1). The enzymes differed not only in molecular size, but also in their sensitivity to inhibitors and in their pH optima. Obviously, in the stroma of Chlamydomonas chloroplasts different peptidases exist for processing of pSS and pOEE1, the latter being converted into an intermediate-sized form, iOEE1, which was found to be further processed to mature OEE1 by a thylakoid-associated protease. To study the species-specificity of the stromal peptidases, stromal extracts from Chlamydomonas and pea chloroplasts were incubated with pSS from either of these organisms. In the heterologous combinations, the precursors were partly hydrolysed, but not to the correct size. In importation assays, pSS from pea (but also the precursor of the ribosomal protein L12 from spinach) could not enter into chloroplasts from Chlamydomonas. In contrast, the algal pSS was imported into chloroplasts from pea, although it was not processed to mature SS. Our results indicate that the importation machinery and the pSS-processing enzymes in higher plants and green algae have different specificities and that in Chlamydomonas several stromal peptidases for different precursor proteins exist.
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Mølgaard, Anne, Jose Arnau, Conni Lauritzen, Sine Larsen, Gitte Petersen, and John Pedersen. "The crystal structure of human dipeptidyl peptidase I (cathepsin C) in complex with the inhibitor Gly-Phe-CHN2." Biochemical Journal 401, no. 3 (January 12, 2007): 645–50. http://dx.doi.org/10.1042/bj20061389.
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hDDPI (human dipeptidyl peptidase I) is a lysosomal cysteine protease involved in zymogen activation of granule-associated proteases, including granzymes A and B from cytotoxic T-lymphocytes and natural killer cells, cathepsin G and neutrophil elastase, and mast cell tryptase and chymase. In the present paper, we provide the first crystal structure of an hDPPI–inhibitor complex. The inhibitor Gly-Phe-CHN2 (Gly-Phe-diazomethane) was co-crystallized with hDPPI and the structure was determined at 2.0 Å (1 Å=0.1 nm) resolution. The structure of the native enzyme was also determined to 2.05 Å resolution to resolve apparent discrepancies between the complex structure and the previously published structure of the native enzyme. The new structure of the native enzyme is, within the experimental error, identical with the structure of the enzyme–inhibitor complex presented here. The inhibitor interacts with three subunits of hDPPI, and is covalently bound to Cys234 at the active site. The interaction between the totally conserved Asp1 of hDPPI and the ammonium group of the inhibitor forms an essential interaction that mimics enzyme–substrate interactions. The structure of the inhibitor complex provides an explanation of the substrate specificity of hDPPI, and gives a background for the design of new inhibitors.
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Chukhontseva, Ksenia N., Vadim V. Salnikov, Oleg S. Morenkov, Sergey V. Kostrov, and Ilya V. Demidyuk. "Protealysin is not Secreted Constitutively." Protein & Peptide Letters 26, no. 3 (March 15, 2019): 221–26. http://dx.doi.org/10.2174/0929866526666181212114907.
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Background:Protealysin, a zinc metalloprotease of Serratia proteamaculans, is the prototype of a new group within the peptidase family M4. Protealysin-like proteases (PLPs) are widely spread in bacteria but are also found in fungi and archaea. The biological functions of PLPs have not been well studied, but published data showed the involvement of enzymes of this group in the interaction of bacteria with higher organisms, and most likely in the pathogenesis. Such functionality requires the release of the proteases from bacterial cells; however, the data on the cellular localization of PLPs are contradictory and no direct data of this kind have been published. </P><P> Objective: Here, the protealysin cellular localization was studied for the first time using immunochemical methods. </P><P> Methods and Results: We have produced polyclonal rabbit antibodies against the protealysin precursor. The enzyme was evaluated in cells and medium of periodic culture of S. proteamaculans 94 using Western blotting as well as the enzyme localization was analysed by immunoelectron microscopy. It was shown that more than 99% of the enzyme is in a cell-associated form. Protealysin is accumulated in cells as an inactive precursor. It matures only after the release from cells (after their lysis). Immunoelectron microscopy analysis of bacterial cells has revealed no specific localization of protealysin; it was evenly distributed in the cytoplasm.Conclusion:The data obtained suggest that S. proteamaculans protealysin and supposedly other protealysin-like proteases are not secreted constitutively and their release from bacteria is likely induced by a certain stimulus such as a contact with a eukaryotic cell. This finding is critical for further studies of the involvement of these enzymes in pathogenesis.
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Ting, Yi Tian, Paul W. R. Harris, Gaelle Batot, Margaret A. Brimble, Edward N. Baker, and Paul G. Young. "Peptide binding to a bacterial signal peptidase visualized by peptide tethering and carrier-driven crystallization." IUCrJ 3, no. 1 (January 1, 2016): 10–19. http://dx.doi.org/10.1107/s2052252515019971.
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Bacterial type I signal peptidases (SPases) are membrane-anchored serine proteases that process the signal peptides of proteins exported via the Sec and Tat secretion systems. Despite their crucial importance for bacterial virulence and their attractiveness as drug targets, only one such enzyme, LepB from Escherichia coli, has been structurally characterized, and the transient nature of peptide binding has stymied attempts to directly visualize SPase–substrate complexes. Here, the crystal structure of SpsB, the type I signal peptidase from the Gram-positive pathogen Staphylococcus aureus, is reported, and a peptide-tethering strategy that exploits the use of carrier-driven crystallization is described. This enabled the determination of the crystal structures of three SpsB–peptide complexes, both with cleavable substrates and with an inhibitory peptide. SpsB–peptide interactions in these complexes are almost exclusively limited to the canonical signal-peptide motif Ala-X-Ala, for which clear specificity pockets are found. Minimal contacts are made outside this core, with the variable side chains of the peptides accommodated in shallow grooves or exposed faces. These results illustrate how high fidelity is retained despite broad sequence diversity, in a process that is vital for cell survival.