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1
Harte, James V., Samantha L. Wakerlin, Andrew J. Lindsay, Justin V. McCarthy, and Caroline Coleman-Vaughan. "Metalloprotease-Dependent S2′-Activation Promotes Cell–Cell Fusion and Syncytiation of SARS-CoV-2." Viruses 14, no. 10 (September 21, 2022): 2094. http://dx.doi.org/10.3390/v14102094.
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SARS-CoV-2 cell–cell fusion and syncytiation is an emerging pathomechanism in COVID-19, but the precise factors contributing to the process remain ill-defined. In this study, we show that metalloproteases promote SARS-CoV-2 spike protein-induced syncytiation in the absence of established serine proteases using in vitro cell–cell fusion assays. We also show that metalloproteases promote S2′-activation of the SARS-CoV-2 spike protein, and that metalloprotease inhibition significantly reduces the syncytiation of SARS-CoV-2 variants of concern. In the presence of serine proteases, however, metalloprotease inhibition does not reduce spike protein-induced syncytiation and a combination of metalloprotease and serine protease inhibition is necessitated. Moreover, we show that the spike protein induces metalloprotease-dependent ectodomain shedding of the ACE2 receptor and that ACE2 shedding contributes to spike protein-induced syncytiation. These observations suggest a benefit to the incorporation of pharmacological inhibitors of metalloproteases into treatment strategies for patients with COVID-19.
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Slapak, Etienne J., JanWillem Duitman, Cansu Tekin, Maarten F. Bijlsma, and C. Arnold Spek. "Matrix Metalloproteases in Pancreatic Ductal Adenocarcinoma: Key Drivers of Disease Progression?" Biology 9, no. 4 (April 18, 2020): 80. http://dx.doi.org/10.3390/biology9040080.
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Pancreatic cancer is a dismal disorder that is histologically characterized by a dense fibrotic stroma around the tumor cells. As the extracellular matrix comprises the bulk of the stroma, matrix degrading proteases may play an important role in pancreatic cancer. It has been suggested that matrix metalloproteases are key drivers of both tumor growth and metastasis during pancreatic cancer progression. Based upon this notion, changes in matrix metalloprotease expression levels are often considered surrogate markers for pancreatic cancer progression and/or treatment response. Indeed, reduced matrix metalloprotease levels upon treatment (either pharmacological or due to genetic ablation) are considered as proof of the anti-tumorigenic potential of the mediator under study. In the current review, we aim to establish whether matrix metalloproteases indeed drive pancreatic cancer progression and whether decreased matrix metalloprotease levels in experimental settings are therefore indicative of treatment response. After a systematic review of the studies focusing on matrix metalloproteases in pancreatic cancer, we conclude that the available literature is not as convincing as expected and that, although individual matrix metalloproteases may contribute to pancreatic cancer growth and metastasis, this does not support the generalized notion that matrix metalloproteases drive pancreatic ductal adenocarcinoma progression.
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Beek, J., H. Nauwynck, D. Maes, and A. Van Soom. "Inhibitors of zinc-dependent metalloproteases hinder sperm passage through the cumulus oophorus during porcine fertilization in vitro." REPRODUCTION 144, no. 6 (December 2012): 687–97. http://dx.doi.org/10.1530/rep-12-0311.
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In this study, we report for the first time on a possible contribution of metalloproteases in sperm passage through the cumulus matrix in pigs. The presence of 20 μM 1,10-phenanthroline (1,10-PHEN), inhibitor of zinc-dependent metalloproteases, strongly inhibited the degree of sperm penetration in cumulus-intact (CI), but not in cumulus-free (CF), porcine oocytes during IVF. The inhibitory effect of 1,10-PHEN was due to the chelation of metal ions as a non-chelating analog (1,7-PHEN) did not affect IVF rates. Furthermore, incubation with 1,10-PHEN did not affect sperm binding to the zona pellucida nor sperm motility, membrane integrity, or acrosomal status. These findings led to the assumption that 1,10-PHEN interacts with a sperm- or cumulus-derived metalloprotease. Metalloproteases are key players in physiological processes involving degradation or remodeling of extracellular matrix. In vivo, their proteolytic activity is regulated by tissue inhibitors of metalloproteases (TIMP1–TIMP4). We tested the effect of TIMP3 on fertilization parameters after porcine IVF. Similar to 1,10-PHEN, TIMP3 inhibited total fertilization rate of CI but not CF oocytes and did not influence sperm quality parameters. Although the inhibitory effect was stronger in CI oocytes, TIMP3 also reduced the degree of sperm penetration in CF oocytes, suggesting the involvement of a metalloprotease in a subsequent step during fertilization. In conclusion, our results indicate the involvement of TIMP3-sensitive, zinc-dependent metalloprotease activity in sperm passage through the cumulus oophorus in pigs. The results should provide the basis for further biochemical research toward the localization and identification of the metalloprotease involved.
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Ramos, O. H. P., and H. S. Selistre-de-Araujo. "Identification of metalloprotease gene families in sugarcane." Genetics and Molecular Biology 24, no. 1-4 (December 2001): 285–90. http://dx.doi.org/10.1590/s1415-47572001000100037.
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Metalloproteases play a key role in many physiological processes in mammals such as cell migration, tissue remodeling and processing of growth factors. They have also been identified as important factors in the patho-physiology of a number of human diseases, including cancer and hypertension. Many bacterial pathogens rely on proteases in order to infect the host. Several classes of metalloproteases have been described in humans, bacteria, snake venoms and insects. However, the presence and characterization of plant metalloproteases have rarely been described in the literature. In our research, we searched the sugarcane expressed sequence tag (SUCEST) DNA library in order to identify, by homology with sequences deposited in other databases, metalloprotease gene families expressed under different conditions. Protein sequences from Arabidopsis thaliana and Glycine max were used to search the SUCEST data bank. Conserved regions corresponding to different metalloprotease domains and sequence motifs were identified in the reads to characterize each group of enzymes. At least four classes of sugarcane metalloproteases have been identified, i.e. matrix metalloproteases, zincins, inverzincins, and ATP-dependent metalloproteases. Each enzyme class was analyzed for its expression in different conditions and tissues.
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Corbett, C. R., M. N. Burtnick, C. Kooi, D. E. Woods, and P. A. Sokol. "An extracellular zinc metalloprotease gene of Burkholderia cepacia." Microbiology 149, no. 8 (August 1, 2003): 2263–71. http://dx.doi.org/10.1099/mic.0.26243-0.
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Burkholderia cepacia produces at least one extracellular zinc metalloprotease that may be involved in virulence. A B. cepacia zinc metalloprotease gene was cloned using a Burkholderia pseudomallei zinc metalloprotease gene as a probe. The predicted amino acid sequences of these B. cepacia and a B. pseudomallei extracellular zinc metalloproteases indicate that they are similar to the thermolysin-like family of metalloproteases (M4 family of metalloendopeptidases) and they are likely to be secreted via the general secretory pathway. zmpA isogenic mutants were constructed in B. cepacia genomovar III strains Pc715j and K56-2 by insertional inactivation of the zmpA genes. The zmpA mutants produced less protease than the parent strains. The B. cepacia strain K56-2 zmpA mutant was significantly less virulent than its parent strain in a chronic respiratory infection model; however, there was no difference between the virulence of B. cepacia strain Pc715j and a Pc715j zmpA mutant. The results indicate that this extracellular zinc metalloprotease may play a greater role in virulence in some strains of B. cepacia.
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Goblirsch, Brandon R., Buenafe T. Arachea, Daniel J. Councell, and Michael C. Wiener. "Phosphoramidon inhibits the integral membrane protein zinc metalloprotease ZMPSTE24." Acta Crystallographica Section D Structural Biology 74, no. 8 (July 24, 2018): 739–47. http://dx.doi.org/10.1107/s2059798318003431.
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The integral membrane protein zinc metalloprotease ZMPSTE24 possesses a completely novel structure, comprising seven long kinked transmembrane helices that encircle a voluminous 14 000 Å3cavity within the membrane. Functionally conserved soluble zinc metalloprotease residues are contained within this cavity. As part of an effort to understand the structural and functional relationships between ZMPSTE24 and soluble zinc metalloproteases, the inhibition of ZMPSTE24 by phosphoramidon [N-(α-rhamnopyranosyl-oxyhydroxyphosphinyl)-Leu-Trp], a transition-state analog and competitive inhibitor of multiple soluble zinc metalloproteases, especially gluzincins, has been characterized functionally and structurally. The functional results, the determination of preliminary IC50values by the use of an intramolecular quenched-fluorescence fluorogenic peptide assay, indicate that phosphoramidon inhibits ZMPSTE24 in a manner consistent with competitive inhibition. The structural results, a 3.85 Å resolution X-ray crystal structure of a ZMPSTE24–phosphoramidon complex, indicate that the overall binding mode observed between phosphoramidon and soluble gluzincins is conserved. Based on the structural data, a significantly lower potency than that observed for soluble gluzincins such as thermolysin and neprilysin is predicted. These results strongly suggest a close relationship between soluble gluzincins and the integral membrane protein zinc metalloprotease ZMPSTE24.
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DE MAERE, V., I. VERCAUTEREN, P. GELDHOF, K. GEVAERT, J. VERCRUYSSE, and E. CLAEREBOUT. "Molecular analysis of astacin-like metalloproteases ofOstertagia ostertagi." Parasitology 130, no. 1 (December 13, 2004): 89–98. http://dx.doi.org/10.1017/s0031182004006274.
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In this study, we describe the molecular analysis of zinc-metalloproteases from the abomasal nematodeOstertagia ostertagiwhich were exclusively recognized by local antibodies of immune cattle. Full-length or partial coding sequences of 4 different zinc-metalloprotease cDNAs ofOstertagia(met-1, -2, -3 and -4) were amplified using gene-specific primers using the 3′- and 5′-Rapid Amplification of cDNA Ends (RACE) technique. Sequence analysis identified the cDNAs as encoding zinc-metalloproteases, which showed between 62% and 70% homology to a metalloprotease 1 precursor ofAncylostoma caninum. The full-length cDNA ofmet-1consists of an open reading frame (ORF) of 586 amino acids which contains 5 potentialN-glycosylation sites and a predicted zinc-binding domain (HEBXHXBGFXHEXXRXDRD). The complete coding sequence ofmet-3contains an ORF of 508 aa and the same conserved zinc-binding domain. These domains are signature sequences of the astacin family of the superfamily of metzincin metalloproteases. The presence of a threonine amino acid after the third histidine in MET-1 and MET-3, however, may place them in a new family or subfamily. Real-time PCR analysis of L3, exsheathed L3, L4and adult cDNA identified transcription of the 4 metalloproteases in different life-stages. The protein MET-1 was expressed in insect cells using the baculovirus expression system but the immunization of calves with this molecule did not lead to protection against challenge infection.
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Wladyka, Benedykt, Michal Bista, Artur J. Sabat, Emilia Bonar, Sabina Grzeszczuk, Waleria Hryniewicz, and Adam Dubin. "A novel member of the thermolysin family, cloning and biochemical characterization of metalloprotease from Staphylococcus pseudintermedius." Acta Biochimica Polonica 55, no. 3 (September 4, 2008): 525–36. http://dx.doi.org/10.18388/abp.2008_3059.
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Thermolysins constitute a family of secreted bacterial metalloproteases expressed, among others, by several pathogens. Strains of Staphylococcus pseudintermedius isolated from diseased dogs and judged as protease-positive, by skim milk agar plate culture, were investigated for protease content. No proteolytic activity was detected when the bacteria were grown in regular liquid media. Unexpectedly, supplementation of the medium with calcium ions resulted in expression of a metalloprotease and profound changes in the profile of extracellular proteins. On the basis of homology to other staphylococcal metalloproteases, the nucleotide sequence of the gene encoding this protease (Pst) and its flanking regions was determined. The full-length pst codes for a protein with an open reading frame of 505 amino acids. The internal region contains the HEXXH catalytic domain that is conserved in members of the thermolysin family. Regardless of the presence of calcium in the medium, the expression of the protease gene was of the same intensity. This suggests that regulation of the metalloprotease production by calcium ions is at a post-transcriptional level. Isolates of S. pseudintermedius exhibit a proteolytic phenotype due to the metalloprotease expression, however only in presence of calcium ions, which most probably stabilize the structure of the protease.
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Guillemet, Elisabeth, Céline Cadot, Seav-Ly Tran, Marie-Hélène Guinebretière, Didier Lereclus, and Nalini Ramarao. "The InhA Metalloproteases of Bacillus cereus Contribute Concomitantly to Virulence." Journal of Bacteriology 192, no. 1 (October 16, 2009): 286–94. http://dx.doi.org/10.1128/jb.00264-09.
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ABSTRACT The virulence of Bacillus cereus requires that bacteria have the capacity to colonize their host, degrade specific tissues, and circumvent the host immune system. To study this aspect of pathogenesis, we focused on three metalloproteases, InhA1, InhA2, and InhA3, which share more than 66% identity. The expression of these metalloprotease genes was assessed by transcriptional fusions with a lacZ reporter gene. The expression profiles suggest a complementary time course of InhA production. Indeed, the genes are simultaneously expressed but are oppositely controlled during stationary phase. We constructed single and multiple inhA mutants and assessed the bacterial locations of the proteins as well as their individual or additive roles in macrophage escape and toxicity, antibacterial-peptide cleavage, and virulence. InhA1, a major component of the spore exosporium, is the only InhA metalloprotease involved in bacterial escape from macrophages. A mutant lacking inhA1, inhA2, and inhA3 shows a strong decrease in the level of virulence for insects. Taken together, these results show that the InhA metalloproteases of B. cereus are important virulence factors that may allow the bacteria to counteract the host immune system.
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Antúnez, Karina, Daniela Arredondo, Matilde Anido, and Pablo Zunino. "Metalloprotease production by Paenibacillus larvae during the infection of honeybee larvae." Microbiology 157, no. 5 (May 1, 2011): 1474–80. http://dx.doi.org/10.1099/mic.0.044321-0.
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American foulbrood is a bacterial disease of worldwide distribution that affects larvae of the honeybee Apis mellifera. The causative agent is the Gram-positive, spore-forming bacterium Paenibacillus larvae. Several authors have proposed that P. larvae secretes metalloproteases that are involved in the larval degradation that occurs after infection. The aim of the present work was to evaluate the production of a metalloprotease by P. larvae during larval infection. First, the complete gene encoding a metalloprotease was identified in the P. larvae genome and its distribution was evaluated by PCR in a collection of P. larvae isolates from different geographical regions. Then, the complete gene was amplified, cloned and overexpressed, and the recombinant metalloprotease was purified and used to generate anti-metalloprotease antibodies. Metalloprotease production was evaluated by immunofluorescence and fluorescence in situ hybridization. The gene encoding a P. larvae metalloprotease was widely distributed in isolates from different geographical origins in Uruguay and Argentina. Metalloprotease was detected inside P. larvae vegetative cells, on the surface of P. larvae spores and secreted to the external growth medium. Its production was also confirmed in vivo, during the infection of honeybee larvae. This protein was able to hydrolyse milk proteins as described for P. larvae, suggesting that could be involved in larval degradation. This work contributes to the knowledge of the pathogenicity mechanisms of a bacterium of great economic significance and is one step in the characterization of potential P. larvae virulence factors.
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Rahman, Fatema, Imin Wushur, Nabin Malla, Ove Alexander Høgmoen Åstrand, Pål Rongved, Jan-Olof Winberg, and Ingebrigt Sylte. "Zinc-Chelating Compounds as Inhibitors of Human and Bacterial Zinc Metalloproteases." Molecules 27, no. 1 (December 22, 2021): 56. http://dx.doi.org/10.3390/molecules27010056.
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Inhibition of bacterial virulence is believed to be a new treatment option for bacterial infections. In the present study, we tested dipicolylamine (DPA), tripicolylamine (TPA), tris pyridine ethylene diamine (TPED), pyridine and thiophene derivatives as putative inhibitors of the bacterial virulence factors thermolysin (TLN), pseudolysin (PLN) and aureolysin (ALN) and the human zinc metalloproteases, matrix metalloprotease-9 (MMP-9) and matrix metalloprotease-14 (MMP-14). These compounds have nitrogen or sulfur as putative donor atoms for zinc chelation. In general, the compounds showed stronger inhibition of MMP-14 and PLN than of the other enzymes, with Ki values in the lower μM range. Except for DPA, none of the compounds showed significantly stronger inhibition of the virulence factors than of the human zinc metalloproteases. TPA and Zn230 were the only compounds that inhibited all five zinc metalloproteinases with a Ki value in the lower μM range. The thiophene compounds gave weak or no inhibition. Docking indicated that some of the compounds coordinated zinc by one oxygen atom from a hydroxyl or carbonyl group, or by oxygen atoms both from a hydroxyl group and a carbonyl group, and not by pyridine nitrogen as in DPA and TPA.
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Ray, JM, and WG Stetler-Stevenson. "The role of matrix metalloproteases and their inhibitors in tumour invasion, metastasis and angiogenesis." European Respiratory Journal 7, no. 11 (November 1, 1994): 2062–72. http://dx.doi.org/10.1183/09031936.94.07112062.
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One critical event of tumour invasion that signals the initiation of the metastatic cascade is thought to be interaction of the tumour cell with the basement membrane. Basement membranes may also pose as barriers to tumour cell invasion at multiple points later in the metastatic cascade, including during the processes of vascular infiltration and extravasation. Thus, an important proteolytic event in the metastatic cascade, and also angiogenesis, appears to be degradation of basement membrane components. A specific class of extracellular matrix degrading metalloenzymes, the matrix metalloproteases, and their endogenous inhibitors, the tissue inhibitors of metalloproteases, are thought to have a role in the creation of the proteolytic defect in basement membrane type IV collagen. We will review the evidence which indicates that matrix metalloproteases and tissue inhibitors of metalloproteases are essential for tumour cell invasion and angiogenesis. The regulation of matrix metalloproteases will be discussed, including gene activation and transcription, messenger ribonucleic acid (mRNA) stability, binding of proenzymes to cell membranes and/or matrix components, proenzyme activation, and inactivation by endogenous inhibitors. We will also discuss the mechanism for tissue inhibitor of metalloproteases-mediated inhibition of tumour invasion and angiogenesis. This appears, at least in part, to be through inhibition of protease activity required for cellular invasion, although recent observations suggest that tissue inhibitors of metalloproteases affect other distinct groups of biological activities through mechanisms other than matrix metalloprotease inhibition.
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KOIKE, Hisashi, Shigeo TOMIOKA, Hiroyuki SORIMACHI, Takaomi C. SAIDO, Kei MARUYAMA, Akira OKUYAMA, Atsuko FUJISAWA-SEHARA, Shigeo OHNO, Koichi SUZUKI, and Shoichi ISHIURA. "Membrane-anchored metalloprotease MDC9 has an α-secretase activity responsible for processing the amyloid precursor protein." Biochemical Journal 343, no. 2 (October 8, 1999): 371–75. http://dx.doi.org/10.1042/bj3430371.
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MDC9, also known as meltrin γ, is a membrane-anchored metalloprotease. MDC9 contains several distinct protein domains: a signal sequence followed by a prodomain and a domain showing sequence similarity to snake venom metalloproteases, a disintegrin-like domain, a cysteine-rich region, an epidermal-growth-factor-like repeat, a transmembrane domain and a cytoplasmic domain. Here we demonstrate that MDC9 expressed in COS cells is cleaved between the prodomain and the metalloprotease domain. Further, when MDC9 was co-expressed in COS cells with amyloid precursor protein (APP695) and treated with phorbol ester, APP695 was digested exclusively at the α-secretory site in MDC9-expressing cells. When an artificial α-secretory site mutant was also co-expressed with MDC9 and treated with phorbol ester, APP secreted by α-secretase was not increased in conditional medium. Inhibition of MDC9 by a hydroxamate-based metalloprotease inhibitor, SI-27, enhanced β-secretase cleavage. These results suggest that MDC9 has an α-secretase-like activity and is activated by phorbol ester.
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Weskamp, G., J. Krätzschmar, M. S. Reid, and C. P. Blobel. "MDC9, a widely expressed cellular disintegrin containing cytoplasmic SH3 ligand domains." Journal of Cell Biology 132, no. 4 (February 15, 1996): 717–26. http://dx.doi.org/10.1083/jcb.132.4.717.
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Cellular disintegrins are a family of proteins that are related to snake venom integrin ligands and metalloproteases. We have cloned and sequenced the mouse and human homologue of a widely expressed cellular disintegrin, which we have termed MDC9 (for metalloprotease/disintegrin/cysteine-rich protein 9). The deduced mouse and human protein sequences are 82% identical. MDC9 contains several distinct protein domains: a signal sequence is followed by a prodomain and a domain with sequence similarity to snake venom metalloproteases, a disintegrin domain, a cysteine-rich region, an EGF repeat, a membrane anchor, and a cytoplasmic tail. The cytoplasmic tail of MDC9 has two proline-rich sequences which can bind the SH3 domain of Src, and may therefore function as SH3 ligand domains. Western blot analysis shows that MDC9 is an approximately 84-kD glycoprotein in all mouse tissues examined, and in NIH 3T3 fibroblast and C2C12 myoblast mouse cell lines. MDC9 can be both cell surface biotinylated and 125I-labeled in NIH 3T3 mouse fibroblasts, indicating that the protein is present on the plasma membrane. Expression of MDC9 in COS-7 cells yields an 84-kD protein, and immunofluorescence analysis of COS-7 cells expressing MDC9 shows a staining pattern that is consistent with a plasma membrane localization. The apparent molecular mass of 84 kD suggests that MDC9 contains a membrane-anchored metalloprotease and disintegrin domain. We propose that MDC9 might function as a membrane-anchored integrin ligand or metalloprotease, or that MDC9 may combine both activities in one protein.
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Clement, Herlinda, Ligia Luz Corrales-García, Damaris Bolaños, Gerardo Corzo, and Elba Villegas. "Immunogenic Properties of Recombinant Enzymes from Bothrops ammodytoides towards the Generation of Neutralizing Antibodies against Its Own Venom." Toxins 11, no. 12 (December 2, 2019): 702. http://dx.doi.org/10.3390/toxins11120702.
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Bothropic venoms contain enzymes such as metalloproteases, serine-proteases, and phospholipases, which acting by themselves, or in synergism, are the cause of the envenomation symptoms and death. Here, two mRNA transcripts, one that codes for a metalloprotease and another for a serine-protease, were isolated from a Bothrops ammodytoides venom gland. The metalloprotease and serine-protease transcripts were cloned on a pCR®2.1-TOPO vector and consequently expressed in a recombinant way in E. coli (strains Origami and M15, respectively), using pQE30 vectors. The recombinant proteins were named rBamSP_1 and rBamMP_1, and they were formed by an N-terminal fusion protein of 16 amino acid residues, followed by the sequence of the mature proteins. After bacterial expression, each recombinant enzyme was recovered from inclusion bodies and treated with chaotropic agents. The experimental molecular masses for rBamSP_1 and rBamMP_1 agreed with their expected theoretical ones, and their secondary structure spectra obtained by circular dichroism were comparable to that of similar proteins. Additionally, equivalent mixtures of rBamSP_1, rBamMP_1 together with a previous reported recombinant phospholipase, rBamPLA2_1, were used to immunize rabbits to produce serum antibodies, which in turn recognized serine-proteases, metalloproteases and PLA2s from B. ammodytoides and other regional viper venoms. Finally, rabbit antibodies neutralized the 3LD50 of B. ammodytoides venom.
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Gusella, Milena, Caterina Bolzonella, Rossella Paolini, Elisabetta Rodella, Laura Bertolaso, Cinzia Scipioni, Silvia Bellini, Antonio Cuneo, Felice Pasini, and Emilio Ramazzina. "Plasma matrix metalloprotease 9 correlates with blood lymphocytosis, leukemic cell invasiveness, and prognosis in B-cell chronic lymphocytic leukemia." Tumor Biology 39, no. 2 (February 2017): 101042831769432. http://dx.doi.org/10.1177/1010428317694325.
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The complex biology underlying chronic lymphocytic leukemia cell migration and tissue invasiveness is not yet completely understood and might provide novel predictive markers and therapeutic targets. A total of 36 patients out of treatment from at least 3 months were enrolled and followed up for a median period of 44.2 months (range: 4.4–99.2). Matrix metalloprotease 9 and tissue inhibitor of metalloproteases 1 plasma levels and production/release from lymphoid cells were measured by zymography and enzyme-linked immunosorbent assay (ELISA) analysis. Malignant and normal lymphocyte mobility and matrix-degradation capability were studied using a Boyden chamber system, with and without autologous plasma. Free matrix metalloprotease 9 plasma levels were related with blood lymphocytosis, especially in more advanced stages (p = 0.003), and higher concentrations were associated with an increased disease progression risk (hazard ratio = 9.0, 95% confidence interval = 1.5–13.8). Leukemic cells expressed and secreted very little matrix metalloprotease 9. On the contrary, normal lymphocytes derived from the same leukemic patients showed matrix metalloprotease 9 intracellular levels that were lower in subjects with higher blood lymphocytosis (p = 0.024) and more advanced stages (p = 0.03); the released quantities were inversely associated with matrix metalloprotease 9 plasma concentrations (p = 0.035). Leukemic cells had a reduced spontaneous mobility and matrix-degradation capability that were stimulated by autologous plasma (p = 0.001) and normal lymphocytes (p = 0.005), respectively. Matrix metalloprotease 9 affected cell invasiveness depending on concentration and disease stage. In conclusion, chronic lymphocytic leukemia cells have a reduced mobility, matrix-degradation capability, and matrix metalloprotease 9 production compared to their own autologous normal lymphocytes. They are exposed to matrix metalloprotease 9 of prevalently systemic origin whose higher levels are associated with both leukemic and normal lymphocyte accumulation in the peripheral blood and have a negative prognostic value.
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Uttarwar, L., F. Peng, D. Wu, S. Kumar, B. Gao, A. J. Ingram, and J. C. Krepinsky. "HB-EGF release mediates glucose-induced activation of the epidermal growth factor receptor in mesangial cells." American Journal of Physiology-Renal Physiology 300, no. 4 (April 2011): F921—F931. http://dx.doi.org/10.1152/ajprenal.00436.2010.
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Glomerular matrix accumulation is a hallmark of diabetic nephropathy. We showed that transactivation of the epidermal growth factor receptor (EGFR) is an important mediator of matrix upregulation in mesangial cells (MC) in response to high glucose (HG). Here, we study the mechanism of EGFR transactivation. In primary MC, EGFR transactivation by 1 h of HG (30 mM) was unaffected by inhibitors of protein kinase C, reactive oxygen species, or the angiotensin II AT1 receptor. However, general metalloprotease inhibition, as well as specific inhibitors of heparin-binding EGF-like growth factor (HB-EGF), prevented both EGFR and downstream Akt activation. HB-EGF was released into the medium by 30 min of HG, and this depended on metalloprotease activity. One of the metalloproteases shown to cleave proHB-EGF is ADAM17 (TACE). HG, but not an osmotic control, activated ADAM17, and its inhibition prevented EGFR and Akt activation and HB-EGF release into the medium. siRNA to either ADAM17 or HB-EGF prevented HG-induced EGFR transactivation. We previously showed that EGFR/Akt signaling increases transforming growth factor (TGF)-β1 transcription through the transcription factor activator protein (AP)-1. HG-induced AP-1 activation, as assessed by EMSA, was abrogated by inhibitors of metalloproteases, HB-EGF and ADAM17. HB-EGF and ADAM17 siRNA also prevented AP-1 activation. Finally, these inhibitors and siRNA prevented TGF-β1 upregulation by HG. Thus, HG-induced EGFR transactivation in MC is mediated by the release of HB-EGF, which requires activity of the metalloprotease ADAM17. The mechanism of ADAM17 activation awaits identification. Targeting upstream mediators of EGFR transactivation including HB-EGF or ADAM17 provides novel therapeutic targets for the treatment of diabetic nephropathy.
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HOWARD, Linda, Xiaohong LU, Sally MITCHELL, Susan GRIFFITHS, and Paul GLYNN. "Molecular cloning of MADM: a catalytically active mammalian disintegrin-metalloprotease expressed in various cell types." Biochemical Journal 317, no. 1 (July 1, 1996): 45–50. http://dx.doi.org/10.1042/bj3170045.
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A peptide sequence of a metalloprotease purified from bovine brain [Chantry, Gregson and Glynn (1989) J. Biol. Chem. 264, 21603–21607] was used to design an oligonucleotide probe for screening a bovine brain cDNA library. A contig of the two overlapping cDNA clones that were isolated encoded a 748-amino-acid polypeptide with similarity to the disintegrin–metalloprotease precursor proteins of haemorrhagic snake venom. The bovine protein has been named MADM, for mammalian disintegrin–metalloprotease. The predicted mature protein has 534 amino acids arrayed as extracellular metalloprotease and disintegrin (potential integrin-binding) domains, a transmembrane helix and a basic/proline-rich cytoplasmic C-terminus. Highly conserved homologues of bovine MADM were found in cDNA libraries of rat brain and a human U937 histiocytic lymphoma cell line. A wide variety of mammalian cell lines expressed low levels of MADM mRNA (4.5 and 3.2 kb transcripts) and mature polypeptide (Mr 62000), as assessed by Northern analysis and Western blotting with an antiserum raised to a peptide within the disintegrin domain. MADM appears to be a rather distantly related member of the reprolysin protein family, which includes both the snake venom disintegrin–metalloproteases and a number of predicted cell-surface disintegrin-containing mammalian proteins.
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Sánchez, E., J. M. Laparra, and Y. Sanz. "Discerning the Role of Bacteroides fragilis in Celiac Disease Pathogenesis." Applied and Environmental Microbiology 78, no. 18 (July 6, 2012): 6507–15. http://dx.doi.org/10.1128/aem.00563-12.
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ABSTRACTCeliac disease (CD) is associated with intestinal dysbiosis, which can theoretically lead to dysfunctions in host-microbe interactions and contribute to the disease. In the present study, possible differences inBacteroidesspp. and their pathogenic features between CD patients and controls were investigated.Bacteroidesclones (n= 274) were isolated, identified, and screened for the presence of the virulence genes (bftandmpII) coding for metalloproteases. The proteolytic activity of selectedBacteroides fragilisstrains was evaluated by zymography and, after gastrointestinal digestion of gliadin, by high-pressure liquid chromatography/electrospray ionization/tandem mass spectrometry. The effects ofB. fragilisstrains on Caco-2 cell culture permeability and inflammatory response to digested gliadin were determined.B. fragiliswas more frequently identified in CD patients than in healthy controls, in contrast toBacteroides ovatus.B. fragilisclones carrying virulence genes coding for metalloproteases were more abundant in CD patients than in controls.B. fragilisstrains, representing the isolated clones and carrying metalloprotease genes, showed gelatinase activity and exerted the strongest adverse effects on the integrity of the Caco-2 cell monolayer. AllB. fragilisstrains also showed gliadin-hydrolyzing activity, and some of them generated immunogenic peptides that preserved or increased inflammatory cytokine production (tumor necrosis factor alpha) and showed increased ability to permeate through Caco-2 cell cultures. These findings suggest that increased abundance ofB. fragilisstrains with metalloprotease activities could play a role in CD pathogenesis, although furtherin vivostudies are required to support this hypothesis.
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Ohtsu, Haruhiko, Peter J. Dempsey, and Satoru Eguchi. "ADAMs as mediators of EGF receptor transactivation by G protein-coupled receptors." American Journal of Physiology-Cell Physiology 291, no. 1 (July 2006): C1—C10. http://dx.doi.org/10.1152/ajpcell.00620.2005.
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A disintegrin and metalloprotease (ADAM) is a membrane-anchored metalloprotease implicated in the ectodomain shedding of cell surface proteins, including the ligands for epidermal growth factor (EGF) receptors (EGFR)/ErbB. It has been well documented that the transactivation of the EGFR plays critical roles for many cellular functions, such as proliferation and migration mediated through multiple G protein-coupled receptors (GPCRs). Recent accumulating evidence has suggested that ADAMs are the key metalloproteases activated by several GPCR agonists to produce a mature EGFR ligand leading to the EGFR transactivation. In this review, we describe the current knowledge on ADAMs implicated in mediating EGFR transactivation. The major focus of the review will be on the possible upstream mechanisms of ADAM activation by GPCRs as well as downstream signal transduction and the pathophysiological significances of ADAM-dependent EGFR transactivation.
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Bajda, Milena, Aleksandra Łoś, Michał Schulz, and Kornel Kasperek. "Mammalian and insect metalloproteases." Medycyna Weterynaryjna 72, no. 7 (2016): 408–12. http://dx.doi.org/10.21521/mw.5538.
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Metalloproteases (metalloproteinases, MMP) digest extracellular matrix proteins. They have zinc ions (Zn2+) in their active site. They are synthesized within cells as proenzymes, to be subsequently activated in the extracellular environment. MMP are active in a neutral or slightly alkaline pH in the presence of Ca2+ ions. Cells that synthesize metalloproteases also produce metalloprotease inhibitors. Until now, 4 of MMP inhibitors as well as 28 endometalloproteases have been discovered, out of which 22 occur in humans. On the other hand, these enzymes have not been well explored in insects, in which only 2 metalloproteases were identified (Ance and ECE). Their optimal activity ranges between pH 7.0 and 9.4. MMP inhibitors control the concentration of metalloproteases in physiological conditions. They fall within two types: specific tissue MMP inhibitors and non-specific plasma MMP inhibitors. Metalloproteases and their inhibitors play an important role in both physiological and pathological processes in the organism. Metalloproteases are cell growth promotors. They inhibit/induce apoptosis, stimulate the development of healthy cells and control the activity of neoplastic cells both in people and in insects. Their activity is increased in skin and periodontal diseases, in arthritis, arteriosclerosis or in the period following myocardial infarction. In insects the activity of MMPs is also increased by environmental pollution, by the use of antibiotics and varroacides. The insect MMPs participate in digestion, biosynthesis of peptide hormones and neurotransmitters, and melanisation. They also affect the development of the reproductive system and the development of larvae and pupae, as well as prevent pathogen invasions. Worthy of special attention is the insect cuticle defensive barrier associated with MMP. Activation of metalloproteases is dependent on the physiological state of the organism, as well as on environmental pressure. Analyzing activities of metalloproteases and their inhibitors enables better monitoring of the pathological conditions in both insects and mammals.
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Guo, Feilong, Lijun Pan, Hongwei Liu, Liangjie Lv, Xiyong Chen, Yuping Liu, Hui Li, Wenwu Ye, and Zengyan Zhang. "Whole-Genome Metalloproteases in the Wheat Sharp Eyespot Pathogen Rhizoctonia cerealis and a Role in Fungal Virulence." International Journal of Molecular Sciences 23, no. 18 (September 14, 2022): 10691. http://dx.doi.org/10.3390/ijms231810691.
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Rhizoctonia cerealis is the causal agent of sharp eyespot, a devastating disease of cereal crops including wheat. Several metalloproteases have been implicated in pathogenic virulence, but little is known about whole-genome metalloproteases in R. cerealis. In this study, a total of 116 metalloproteases-encoding genes were identified and characterized from the R. cerealis Rc207 genome. The gene expression profiles and phylogenetic relationship of 11 MEP36/fungalysin metalloproteases were examined during the fungal infection to wheat, and function of an upregulated secretory MEP36 named RcFL1 was validated. Of 11 MEP36 family metalloproteases, ten, except RcFL5, were predicted to be secreted proteins and nine encoding genes, but not RcFL5 and RcFL2, were expressed during the R. cerealis infection process. Phylogenetic analysis suggested that MEP36 metalloproteases in R. cerealis were closely related to those of Rhizoctonia solani but were remote to those of Bipolaris sorokiniana, Fusarium graminearum, F. pseudograminearum, and Pyricularia oryzae. Expression of RcFL1 was significantly upregulated during the infection process and induced plant cell death in wheat to promote the virulence of the pathogen. The MEP36 domain was necessary for the activities of RcFL1. Furthermore, RcFL1 could repress the expression of wheat genes coding for the chitin elicitor receptor kinase TaCERK1 and chitinases. These results suggest that this MEP36 metalloprotease RcFL1 may function as a virulence factor of R. cerealis through inhibiting host chitin-triggered immunity and chitinases. This study provides insights on pathogenic mechanisms of R. cerealis. RcFL1 likely is an important gene resource for improving resistance of wheat to R. cerealis through host-induced gene silencing strategy.
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Hasegawa, Hiroaki, Erin J. Lind, Markus A. Boin, and Claudia C. Häse. "The Extracellular Metalloprotease of Vibrio tubiashii Is a Major Virulence Factor for Pacific Oyster (Crassostrea gigas) Larvae." Applied and Environmental Microbiology 74, no. 13 (May 2, 2008): 4101–10. http://dx.doi.org/10.1128/aem.00061-08.
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ABSTRACT Vibrio tubiashii is a recently reemerging pathogen of larval bivalve mollusks, causing both toxigenic and invasive disease. Marine Vibrio spp. produce an array of extracellular products as potential pathogenicity factors. Culture supernatants of V. tubiashii have been shown to be toxic to oyster larvae and were reported to contain a metalloprotease and a cytolysin/hemolysin. However, the structural genes responsible for these proteins have yet to be identified, and it is uncertain which extracellular products play a role in pathogenicity. We investigated the effects of the metalloprotease and hemolysin secreted by V. tubiashii on its ability to kill Pacific oyster (Crassostrea gigas) larvae. While V. tubiashii supernatants treated with metalloprotease inhibitors severely reduced the toxicity to oyster larvae, inhibition of the hemolytic activity did not affect larval toxicity. We identified structural genes of V. tubiashii encoding a metalloprotease (vtpA) and a hemolysin (vthA). Sequence analyses revealed that VtpA shared high homology with metalloproteases from a variety of Vibrio species, while VthA showed high homology only to the cytolysin/hemolysin of Vibrio vulnificus. Compared to the wild-type strain, a VtpA mutant of V. tubiashii not only produced reduced amounts of protease but also showed decreased toxicity to C. gigas larvae. Vibrio cholerae strains carrying the vtpA or vthA gene successfully secreted the heterologous protein. Culture supernatants of V. cholerae carrying vtpA but not vthA were highly toxic to Pacific oyster larvae. Together, these results suggest that the V. tubiashii extracellular metalloprotease is important in its pathogenicity to C. gigas larvae.
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Sammel, Peters, Lokau, Scharfenberg, Werny, Linder, Garbers, Rose-John, and Becker-Pauly. "Differences in Shedding of the Interleukin-11 Receptor by the Proteases ADAM9, ADAM10, ADAM17, Meprin α, Meprin β and MT1-MMP." International Journal of Molecular Sciences 20, no. 15 (July 26, 2019): 3677. http://dx.doi.org/10.3390/ijms20153677.
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Interleukin-11 (IL-11) has been associated with inflammatory conditions, bone homeostasis, hematopoiesis, and fertility. So far, these functions have been linked to classical IL-11 signaling via the membrane bound receptor (IL-11R). However, a signaling cascade via the soluble IL-11R (sIL-11R), generated by proteolytic cleavage, can also be induced. This process is called IL-11 trans-signaling. A disintegrin and metalloprotease 10 (ADAM10) and neutrophil elastase were described as ectodomain sheddases of the IL-11R, thereby inducing trans-signaling. Furthermore, previous studies employing approaches for the stimulation and inhibition of endogenous ADAM-proteases indicated that ADAM10, but not ADAM17, can cleave the IL-11R. Herein, we show that several metalloproteases, namely ADAM9, ADAM10, ADAM17, meprin β, and membrane-type 1 matrix metalloprotease/matrix metalloprotease-14 (MT1-MMP/MMP-14) when overexpressed are able to shed the IL-11R. All sIL-11R ectodomains were biologically active and capable of inducing signal transducer and activator of transcription 3 (STAT3) phosphorylation in target cells. The difference observed for ADAM10/17 specificity compared to previous studies can be explained by the different approaches used, such as stimulation of protease activity or making use of cells with genetically deleted enzymes.
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Malecki, Michael J., Cheryll Sanchez-Irizarry, Jennifer L. Mitchell, Gavin Histen, Mina L. Xu, Jon C. Aster, and Stephen C. Blacklow. "Leukemia-Associated Mutations within the NOTCH1 Heterodimerization Domain Fall into at Least Two Distinct Mechanistic Classes." Molecular and Cellular Biology 26, no. 12 (June 15, 2006): 4642–51. http://dx.doi.org/10.1128/mcb.01655-05.
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ABSTRACT The NOTCH1 receptor is cleaved within its extracellular domain by furin during its maturation, yielding two subunits that are held together noncovalently by a juxtamembrane heterodimerization (HD) domain. Normal NOTCH1 signaling is initiated by the binding of ligand to the extracellular subunit, which renders the transmembrane subunit susceptible to two successive cleavages within and C terminal to the heterodimerization domain, catalyzed by metalloproteases and γ-secretase, respectively. Because mutations in the heterodimerization domain of NOTCH1 occur frequently in human T-cell acute lymphoblastic leukemia (T-ALL), we assessed the effect of 16 putative tumor-associated mutations on Notch1 signaling and HD domain stability. We show here that 15 of the 16 mutations activate canonical NOTCH1 signaling. Increases in signaling occur in a ligand-independent fashion, require γ-secretase activity, and correlate with an increased susceptibility to cleavage by metalloproteases. The activating mutations cause soluble NOTCH1 heterodimers to dissociate more readily, either under native conditions (n = 3) or in the presence of urea (n = 11). One mutation, an insertion of 14 residues immediately N terminal to the metalloprotease cleavage site, increases metalloprotease sensitivity more than all others, despite a negligible effect on heterodimer stability by comparison, suggesting that the insertion may expose the S2 site by repositioning it relative to protective NOTCH1 ectodomain residues. Together, these studies show that leukemia-associated HD domain mutations render NOTCH1 sensitive to ligand-independent proteolytic activation through two distinct mechanisms.
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Layfield, Harry J., Harry F. Williams, Divyashree Ravishankar, Amita Mehmi, Medha Sonavane, Anika Salim, Rajendran Vaiyapuri, et al. "Repurposing Cancer Drugs Batimastat and Marimastat to Inhibit the Activity of a Group I Metalloprotease from the Venom of the Western Diamondback Rattlesnake, Crotalus atrox." Toxins 12, no. 5 (May 9, 2020): 309. http://dx.doi.org/10.3390/toxins12050309.
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Snakebite envenomation causes over 140,000 deaths every year, predominantly in developing countries. As a result, it is one of the most lethal neglected tropical diseases. It is associated with incredibly complex pathophysiology due to the vast number of unique toxins/proteins present in the venoms of diverse snake species found worldwide. Here, we report the purification and functional characteristics of a Group I (PI) metalloprotease (CAMP-2) from the venom of the western diamondback rattlesnake, Crotalus atrox. Its sensitivity to matrix metalloprotease inhibitors (batimastat and marimastat) was established using specific in vitro experiments and in silico molecular docking analysis. CAMP-2 shows high sequence homology to atroxase from the venom of Crotalus atrox and exhibits collagenolytic, fibrinogenolytic and mild haemolytic activities. It exerts a mild inhibitory effect on agonist-induced platelet aggregation in the absence of plasma proteins. Its collagenolytic activity is completely inhibited by batimastat and marimastat. Zinc chloride also inhibits the collagenolytic activity of CAMP-2 by around 75% at 50 μM, while it is partially potentiated by calcium chloride. Molecular docking studies have demonstrated that batimastat and marimastat are able to bind strongly to the active site residues of CAMP-2. This study demonstrates the impact of matrix metalloprotease inhibitors in the modulation of a purified, Group I metalloprotease activities in comparison to the whole venom. By improving our understanding of snake venom metalloproteases and their sensitivity to small molecule inhibitors, we can begin to develop novel and improved treatment strategies for snakebites.
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Yu, Yuen-Tsu Nicco, and Lee Kroos. "Evidence that SpoIVFB Is a Novel Type of Membrane Metalloprotease Governing Intercompartmental Communication duringBacillus subtilis Sporulation." Journal of Bacteriology 182, no. 11 (June 1, 2000): 3305–9. http://dx.doi.org/10.1128/jb.182.11.3305-3309.2000.
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ABSTRACT Processing of pro-ςK in the mother cell compartment of sporulating Bacillus subtilis involves SpoIVFB and is governed by a signal from the forespore. SpoIVFB has an HEXXH motif characteristic of metalloproteases embedded in one of its transmembrane segments. Several conservative single amino acid changes in the HEXXH motif abolished function. However, changing the glutamic acid residue to aspartic acid, or changing the isoleucine residue that precedes the motif to proline, permitted SpoIVFB function. Only one other putative metalloprotease, site 2 protease has been shown to tolerate aspartic acid rather than glutamic acid in its HEXXH sequence. Site 2 protease and SpoIVFB share a second region of similarity with a family of putative membrane metalloproteases. A conservative change in this region of SpoIVFB abolished function. Interestingly, SpoIVFA increased the accumulation of certain mutant SpoIVFB proteins but was unnecessary for accumulation of wild-type SpoIVFB.
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Jeng, Arco Y., Paul Mulder, Aij-Lie Kwan, and Bruno Battistini. "Nonpeptidic endothelin-converting enzyme inhibitors and their potential therapeutic applications." Canadian Journal of Physiology and Pharmacology 80, no. 5 (May 1, 2002): 440–49. http://dx.doi.org/10.1139/y02-025.
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Endothelins (ETs) are potent vasoconstrictors, promitogens, and inflammatory mediators. They have been implicated in the pathogenesis of various cardiovascular, renal, pulmonary, and central nervous system diseases. Since the final step of the biosynthesis of ETs is catalyzed by a family of endothelin-converting enzymes (ECEs), inhibitors of these enzymes may represent novel therapeutic agents. Currently, seven isoforms of these metalloproteases have been identified; they all share a significant amino acid sequence identity with neutral endopeptidase 24.11 (NEP), another metalloprotease. Therefore, it is not surprising that the majority of ECE inhibitors also possess potent NEP inhibitory activity. To date, three classes of ECE inhibitors have been synthesized: dual ECE/NEP inhibitors, triple ECE/NEP/ACE inhibitors, and selective ECE inhibitors. Potential clinical applications of these compounds in hypertension, chronic heart failure, restenosis, renal failure, and cerebral vasospasm deduced from studies with relevant animal models are reviewed.Key words: endothelin-converting enzyme, ECE, inhibitors, phosphoramidon, CGS 26303, CGS 35066, FR 901533, SCH 54470, metalloprotease.
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Neuman, Manuela G., Hemda Schmilovitz-Weiss, Nir Hilzenrat, Marc Bourliere, Patrick Marcellin, Cristhian Trepo, Tony Mazulli, et al. "Markers of Inflammation and Fibrosis in Alcoholic Hepatitis and Viral Hepatitis C." International Journal of Hepatology 2012 (2012): 1–10. http://dx.doi.org/10.1155/2012/231210.
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High levels of profibrinogenic cytokine transforming factor beta (TGF-β), metalloprotease (MMP2), and tissue inhibitor of matrix metalloprotease 1 (TIMP1) contribute to fibrogenesis in hepatitis C virus (HCV) infection and in alcohol-induced liver disease (ALD). The aim of our study was to correlate noninvasive serum markers in ALD and HCV patients with various degrees of inflammation and fibrosis in their biopsies.Methods. Serum cytokines levels in HCV-infected individuals in the presence or absence of ALD were measured. Student's-t-test with Bonferroni correction determined the significance between the groups.Results. Both tumor-necrosis-factor- (TNF)-αand TGF-βlevels increased significantly with the severity of inflammation and fibrosis. TGF-βlevels increased significantly in ALD patients versus the HCV patients. Proinflammatory cytokines’ responses to viral and/or toxic injury differed with the severity of liver inflammation. A combination of these markers was useful in predicting and diagnosing the stages of inflammation and fibrosis in HCV and ALD.Conclusion. Therapeutic monitoring of TGF-βand metalloproteases provides important insights into fibrosis.
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Veloso, Laura Barral, Flávia de Oliveira Cardoso, Karen dos Santos Charret, Matheus Pereira de Sá Silva, Luzia Monteiro de Castro Côrtes, Kátia da Silva Calabrese, Franklin Souza da Silva, Joel Fontes de Sousa, Marília Fonseca Rocha, and Carlos Roberto Alves. "Detection of Metalloproteases and Cysteine Proteases RNA Transcripts of Leishmania (Leishmania) infantum in Ear Edge Skin of Naturally Infected Dogs." BioMed Research International 2020 (June 24, 2020): 1–8. http://dx.doi.org/10.1155/2020/2615787.
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Leishmania spp. proteases have been proposed as virulence factors contributing to adaptive success these parasites to the mammalian hosts. Since these enzymes are poorly studied in naturally infected dogs, this work aims to show the differences in metalloprotease and cysteine proteases gene expression in ear edge skin of dogs naturally infected by Leishmania (Leishmania) infantum. A cohort of dogs (n=20) naturally infected by L. (L.) infantum was clinically classified as asymptomatic, oligosymptomatic, and polysymptomatic and the parasite load range estimated. The analysis of proteases expression by RT-PCR in the ear edge skin was also assessed, suggesting more transcripts of proteases in cDNA samples from polysymptomatic dogs than oligosymptomatic and asymptomatic ones. Metalloprotease RT-PCR assays yielded products (202 bp) in all assessed cDNA dog samples. In contrast, cysteine proteases transcripts (227 bp) had shown to be better detected in cDNA samples of polysymptomatic dogs, compared with cDNA samples from asymptomatic and oligosymptomatic dogs. Predictive in silico assays suggested that secondary structures of metalloproteasee mRNAs can be more stable than cysteine proteases at the skin temperature of dogs. Evidence is presented that during natural infection of dogs by L. (L.) infantum, this parasite produces transcripts of metalloprotease and cysteine protease RNA in the skin from asymptomatic, oligosymptomatic, and polysymptomatic dogs.
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Kuliopulos, Athan. "Matrix Metalloproteases and PAR Activation." Blood 118, no. 21 (November 18, 2011): SCI—43—SCI—43. http://dx.doi.org/10.1182/blood.v118.21.sci-43.sci-43.
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Abstract Abstract SCI-43 Myocardial infarction due to rupture of atherosclerotic plaques is a leading contributor to morbidity and mortality in the United States, Europe, and other industrialized nations. Although pathoanatomic studies of human atherosclerotic lesions suggest that large plaques can cause ischemic symptoms, a key contributing factor to the morbidity and mortality associated with atherosclerosis is excessive platelet thrombus formation on exposed collagen surfaces following acute plaque rupture. Following their initial tethering to subendothelial collagen and matrix proteins, activation of transiently adhered platelets by autocrine mediators is critical for propagation of the platelet thrombus. Reinforcement of the transient adhesive contacts by activating G protein-dependent shape change, granule release, and integrins permits growth of a stable thrombus that is resistant to the high shear stress of arterial blood flow. Drugs that target the secondary autocrine mediators of platelet thrombus formation such as aspirin and thienopyridines have proven to be beneficial; however, many patients taking these drugs still sustain thrombotic events and might benefit from new therapeutics that interfere with matrix-dependent platelet activation. Matrix metalloproteases (MMPs) have recently emerged as important mediators of platelet function and vascular biology. Initially described as matrix remodeling enzymes involved in tissue repair and cancer invasion, a renewed focus has centered on MMPs and the related metalloprotease disintegrins because of their prominence in vascular wall inflammation and thrombotic thrombocytopenic purpura. Endogenous platelet metalloproteases have been shown to damage platelet function by cleaving cell surface receptors and broad-spectrum metalloprotease inhibitors improve post-transfusion recovery of platelet concentrates. Platelets express several metalloproteases including MMP-1, MMP-2, MMP-3, and MMP-14 on their surface but their roles in platelet aggregation are not well understood. It was recently shown that the G protein-coupled receptor, PAR1, is directly cleaved and activated on the surface of cancer cells by fibroblast-derived MMP-1. PAR1 is the major thrombin receptor of human platelets and is an important mediator of platelet aggregation following tissue factor (TF)-dependent generation of thrombin. However, under pathophysiologic conditions of acute plaque rupture, exposed collagen is the most efficient stimulus of the critical early events of platelet recruitment and propagation under arterial flow, which could trigger metalloprotease activation on the platelet surface. We found that exposure of platelets to collagen caused activation of MMP-1, which in turn directly cleaved PAR1 on the surface of platelets. Unexpectedly, MMP-1 cleaved the N-terminal extracellular domain of PAR1 at a distinct site from the thrombin cleavage site. This cleavage event generated a longer tethered peptide ligand, which was an agonist of platelet activation and PAR1 signaling. Blocking the MMP1-PAR1 pathway inhibited collagen-dependent thrombogenesis, arterial thrombosis and clot retraction, suggesting that therapeutics that target this metalloprotease-receptor system could be a new strategy in the treatment of patients with acute coronary syndromes. Disclosures: No relevant conflicts of interest to declare.
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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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Fedhila, Sinda, Patricia Nel, and Didier Lereclus. "The InhA2 Metalloprotease of Bacillus thuringiensis Strain 407 Is Required for Pathogenicity in Insects Infected via the Oral Route." Journal of Bacteriology 184, no. 12 (June 15, 2002): 3296–304. http://dx.doi.org/10.1128/jb.184.12.3296-3304.2002.
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ABSTRACT The entomopathogenic bacterium Bacillus thuringiensis is known to secrete a zinc metalloprotease (InhA) that specifically cleaves antibacterial peptides produced by insect hosts. We identified a second copy of the inhA gene, named inhA2, in B. thuringiensis strain 407 Cry−. The inhA2 gene encodes a putative polypeptide showing 66.2% overall identity with the InhA protein and harboring the zinc-binding domain (HEXXH), which is characteristic of the zinc-requiring metalloproteases. We used a transcriptional inhA2′-lacZ fusion to show that inhA2 expression is induced at the onset of the stationary phase and is overexpressed in a Spo0A minus background. The presence of a reverse Spo0A box in the promoter region of inhA2 suggests that Spo0A directly regulates the transcription of inhA2. To determine the role of the InhA and InhA2 metalloproteases in pathogenesis, we used allelic exchange to isolate single and double mutant strains for the two genes. Spores and vegetative cells of the mutant strains were as virulent as those of the parental strain in immunized Bombyx mori larvae infected by the intrahemocoelic route. Exponential phase cells of all the strains displayed the same in vitro potential for colonizing the vaccinated hemocoel. We investigated the synergistic effect of the mutant strain spores on the toxicity of Cry1C proteins against Galleria mellonella larvae infected via the oral pathway. The spores of ΔinhA2 mutant strain were ineffective in providing synergism whereas those of the ΔinhA mutant strain were not. These results indicate that the B. thuringiensis InhA2 zinc metalloprotease has a vital role in virulence when the host is infected via the oral route.
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Gunas, Valery, Oleksandr Maievskyi, Nataliia Raksha, Tetiana Vovk, Oleksiy Savchuk, Serhii Shchypanskyi, and Igor Gunas. "The Activity of Metalloproteases and Serine Proteases in Various Organs after Leiurus macroctenus Envenomation." Journal of Toxicology 2023 (February 20, 2023): 1–8. http://dx.doi.org/10.1155/2023/5262729.
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Background. Scorpion stings may be life-threatening since their venoms are comprised of a wide range of toxins and other bioactive molecules, such as enzymes. At the same time, scorpion envenomation may increase matrix metalloproteases (MMPs) levels, which enhance proteolytic tissue destruction by venom. However, investigations on the impact of many scorpions’ venoms, such as those of Leiurus macroctenus, on tissue proteolytic activity and MMP levels have not yet been conducted. Methods and Results. The present study aimed to examine the total proteolysis levels in various organs after Leiurus macroctenus envenomation and evaluate the metalloproteases and serine proteases’ contributions to the total proteolytic activity. Changes in MMPs and TIMP-1 levels were tested as well. Envenomation led to a significant increase in proteolytic activity levels in all assessed organs, mostly in the heart (by 3.34 times) and lungs (by 2.25 times). Conclusions. Since EDTA presence showed a noticeable decrease in total proteolytic activity level, metalloproteases appeared to play a prominent role in total proteolytic activity. At the same time, MMPs and TIMP-1 levels were increased in all assessed organs, suggesting that Leiurus macroctenus envenomation causes systemic envenomation, which may induce multiple organ abnormalities, mostly because of the uncontrolled metalloprotease activity.
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Klein, Theo, and Rainer Bischoff. "Active Metalloproteases of the A Disintegrin And Metalloprotease (ADAM) Family: Biological Function and Structure." Journal of Proteome Research 10, no. 1 (January 7, 2011): 17–33. http://dx.doi.org/10.1021/pr100556z.
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Bryant, Jack A., Ian T. Cadby, Zhi-Soon Chong, Gabriela Boelter, Yanina R. Sevastsyanovich, Faye C. Morris, Adam F. Cunningham, et al. "Structure-Function Characterization of the Conserved Regulatory Mechanism of the Escherichia coli M48 Metalloprotease BepA." Journal of Bacteriology 203, no. 2 (October 26, 2020): e00434-20. http://dx.doi.org/10.1128/jb.00434-20.
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ABSTRACTThe asymmetric Gram-negative outer membrane (OM) is the first line of defense for bacteria against environmental insults and attack by antimicrobials. The key component of the OM is lipopolysaccharide, which is transported to the surface by the essential lipopolysaccharide transport (Lpt) system. Correct folding of the Lpt system component LptD is regulated by a periplasmic metalloprotease, BepA. Here, we present the crystal structure of BepA from Escherichia coli, solved to a resolution of 2.18 Å, in which the M48 protease active site is occluded by an active-site plug. Informed by our structure, we demonstrate that free movement of the active-site plug is essential for BepA function, suggesting that the protein is autoregulated by the active-site plug, which is conserved throughout the M48 metalloprotease family. Targeted mutagenesis of conserved residues reveals that the negative pocket and the tetratricopeptide repeat (TPR) cavity are required for function and degradation of the BAM complex component BamA under conditions of stress. Last, we show that loss of BepA causes disruption of OM lipid asymmetry, leading to surface exposed phospholipid.IMPORTANCE M48 metalloproteases are widely distributed in all domains of life. E. coli possesses four members of this family located in multiple cellular compartments. The functions of these proteases are not well understood. Recent investigations revealed that one family member, BepA, has an important role in the maturation of a central component of the lipopolysaccharide (LPS) biogenesis machinery. Here, we present the structure of BepA and the results of a structure-guided mutagenesis strategy, which reveal the key residues required for activity that inform how all M48 metalloproteases function.
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Miyoshi, Shin-ichi, Hiromi Nakazawa, Koji Kawata, Ken-ichi Tomochika, Kazuo Tobe, and Sumio Shinoda. "Characterization of the Hemorrhagic Reaction Caused by Vibrio vulnificus Metalloprotease, a Member of the Thermolysin Family." Infection and Immunity 66, no. 10 (October 1, 1998): 4851–55. http://dx.doi.org/10.1128/iai.66.10.4851-4855.1998.
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ABSTRACT Vibrio vulnificus is an opportunistic human pathogen causing wound infections and septicemia, characterized by hemorrhagic and edematous damage to the skin. This human pathogen secretes a metalloprotease (V. vulnificus protease [VVP]) as an important virulence determinant. When several bacterial metalloproteases including VVP were injected intradermally into dorsal skin, VVP showed the greatest hemorrhagic activity. The level of the in vivo hemorrhagic activity of the bacterial metalloproteases was significantly correlated with that of the in vitro proteolytic activity for the reconstituted basement membrane gel. Of two major basement membrane components (laminin and type IV collagen), only type IV collagen was easily digested by VVP. Additionally, the immunoglobulin G antibody against type IV collagen, but not against laminin, showed sufficient protection against the hemorrhagic reaction caused by VVP. Capillary vessels are known to be stabilized by binding of the basal surface of vascular endothelial cells to the basement membrane. Therefore, specific degradation of type IV collagen may cause destruction of the basement membrane, breakdown of capillary vessels, and leakage of blood components including erythrocytes.
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Müllberg, J., F. H. Durie, C. Otten-Evans, M. R. Alderson, S. Rose-John, D. Cosman, R. A. Black, and K. M. Mohler. "A metalloprotease inhibitor blocks shedding of the IL-6 receptor and the p60 TNF receptor." Journal of Immunology 155, no. 11 (December 1, 1995): 5198–205. http://dx.doi.org/10.4049/jimmunol.155.11.5198.
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Abstract Many cytokines and soluble cytokine receptors are generated by limited proteolysis of membrane-bound precursors. We have examined the ability of the recently described metalloprotease inhibitor, TNF-alpha protease inhibitor (TAPI), and other protease inhibitors to modulate shedding. The membrane-bound forms of the ligands TNF-alpha and CSF-1, the p60 TNFR and the IL-6R, were expressed in COS-7 cells. As expected, TAPI blocked the spontaneous and PMA-induced release of TNF-alpha from transfected cells. Interestingly, TAPI also inhibited the release of soluble forms of p60 TNFR and IL-6R in COS-7 cells. However, the processing of CSF-1, which also requires proteolytic cleavage of a membrane protein, was not affected. The ability of TAPI to inhibit shedding was unique, since several other classes of protease inhibitors, including three other metalloprotease inhibitors, did not inhibit shedding of IL-6R. To determine whether TAPI would prevent shedding under more physiologic conditions, we demonstrated that TAPI was able to prevent unstimulated and PMA-induced release of the soluble forms of TNF-alpha, p60 TNFR, and IL-6R from the monocytic cell line, THP-1, and from human peripheral blood monocytes. In addition, TAPI was able to inhibit LPS-induced shedding of the p60 TNFR and TNF-alpha from monocytes. In summary, our results indicate that a metalloprotease or group of related metalloproteases is responsible for the proteolytic cleavage of several cell surface proteins.
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BOWLES, V. M., A. R. YOUNG, and S. C. BARKER. "Metalloproteases and egg-hatching in Pediculus humanus, the body (clothes) louse of humans (Phthiraptera: Insecta)." Parasitology 135, no. 1 (September 25, 2007): 125–30. http://dx.doi.org/10.1017/s0031182007003587.
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SUMMARYTo investigate the biochemical components of egg-hatch in the body louse, Pediculus humanus, egg-shell-washings (ESW) were collected during the first 2 h post-hatching and analysed by gelatin SDS-PAGE. These ESW contained proteases with molecular mass in the range of 25–100 kDa; the most abundant proteases were ~25 kDa. The 3 main regions of protease activity in the one-dimensional gelatin SDS-PAGE gels resolved to at least 23 distinct regions of protease activity when analysed by two-dimensional gelatin SDS-PAGE, with iso-electric points spread over the entire 3 to 10 pH range. Mechanistic characterization indicated that the ESW contained proteases of the metallo-class, inhibited by both 1,10-phenanthroline and EDTA. Several protease inhibitors were tested for their ability to inhibit louse egg-hatch in vitro. The metalloprotease inhibitor 1,10-phenanthroline and the aminopeptidase inhibitor bestatin significantly inhibited (P<0·05) louse egg-hatch (100% and 58%, respectively). The presence of metalloproteases at the time of egg-hatch and the inhibition of egg-hatch in P. humanus by metalloprotease inhibitors suggests a crucial role for these proteases in the hatching of this medically important parasite.
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Geraghty, Patrick, Catherine M. Greene, Michael O'Mahony, Shane J. O'Neill, Clifford C. Taggart, and Noel G. McElvaney. "Secretory Leucocyte Protease Inhibitor Inhibits Interferon-γ-induced Cathepsin S Expression." Journal of Biological Chemistry 282, no. 46 (September 18, 2007): 33389–95. http://dx.doi.org/10.1074/jbc.m706884200.
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We have demonstrated that bronchoalveolar lavage fluid from chronic obstructive pulmonary disease patients contains higher levels of interferon-γ compared with controls. Interferon-γ is a potent inducer of various cathepsins and matrix metalloproteases. Therefore, we postulated that interferon-γ could induce protease expression by macrophages in acute and chronic lung disease. Chronic obstructive pulmonary disease patients had greater levels of cathepsin S and matrix metalloprotease-12 in their bronchoalveolar lavage fluid. Macrophages incubated with chronic obstructive pulmonary disease bronchoalveolar lavage fluid exhibited increased expression of cathepsin S and matrix metalloprotease-12, which was inhibited by the addition of interferon-γ-neutralizing immunoglobulin. Human secretory leukocyte protease inhibitor is an 11.7-kDa cationic non-glycosylated antiprotease synthesized and secreted by cells at the site of inflammation. We have demonstrated that secretory leukocyte protease inhibitor can inhibit interferon-γ-induced cathepsin S production by macrophages. Pretreatment of macrophages with secretory leukocyte protease inhibitor inhibited interferon-γ-induced inhibitor κB β degradation and activation of nuclear factor κB. Secretory leukocyte protease inhibitor may prove to be therapeutically important as a potential inhibitor of protease expression in chronic obstructive pulmonary disease.
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Leca, G., S. E. Mansur, and A. Bensussan. "Expression of VCAM-1 (CD106) by a subset of TCR gamma delta-bearing lymphocyte clones. Involvement of a metalloprotease in the specific hydrolytic release of the soluble isoform." Journal of Immunology 154, no. 3 (February 1, 1995): 1069–77. http://dx.doi.org/10.4049/jimmunol.154.3.1069.
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Abstract The cytokine-inducible vascular cell adhesion molecule 1/CD106 is widely distributed in endothelial, epithelial, macrophage, and dentritic cells. We previously have reported a mAb termed sTA that recognizes the CD106 molecule on various TCR gamma delta T cell clones that do not proliferate in response to an anti-CD3 stimulation. In the present report, further biochemical analysis reveals two intracellular precursors (82 and 98 kDa) of the membrane-bound 110-kDa form of CD106. In addition, we detect a 100-kDa soluble form in the culture supernatant of the specific cloned lymphocytes. Phorbol ester raises the amount of the soluble CD106 in the supernatant while simultaneously inducing the disappearance of the membrane-bound form. We show that the membrane-anchored form of CD106 is converted to soluble form by a regulated proteolytic cleavage process involving a metalloprotease. EDTA and 1,10-phenanthroline, two potent inhibitors of metalloproteases, specifically inhibit the conversion of the membrane anchored to the soluble form of the CD106 molecule. In fact, these results implicate a Zn(2+)-activated metalloprotease in the regulation of CD106 expression in a subset of T cells and, therefore, represent a novel pathway of T cell functions.
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Thwaite, Joanne E., Stephen Hibbs, Richard W. Titball, and Timothy P. Atkins. "Proteolytic Degradation of Human Antimicrobial Peptide LL-37 by Bacillus anthracis May Contribute to Virulence." Antimicrobial Agents and Chemotherapy 50, no. 7 (July 2006): 2316–22. http://dx.doi.org/10.1128/aac.01488-05.
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ABSTRACTIn this paper we report on the susceptibilities of a range ofBacillusspecies to the human antimicrobial peptide LL-37.B. subtilisshowed a low level of resistance to killing by LL-37 (50% growth-inhibitory concentration [GI50], 1 μg/ml).B. cereusandB. thuringiensisshowed intermediate levels of resistance to killing (GI50s, 33 μg/ml and 37 μg/ml, respectively).B. anthracisshowed the highest level of resistance (GI50s, 40 to 66 μg/ml). The degradation of LL-37 byB. anthracisculture supernatant was blocked by the metalloprotease inhibitors EDTA and 1,10-phenanthroline, and the gene encoding the protease responsible for LL-37 degradation was not plasmid borne. Our findings suggest that alongside the classical plasmid-based virulence determinants, extracellular metalloproteases ofB. anthracismay play a role in survival in the host.
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Gavert, Nancy, Maralice Conacci-Sorrell, Daniela Gast, Annette Schneider, Peter Altevogt, Thomas Brabletz, and Avri Ben-Ze'ev. "L1, a novel target of β-catenin signaling, transforms cells and is expressed at the invasive front of colon cancers." Journal of Cell Biology 168, no. 4 (February 14, 2005): 633–42. http://dx.doi.org/10.1083/jcb.200408051.
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Aberrant β-catenin-TCF target gene activation plays a key role in colorectal cancer, both in the initiation stage and during invasion and metastasis. We identified the neuronal cell adhesion molecule L1, as a target gene of β-catenin-TCF signaling in colorectal cancer cells. L1 expression was high in sparse cultures and coregulated with ADAM10, a metalloprotease involved in cleaving and shedding L1's extracellular domain. L1 expression conferred increased cell motility, growth in low serum, transformation and tumorigenesis, whereas its suppression in colon cancer cells decreased motility. L1 was exclusively localized in the invasive front of human colorectal tumors together with ADAM10. The transmembrane localization and shedding of L1 by metalloproteases could be useful for detection and as target for colon cancer therapy.
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Sakovich, V. V. "METALLOPROTEASE FROM THE CULTURAL LIQUID OF Pleurotus osreatus." Biotechnologia Acta 12, no. 6 (December 2019): 35–45. http://dx.doi.org/10.15407/biotech12.06.035.
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Coleman, James L. J., Tony Ngo, Johannes Schmidt, Nadine Mrad, Chu Kong Liew, Nicole M. Jones, Robert M. Graham, and Nicola J. Smith. "Metalloprotease cleavage of the N terminus of the orphan G protein–coupled receptor GPR37L1 reduces its constitutive activity." Science Signaling 9, no. 423 (April 12, 2016): ra36. http://dx.doi.org/10.1126/scisignal.aad1089.
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Little is known about the pharmacology or physiology of GPR37L1, a G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptor that is abundant in the cerebellum. Mice deficient in this receptor exhibit precocious cerebellar development and hypertension. We showed that GPR37L1 coupled to the G protein Gαs when heterologously expressed in cultured cells in the absence of any added ligand, whereas a mutant receptor that lacked the amino terminus was inactive. Conversely, inhibition of ADAMs (a disintegrin and metalloproteases) enhanced receptor activity, indicating that the presence of the amino terminus is necessary for GPR37L1 signaling. Metalloprotease-dependent processing of GPR37L1 was evident in rodent cerebellum, where we detected predominantly the cleaved, inactive form. However, comparison of the accumulation of cAMP (adenosine 3′,5′-monophosphate) in response to phosphodiesterase inhibition in cerebellar slice preparations from wild-type and GPR37L1-null mice showed that some constitutive signaling remained in the wild-type mice. In reporter assays of Gαs or Gαi signaling, the synthetic, prosaposin-derived peptide prosaptide (TX14A) did not increase GPR37L1 activity. Our data indicate that GPR37L1 may be a constitutively active receptor, or perhaps its ligand is present under the conditions that we used for analysis, and that the activity of this receptor is instead controlled by signals that regulate metalloprotease activity in the tissue.
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TAYLOR, Kathryn M., Helen E. MORGAN, Andrea JOHNSON, Lisa J. HADLEY, and Robert I. NICHOLSON. "Structure–function analysis of LIV-1, the breast cancer-associated protein that belongs to a new subfamily of zinc transporters." Biochemical Journal 375, no. 1 (October 1, 2003): 51–59. http://dx.doi.org/10.1042/bj20030478.
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The LIV-1 gene has been previously associated with oestrogen-positive breast cancer and its metastatic spread to the regional lymph nodes. We have investigated the protein product of this gene as a marker for disease progression of breast cancer. The protein sequence contains a potential metalloprotease motif (HEXPHEXGD), which fits the consensus sequence for the catalytic zinc-binding site motif of the zincin metalloproteases. This motif has identified a new subfamily of ZIP (Zrt-, Irt-like proteins) zinc transporters, which we have termed LZT (LIV-1 subfamily of ZIP zinc transporters). Expression of recombinant LIV-1 in Chinese-hamster ovary cells confirmed the prediction that LZT proteins can act as zinc-influx transporters. Zinc is essential for growth and zinc transporters have an important role in maintaining intracellular zinc homoeostasis, aberrations of which could lead to diseases such as cancer. This is the first report of the expression of a recombinant human LZT protein in mammalian cells. Recombinant LIV-1 locates to the plasma membrane, concentrated in lamellipodiae, similar to membrane-type metalloproteases. Examination of LIV-1 tissue expression located it mainly to hormonally controlled tissues with widespread expression in the brain. Interestingly, the LIV-1 sequence contains a strong PEST site and other potential degradation motifs, which, combined with our evidence that recombinant LIV-1 associates with ubiquitin, may explain the low-level expression of LIV-1. Combining the crucial role that zinc plays in cell growth and the proven role of metalloproteases in metastasis presents an exciting indication of how LIV-1 plays a role in breast cancer progression.
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HOWARD, Linda, Rose A. MACIEWICZ, and Carl P. BLOBEL. "Cloning and characterization of ADAM28: evidence for autocatalytic pro-domain removal and for cell surface localization of mature ADAM28." Biochemical Journal 348, no. 1 (May 9, 2000): 21–27. http://dx.doi.org/10.1042/bj3480021.
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The metalloprotease disintegrins are a family of membrane-anchored glycoproteins with diverse functions in fertilization, myoblast fusion, neurogenesis and protein ectodomain shedding. Here we report a cDNA sequence, encoding a metalloprotease disintegrin, termed ADAM28 (‘a disintegrin and metalloprotease 28’), which was cloned from mouse lung. From protein sequence comparisons, ADAM28 is more closely related to snake venom metalloproteases (SVMPs) than to other ADAMs, and hence may cleave similar substrates to SVMPs, perhaps including components of the extracellular matrix. Northern blot analysis of selected mouse tissues revealed that ADAM28 is expressed highly and in alternatively spliced forms in the epididymis, suggesting a possible role in sperm maturation, and at lower levels in lung. The intracellular maturation of ADAM28 expressed in COS-7 cells resembles that of other ADAMs, in that ADAM28 is made as a precursor and processed to a mature form in a late Golgi compartment of the secretory pathway. Most or all of the mature, and thus presumably catalytically active, form of ADAM28 in COS-7 cells is accessible to cell surface trypsinization, suggesting that ADAM28 functions mainly on the cell surface. A mutation converting the catalytic-site glutamate residue into alanine abolishes pro-domain removal, even though this mutant form of ADAM28 can be transported to the cell surface in a manner similar to the wild-type protein. This suggests that pro-domain removal and maturation of ADAM28 may be, at least in part, autocatalytic. This is in contrast with several other ADAMs, for which furin-like proprotein convertases are involved in pro-domain removal, and in which a glutamate-to-alanine mutation in the catalytic site does not alter pro-domain removal.
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Levine, Stewart J., Barbara Adamik, Feras I. Hawari, Aminul Islam, Zu-Xi Yu, Da-Wei Liao, Jing Zhang, Xinle Cui, and Farshid N. Rouhani. "Proteasome inhibition induces TNFR1 shedding from human airway epithelial (NCI-H292) cells." American Journal of Physiology-Lung Cellular and Molecular Physiology 289, no. 2 (August 2005): L233—L243. http://dx.doi.org/10.1152/ajplung.00469.2004.
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The type 1 55-kDa TNF receptor (TNFR1) is an important modulator of lung inflammation. Here, we hypothesized that the proteasome might regulate TNFR1 shedding from human airway epithelial cells. Treatment of NCI-H292 human airway epithelial cells for 2 h with the specific proteasome inhibitor clasto-lactacystin β-lactone induced the shedding of proteolytically cleaved TNFR1 ectodomains. Clasto-lactacystin β-lactone also induced soluble TNFR1 (sTNFR1) release from the A549 pulmonary epithelial cell line, as well as from primary cultures of human small airway epithelial cells and human umbilical vein endothelial cells. Furthermore, sTNFR1 release induced by clasto-lactacystin β-lactone was not a consequence of apoptosis or the extracellular release of TNFR1 exosome-like vesicles. The clasto-lactacystin β-lactone-induced increase in TNFR1 shedding was associated with reductions in cell surface receptors and intracytoplasmic TNFR1 stores that were primarily localized to vesicular structures. As expected, the broad-spectrum zinc metalloprotease inhibitor TNF-α protease inhibitor 2 (TAPI-2) attenuated clasto-lactacystin β-lactone-mediated TNFR1 shedding, which is consistent with its ability to inhibit the zinc metalloprotease-catalyzed cleavage of TNFR1 ectodomains. TAPI-2 also reduced TNFR1 on the cell surface and attenuated the clasto-lactacystin β-lactone-induced reduction of intracytoplasmic TNFR1 vesicles. This suggests that TNFR1 shedding induced by clasto-lactacystin β-lactone involves the zinc metalloprotease-dependent trafficking of intracytoplasmic TNFR1 vesicles to the cell surface. Together, these data are consistent with the conclusion that proteasomal activity negatively regulates TNFR1 shedding from human airway epithelial cells, thus identifying previously unrecognized roles for the proteasome and zinc metalloproteases in modulating the generation of sTNFRs.
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Pipan, Maja Zakošek, Marjan Kosec, Janko Mrkun, and Petra Zrimšek. "Gelatinases in Boar Seminal Plasma and Their Relation to Semen Indicators." Acta Veterinaria Brno 79, no. 3 (2010): 491–96. http://dx.doi.org/10.2754/avb201079030491.
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Matrix metalloproteinases were detected in reproductive tissues and seminal plasma of various animal species. The aim of this study was to determine for the first time the presence of gelatinases and metalloproteases in boar seminal plasma and to correlate the results with semen indicators. Gelatin zymography was used for simultaneous identification and measurement of gelatinase enzyme activity associated with their molecular weights. Several gelatinase forms were identified in seminal plasma of boars. Those that were stimulated by CaCl2 and inhibited by EDTA and phenanthroline were considered as metalloproteases. Negative correlation between semen indicators (sperm index, sperm concentration and concentration of progressive motile sperm) and the concentrations of metalloprotease at 78 kDa and 66 kDa means that higher values of semen indicators correlate with lower concentrations of these metaloproteases in seminal plasma. Gelatinases with molecular weight of 225, 78 and 66 kDa correlated with higher levels of acrosome damage. Samples with sperm index above 110 M/ml contained gelatinases of significantly lower band intensities at 78 and 66 kDa compared to samples with SI less than 110 M/ml. Bands with 225, 78 and 66 kDa are suggested to belong to a dimer of MMP-9, proMMP-2 and mature MMP-2.
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Kothary, M. H., B. A. McCardell, C. D. Frazar, D. Deer, and B. D. Tall. "Characterization of the Zinc-Containing Metalloprotease Encoded by zpx and Development of a Species-Specific Detection Method for Enterobacter sakazakii." Applied and Environmental Microbiology 73, no. 13 (May 4, 2007): 4142–51. http://dx.doi.org/10.1128/aem.02729-06.
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ABSTRACT Enterobacter sakazakii causes a severe form of neonatal meningitis that occurs as sporadic cases as well as outbreaks. The disease has been epidemiologically associated with consumption of reconstituted, dried infant formulas. Very little information is available regarding pathogenicity of the organism and production of virulence factors. Clinical and environmental strains were screened for production of factors which have activity against Chinese hamster ovary (CHO) cells in tissue culture. Polymyxin B lysate and sonicate preparations but not culture supernatants from the strains caused “rounding” of CHO cells. Subsequent studies showed that the CHO cell-rounding factor is a proteolytic enzyme that has activity against azocasein. The cell-bound protease was isolated by using a combination of polymyxin B lysis, followed by sonication of cells harvested from tryptone broth. The protease was purified to homogeneity by sequential ammonium sulfate precipitation, gel filtration chromatography with Sephadex G-100, hydrophobic interaction chromatography with phenyl-Sepharose CL-4B, and a second gel filtration with Sephadex G-100. In addition to activity against azocasein, the purified protease also exhibits activity against azocoll and insoluble casein but not elastin. The protease has a molecular weight of 38,000 and an isoelectric point of 4.4. It is heat labile and for maximal activity against azocasein has an optimum temperature of 37°C and a pH range of 5 to 7. Proteolytic activity is inhibited by ortho-phenanthroline and Zincov but is not affected by phenylmethylsulfonyl fluoride, N-ethylmaleimide, and trypsin inhibitors, which demonstrates that the protease is a zinc-containing metalloprotease. The metalloprotease does not hemagglutinate chicken or sheep erythrocytes. Twenty-three to 27 of the first 42 N-terminal amino acid residues of the metalloprotease are identical to proteases produced by Serratia proteamaculans, Pectobacterium carotovorum, and Anabaena sp. PCR analysis using primers designed from a consensus nucleotide sequence showed that 135 E. sakazakii strains possessed the metalloprotease gene, zpx, and 25 non-E. sakazakii strains did not. The cloned zpx gene of strain 29544 consists of 1,026 nucleotides, and the deduced amino acid sequence of the metalloprotease has 341 amino acid residues, which corresponds to a theoretical protein size of 37,782 with a theoretical pI of 5.23. The sequence possesses three well-characterized zinc-binding and active-site motifs present in other bacterial zinc metalloproteases.