Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.5 (thrombin)
 33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In previous studies, Yersinia pestis YopM has been shown through mutational analysis to be necessary for virulence in mice and found to have homology with the thrombin-binding domain of the platelet receptor GPIb alpha. In this study, YopM was purified and shown by dot blot and chemical cross-linking tests to bind to human alpha-thrombin. No cross-linked product could be detected when human prothrombin was incubated with YopM. As a functional test of thrombin binding, it was shown that native but not boiled YopM inhibits thrombin-induced aggregation of human platelets. Control tests showed that YopM did not inactivate the platelets themselves, nor was its effect a nonspecific consequence of its very acidic isoelectric point. Microsequencing of YopM revealed an intact N terminus, indicating that functional YopM is not processed at the N terminus or secreted by a mechanism involving a cleavable signal sequence. Further characterization was made of an interesting effect on yopM expression that had been noticed in a previous study. A 1.5-kb HaeIII subclone overexpressed YopM in both Y. pestis and Escherichia coli compared with a larger clone containing the 5.3-kb HindIII-F fragment. To search for a possible regulator of YopM expression, the HindIII-F fragment was sequenced, revealing several open reading frames and three large repeated sequences. Deletional analysis showed that these were not involved in regulation of yopM. The data implicated a DNA structure 5' to yopM in moderating yopM expression.
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PMID:Yersinia pestis YopM: thrombin binding and overexpression. 145 57

In Yersinia pestis KIM there are 11 Yops (yersinial outer membrane proteins) encoded by the low-Ca2+ response virulence plasmid pCD1. Only YopM and YopN are found in easily detectable amounts in the culture medium. In our previous work, we characterized the yopM gene. In the present study, we constructed a YopM- mutant to elucidate the role of YopM in the virulence of Y. pestis. A lacZYA sequence was inserted 126 base pairs downstream from the start codon of the yopM gene in pCD1. The YopM- mutant had the same growth properties as the parent, Y. pestis KIM5-3001. The inserted lacZ gene was regulated by the promoter of the yopM gene. Accordingly, it was expressed strongly at 37 degrees C in the absence of Ca2+ and was decreased in expression when Ca2+ was present. Northern blot (RNA blot) analysis revealed that the yopM gene was in a monocistronic operon, suggesting that the yopM insertion mutation was unlikely to have polar effects on other genes. The YopM- mutant had strongly decreased virulence in mice, with a 50% lethal dose of 3.4 x 10(5) CFU. Virulence was restored by the cloned yopM-containing 5.5-kilobase HindIII F fragment of pCD1. However, supplying a cloned 1.57-kilobase fragment containing little more than the yopM structural gene caused the yopM mutant to significantly overexpress YopM and failed to restore virulence. The infection kinetics of the YopM- mutant revealed growth in both spleens and livers from days 2 to 4 after infection, followed by a precipitous clearance of the bacteria. YopM-containing supernatant proteins of Y. pestis inhibited thrombin- or ristocetin-induced platelet aggregation, whereas there was no inhibition by supernatant proteins from the YopM- Y. pestis mutant. Accordingly, YopM may prevent platelet-mediated events and serve as an important strategy for the yersiniae in the initial stages of a plague infection.
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PMID:YopM inhibits platelet aggregation and is necessary for virulence of Yersinia pestis in mice. 240 64

In Yersinia pestis KIM, there are 11 Yops (yersinial outer membrane proteins) encoded by the low-Ca2+ response virulence plasmid pCD1. Only Yops M and N are found in easily detectable amounts in the culture medium. In this study, we located and characterized the yopM gene to obtain clues about its role in the virulence of Y. pestis. Rabbit antibody was raised against Yops M and H, copurified from the supernatant of Y. pseudotuberculosis 43(pGW600, pCD1 yopE::Mu dI1[Apr lac]). This antiserum was adsorbed with an Escherichia coli clone that strongly expressed YopH. The resulting YopM-specific antibody was used to screen a HindIII library of pCD1. HindIII-F and several subclones from it expressed YopM in E. coli minicells. A DNA fragment of 1.39 kilobases from HindIII-F was sequenced and found to contain a 367-amino-acid open reading frame capable of encoding a protein with molecular mass (41,566 daltons) and isoelectric point (4.06) similar to those of YopM. The +1 site of the yopM gene was determined by primer extension. The DNA sequence contained repeating structures: 11 pairs of exact direct repeats, two exact inverted repeats, and three palindromes, ranging from 10 to 42 bases in size. One consensus 14-amino-acid sequence was repeated six times in the predicted protein sequence. The YopM sequence shares some significant homology with the von Willebrand factor- and thrombin-binding domain of the alpha chain of human platelet membrane glycoprotein Ib. These findings suggested a testable hypothesis for the function of YopM.
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PMID:The yopM gene of Yersinia pestis encodes a released protein having homology with the human platelet surface protein GPIb alpha. 267 Aug 88

Yersinia pestis toxin (II fraction by E. Baker) inhibited aggregation of human platelets as well as elevation of Ca2+, induced by different agonists ADP, PAF, thrombin. Agonist-induced Ca2+ mobilization and Ca2+ influx were dose-dependently inhibited by the toxin. The effect was rapid, developing during the first minute of incubation with the toxin. In contrast to murine lethal protein the platelet inhibitory activity was thermostable. The action of thermostable factor on platelets was accompanied by elevation of cellular cGMP level. This factor of Y. pestis activated guanylate cyclase in human platelets.
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PMID:[Features of regulatory system function in platelets under the effect of plague toxin]. 761 91

Yersinia enterocolitica infection in humans causes a broad spectrum of diseases ranging from acute bowel disease to extraintestinal manifestations such as reactive arthritis, erythema nodosum and uveitis. During the last decade a fascinating part of the molecular biology of the pathogenicity of human pathogenic Yersinia species has been unraveled. Pathogenicity factors such as protein tyrosine phosphatase, protein kinase, thrombin- and collagen-binding factors have been identified and characterized on the molecular level. In contrast to many animal models for human enteropathogenic microorganisms, experimental Y. enterocolitica infection in rodents resembles yersiniosis in humans and thus offers extraordinary opportunities to study the sequential steps of the infectious process. Rabbits are suitable animals in which to study Yersinia-induced enteritis (enterotoxin-mediated) and the humoral immune response after oral infection. The role of Peyer's patches (PP) in the entry of enteropathogenic Yersinia species has been elucidated in mice and rabbits. M cells are probably the primary target cells of invading Yersiniae. Surprisingly, after penetration of the mucosal epithelial cell layer Yersinia bacilli were visualized to be exclusively extracellular in PP tissue. Obviously neutrophils within PP were unable to phagocytize the invading microorganisms. Presently, it is not clear how the microorganisms disseminate from PP into lymph nodes, spleen, liver and lung of mice where they form abscesses and granuloma-like lesions. Immunohistologically the involvement of macrophages and T cells could be demonstrated in Yersinia-induced lesions of mice. Direct evidence for the role of T cells and cytokine-activated macrophages in the host defense reaction against a primary Yersinia infection in mice could be obtained from experiments including adoptive transfer of Yersinia-specific T cells and in vivo neutralization of TNF-alpha and IFN-gamma. The experimental rat model turned out to be a suitable model for studying Yersinia-induced aseptic arthritis. Lewis- and SHR rats proved to be arthritis-susceptible. Arthritogenicity of Yersinia for rats appeared to be restricted to Y. enterocolitica of serotype 08 and correlated with the virulence potential of this serotype. Surprisingly, expression of YadA, the collagen-binding factor, was not necessary for arthritis induction. A close association between both susceptibility to arthritis induction and Yersinia infection could be demonstrated in various rat strains. Depletion of alpha/beta T-cell receptor (alpha beta-TCR)-positive T cells by treatment with alpha beta-TCR-specific antibody revealed that T cells were required for clearance of the pathogen.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Experimental Yersinia enterocolitica infection in rodents: a model for human yersiniosis. 836 22

YopM, a 41.5 kDa virulence protein of Yersinia pestis is believed to have an anti-inflammatory role in bubonic plague. It has been shown previously that YopM binds human alpha-thrombin but not prothrombin and inhibits thrombin-induced platelet aggregation in vitro. In the present studies we carried out crosslinking reactions between purified YopM and alpha-thrombin or its blocked form FPR-alpha-thrombin in the presence of various competitors to identify where on thrombin YopM binds. We found that thrombin cleaves YopM at the C-terminus, indicating that this part of YopM must interact with thrombin's catalytic site. Hirudin, a 65 amino acid natural thrombin inhibitor, prevents both the YopM degradation and the formation of a ca. 75 kDa crosslinking complex between YopM and alpha-thrombin. A similar effect is observed when hirugen, a short peptide corresponding to hirudin's C-terminus (amino acids 58-64), is used as a synthetic thrombin inhibitor. A 15 bp long specific oligonucleotide known to block alpha-thrombin successfully competes with YopM for the thrombin-binding site, whereas a control, scrambled sequence aptamer does not. As these competitors block a fibrinogen binding site (also called anion binding exosite I), our crosslinking data indicate that YopM binds not only to the active site of alpha-thrombin but also to the abeI.
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PMID:Interaction between Yersinia pestis YopM protein and human alpha-thrombin. 888 45

The Yersinia Yop virulon is an anti-host system made up of four elements: (i) a type III secretion system called Ysc; (ii) a system designed to deliver bacterial proteins into eukaryotic target cells (YopB, YopD); (iii) a control element (YopN); and (iv) a set of intracellularly delivered proteins designed to disarm these cells or disrupt their communications (YopE, YopH and possibly others). YopM, another Yop protein, binds thrombin and is thus presumed to act as an extracellular effector. Here, we analyzed YopM from Y.enterocolitica and we wondered whether it could also be delivered inside eukaryotic cells. To answer this question we applied the Yop-Cya reporter strategy. Hybrids made of 141 or 100 N-terminal residues of YopM fused to Cya were delivered inside PU5-1.8 macrophages by recombinant Y.enterocolitica strains. YopB and YopD were required as translocators. Leakage of the reporters into the macrophage culture supernatant during the bacterial infection increased strongly when YopN was missing, showing that YopN is involved in the control of delivery of YopM inside eukaryotic cells. YopN itself was not delivered into the macrophages. In conclusion, YopM is translocated inside the eukaryotic cells and its physiopathological role should be revised or completed.
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PMID:Status of YopM and YopN in the Yersinia Yop virulon: YopM of Y.enterocolitica is internalized inside the cytosol of PU5-1.8 macrophages by the YopB, D, N delivery apparatus. 889 64

YopM of Yersinia pestis has previously been shown to be necessary for full virulence in mice and to be able to bind human alpha-thrombin. This activity prompted the hypothesis that YopM, functioning extracellularly during plague, might be accessible to neutralization by antibody and hence might be a protective antigen. This study tested this hypothesis and found that YopM was not protective, either by passive or active immunization, in inbred or outbred mice. These findings showed that either YopM-specific antibody does not have access to YopM during experimental plague or the function of extracellular YopM is not neutralizable by antibody. Exogenously supplied YopM partially restored virulence to a YopM- strain of Y. pestis while having no effect on lethality of Listeria monocytogenes. These findings indicate that YopM does not significantly alter host defenses important for resistance against heterologous infection (Listeria monocytogenes) but raise the possibility that YopM has a minor extracellular function specific to homologous infection (Y. pestis).
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PMID:Effect of Yersinia pestis YopM on experimental plague. 903 98

The YopM virulence protein of Yersinia pestis has been described as binding human alpha-thrombin and inhibiting thrombin-induced platelet aggregation in vitro. However, recent studies have shown that a YopM-CyaA fusion protein could be targeted vectorially into eukaryotic cells through the Yersinia type III secretion system. In this study, our objective was to characterize YopM's fate in more detail. We followed YopM in the culture medium and inside infected HeLa cells. We confirmed that the native YopM is targeted into HeLa cells, where it is insensitive to exogenous trypsin. The bacteria must be surface located to target YopM, and YopB and YopD are necessary, whereas the LcrE protein (called also YopN) makes this process more efficient. Immunofluorescence localization revealed that YopM, in contrast to YopE, is not only targeted to the cytoplasm but also trafficks to the cell's nucleus by means of a vesicle-associated pathway that is strongly inhibited by brefeldin A, perturbed by monensin or bafilomycin A1 and dependent upon microtubules (decreased by colchicine and nocodazole). These findings revealed a novel interaction of Yersinia pestis with its eukaryotic host.
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PMID:Targeting of the Yersinia pestis YopM protein into HeLa cells and intracellular trafficking to the nucleus. 998 81

Yersinia spp. inject effector proteins (Yersinia outer proteins, Yops) into target cells via a type III secretion apparatus. The effector YopE was recently shown to possess GAP activity towards the Rho GTPases RhoA, Rac and CDC42 in vitro. To investigate the intracellular, 'in vivo' targets of YopE we generated a Yersinia enterocolitica strain [WA(pYLCR+E)] that injects 'life-like' amounts of YopE as only effector. Primary human umbilical vein endothelial cells (HUVEC) were infected with WA(pYLCR+E) and were then stimulated with: (i) bradykinin to induce actin microspikes followed by ruffles as an assay for CDC42 activity followed by CDC42 stimulated Rac activity; (ii) sphingosine-1-phosphate to form ruffles by direct Rac activation; or (iii) thrombin to generate actin stress fibres through Rho activation. In WA(pYLCR+E)-infected HUVEC microspike formation stimulated with bradykinin remained intact but the subsequent development of ruffles was abolished. Furthermore, ruffle formation after stimulation with sphingosine-1-phosphate or thrombin induced production of stress fibres was unaltered in the infected cells. These data suggest that YopE is able to inhibit Rac- but not Rho- or CDC42-regulated actin structures and, more specifically, that YopE is capable of blocking CDC42Hs dependent Rac activation but not direct Rac activation in HUVEC. This provides evidence for a considerable specificity of YopE towards selective Rac-mediated signalling pathways in primary target cells of Yersinia.
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PMID:YopE of Yersinia, a GAP for Rho GTPases, selectively modulates Rac-dependent actin structures in endothelial cells. 1129 53


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