Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.12.2 (MEK)
18,161 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Anthrax lethal toxin triggers death in some cell types, such as macrophages, and causes a variety of cellular dysfunctions in others. Collectively, these effects dampen the innate and adaptive immune systems to allow Bacillus anthracis to survive and proliferate in the mammalian host. The diverse effects caused by the toxin have in part been attributed to its interference with signaling pathways in target cells. Lethal factor (LF) is the proteolytic component of the toxin, and cleaves six members of the mitogen-activated protein kinase kinase family after being delivered to the cytosol by the cell-binding component of the toxin, protective antigen. The effect of cleaving these mitogen-activated protein kinase kinases is to interfere with extracellular signal-related kinase (ERK), p38 and c-Jun N-terminal kinase signaling. Here, we characterized an LF mutant, LF-K518E/E682G, that was defective at causing pyroptosis in RAW 264.7 cells and at activating the Nlrp1b inflammasome in a heterologous expression system. LF-K518E/E682G did not exhibit an overall impairment of function, however, because it was able to downregulate the ERK pathway, but not the p38 or c-Jun N-terminal kinase pathways. Furthermore, LF-K518E/E682G efficiently killed melanoma cells, which were shown previously to undergo apoptosis in response to lethal toxin or to pharmacological inhibition of the ERK pathway. Our results suggest that LF-K518E/E682G is defective at cleaving a substrate involved in the activation of the Nlrp1b inflammasome.
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PMID:An anthrax lethal factor mutant that is defective at causing pyroptosis retains proapoptotic activity. 1992 72

AB-type toxins, like other bacterial toxins, are notably opportunistic molecules. They rely on target cell receptors to reach the appropriate location within the target cell where translocation of their enzymatic subunits occurs. The anthrax toxin, however, times its own uptake, suggesting that toxin binding triggers specific signaling events. Here we show that the anthrax toxin triggers tyrosine phosphorylation of its own receptors, capillary morphogenesis gene 2 and tumor endothelial marker 8, which are not endowed with intrinsic kinase activity. This is required for efficient toxin uptake because endocytosis of the mutant receptor lacking the cytoplasmic tyrosine residues is strongly delayed. Phosphorylation of the receptors was dependent on src-like kinases, which where activated upon toxin binding. Importantly, src-dependent phosphorylation of the receptor was required for its subsequent ubiquitination, which in turn was required for clathrin-mediated endocytosis. Consistently, we found that uptake of the anthrax toxin and processing of the lethal factor substrate MEK1 are inhibited by silencing of src and fyn, as well as in src and fyn knockout cells.
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PMID:Anthrax toxin triggers the activation of src-like kinases to mediate its own uptake. 2008 Jun 40

Anthrax lethal toxin (LeTx) stands for the major virulence factor of the anthrax disease. It comprises a 90kDa highly specific metalloprotease, the anthrax lethal factor (LF). LF possesses a catalytic Zn(2+) binding site and is highly specific against MAPK kinases, thus representing the most potent native biomolecule to alter and inactivate MKK [MAPK (mitogen-activated protein kinase) kinases] signalling pathways. Given the importance of the interaction between LF and substrate for the development of anti-anthrax agents as well as the potential treatment of nascent tumours, the analysis of the structure and dynamic properties of the LF catalytic site are essential to elucidate its enzymatic properties. Here we report the recombinant expression and purification of a C-terminal part of LF (LF(672-776)) that harbours the enzyme's core protease domain. The biophysical characterization and backbone assignments ((1)H, (13)C, (15)N) of the polypeptide revealed a stable, well folded structure even in the absence of Zn(2+), suitable for high resolution structural analysis by NMR.
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PMID:Purification and biophysical characterization of the core protease domain of anthrax lethal factor. 2043 2

Anthrax lethal toxin (LT) increases vascular leakage in a number of mammalian models and in human anthrax disease. Using a zebrafish model, we determined that vascular delivery of LT increased permeability, which was phenocopied by treatment with a selective chemical inhibitor of MEK1 and MEK2 (also known as mitogen-activated protein kinase [MAPK] kinase, MEK, or MKK). Here we investigate further the role of MEK1/phospho-ERK (pERK) in the action of LT. Overexpression of wild-type zebrafish MEK1 at high levels did not induce detrimental effects. However, a constitutively activated version, MEK1(S219D,S223D) (MEK1DD), induced early defects in embryonic development that correlated with increased ERK/MAPK phosphorylation. To bypass these early developmental defects and to provide a genetic tool for examining the action of lethal factor (LF), we generated inducible transgenic zebrafish lines expressing either wild-type or activated MEK1 under the control of a heat shock promoter. Remarkably, induction of MEK1DD transgene expression prior to LT delivery prevented vascular damage, while the wild-type MEK1 line did not. In the presence of both LT and MEK1DD transgene expression, cardiovascular development and function proceeded normally in most embryos. The resistance to microsphere leakage in transgenic animals demonstrated a protective role against LT-induced vascular permeability. A consistent increase in ERK phosphorylation among LT-resistant MEK1DD transgenic animals provided additional confirmation of transgene activation. These findings provide a novel genetic approach to examine mechanism of action of LT in vivo through one of its known targets. This approach may be generally applied to investigate additional pathogen-host interactions and to provide mechanistic insights into host signaling pathways affected by pathogen entry.
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PMID:Constitutive MEK1 activation rescues anthrax lethal toxin-induced vascular effects in vivo. 2085 11

Anthrax lethal toxin (LT) is the major virulence factor for Bacillus anthracis. The lethal factor (LF) component of this bipartite toxin is a protease which, when transported into the cellular cytoplasm, cleaves mitogen-activated protein kinase kinase (MEK) family proteins and induces rapid toxicity in mouse macrophages through activation of the Nlrp1b inflammasome. A high-throughput screen was performed to identify synergistic LT-inhibitory drug combinations from within a library of approved drugs and molecular probes. From this screen we discovered that auranofin, an organogold compound with anti-inflammatory activity, strongly inhibited LT-mediated toxicity in mouse macrophages. Auranofin did not inhibit toxin transport into cells or MEK cleavage but inhibited both LT-mediated caspase-1 activation and caspase-1 catalytic activity. Thus, auranofin inhibited LT-mediated toxicity by preventing activation of the Nlrp1b inflammasome and the downstream actions that occur in response to the toxin. Idebenone, an analog of coenzyme Q, synergized with auranofin to increase its protective effect. We found that idebenone functions as an inhibitor of voltage-gated potassium channels and thus likely mediates synergy through inhibition of the potassium fluxes which have been shown to be required for Nlrp1b inflammasome activation.
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PMID:Auranofin protects against anthrax lethal toxin-induced activation of the Nlrp1b inflammasome. 2114 29

Mitogen-activated protein kinase kinases (MKK or MEK) 1 and 2 are usually treated as redundant kinases. However, in assessing their relative contribution towards ERK-mediated biologic response investigators have relied on tests of necessity, not sufficiency. In response we developed a novel experimental model using lethal toxin (LeTx), an anthrax toxin-derived pan-MKK protease, and genetically engineered protease resistant MKK mutants (MKKcr) to test the sufficiency of MEK signaling in melanoma SK-MEL-28 cells. Surprisingly, ERK activity persisted in LeTx-treated cells expressing MEK2cr but not MEK1cr. Microarray analysis revealed non-overlapping downstream transcriptional targets of MEK1 and MEK2, and indicated a substantial rescue effect of MEK2cr on proliferation pathways. Furthermore, LeTx efficiently inhibited the cell proliferation and anchorage-independent growth of SK-MEL-28 cells expressing MKK1cr but not MEK2cr. These results indicate in SK-MEL-28 cells MEK1 and MEK2 signaling pathways are not redundant and interchangeable for cell proliferation. We conclude that in the absence of other MKK, MEK2 is sufficient for SK-MEL-28 cell proliferation. MEK1 conditionally compensates for loss of MEK2 only in the presence of other MKK.
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PMID:MEK2 is sufficient but not necessary for proliferation and anchorage-independent growth of SK-MEL-28 melanoma cells. 2136 9

The responses of macrophages to Bacillus anthracis infection are important for the survival of the host, since macrophages are required for the germination of B. anthracis spores in lymph nodes, and macrophage death exacerbates anthrax lethal toxin (LeTx)-induced organ collapse. To elucidate the mechanism of macrophage cell death induced by LeTx, we performed a genetic screen to search for genes associated with LeTx-induced macrophage cell death. RAW264.7 cells, a macrophage-like cell line sensitive to LeTx-induced death, were randomly mutated and LeTx-resistant mutant clones were selected. AMP deaminase 3 (AMPD3), an enzyme that converts AMP to IMP, was identified to be mutated in one of the resistant clones. The requirement of AMPD3 in LeTx-induced cell death of RAW 264.7 cells was confirmed by the restoration of LeTx sensitivity with ectopic reconstitution of AMPD3 expression. AMPD3 deficiency does not affect LeTx entering cells and the cleavage of mitogen-activated protein kinase kinase (MKK) by lethal factor inside cells, but does impair an unknown downstream event that is linked to cell death. Our data provides new information regarding LeTx-induced macrophage death and suggests that there is a key regulatory site downstream of or parallel to MKK cleavage that controls the cell death in LeTx-treated macrophages.
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PMID:AMPD3 is involved in anthrax LeTx-induced macrophage cell death. 2182 1

Binary toxins are among the most potent bacterial protein toxins performing a cooperative mode of translocation and exhibit fatal enzymatic activities in eukaryotic cells. Anthrax and C2 toxin are the most prominent examples for the AB(7/8) type of toxins. The B subunits bind both host cell receptors and the enzymatic A polypeptides to trigger their internalization and translocation into the host cell cytosol. C2 toxin is composed of an actin ADP-ribosyltransferase (C2I) and C2II binding subunits. Anthrax toxin is composed of adenylate cyclase (EF) and MAPKK protease (LF) enzymatic components associated to protective antigen (PA) binding subunit. The binding and translocation components anthrax protective antigen (PA(63)) and C2II of C2 toxin share a sequence homology of about 35%, suggesting that they might substitute for each other. Here we show by conducting in vitro measurements that PA(63) binds C2I and that C2II can bind both EF and LF. Anthrax edema factor (EF) and lethal factor (LF) have higher affinities to bind to channels formed by C2II than C2 toxin's C2I binds to anthrax protective antigen (PA(63)). Furthermore, we could demonstrate that PA in high concentration has the ability to transport the enzymatic moiety C2I into target cells, causing actin modification and cell rounding. In contrast, C2II does not show significant capacity to promote cell intoxication by EF and LF. Together, our data unveiled the remarkable flexibility of PA in promoting C2I heterologous polypeptide translocation into cells.
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PMID:Cross-reactivity of anthrax and C2 toxin: protective antigen promotes the uptake of botulinum C2I toxin into human endothelial cells. 2185 Feb 57

The anthrax lethal toxin (LT) enters host cells and enzymatically cleaves MAPKKs or MEKs. How these molecular events lead to death from anthrax remains poorly understood, but published reports suggest a direct effect of LT on vascular permeability. We have found that LT challenge in mice disrupts signaling through Tie-2, a tonically activated receptor tyrosine kinase in the endothelium. Genetic manipulations favoring Tie-2 activation enhanced interendothelial junctional contacts, prevented vascular leakage, and promoted survival following a lethal dose of LT. Cleavage of MEK1/2 was necessary for LT to induce endothelial barrier dysfunction, and activated Tie-2 signaled through the uncleaved fraction of MEKs to prevent LT's effects on the endothelium. Finally, primates infected with toxin-secreting Bacillus anthracis bacilli developed a rapid and marked imbalance in the endogenous ligands that signal Tie-2, similar to that seen in LT-challenged mice. Our results show that B. anthracis LT blunts signaling through Tie-2, thereby weakening the vascular barrier and contributing to lethality of the disease. Measurement of circulating Tie-2 ligands and manipulation of Tie-2 activity may represent future prognostic and therapeutic avenues for humans exposed to B. anthracis.
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PMID:Impaired function of the Tie-2 receptor contributes to vascular leakage and lethality in anthrax. 2266 99

Protease inhibitor discovery has focused almost exclusively on compounds that bind to the active site. Inhibitors targeting protease exosites, regions outside of the active site that influence catalysis, offer potential advantages of increased specificity but are difficult to systematically discover. Here, we describe an assay suitable for detecting exosite-targeting inhibitors of the metalloproteinase anthrax lethal factor (LF) based on cleavage of a full-length mitogen-activated protein kinase kinase (MKK) substrate. We used this assay to screen a small-molecule library and then subjected hits to a secondary screen to exclude compounds that efficiently blocked cleavage of a peptide substrate. We identified a compound that preferentially inhibited cleavage of MKKs compared with peptide substrates and could suppress LF-induced macrophage cytolysis. This approach should be generally applicable to the discovery of exosite-targeting inhibitors of many additional proteases.
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PMID:Identification of exosite-targeting inhibitors of anthrax lethal factor by high-throughput screening. 2284 Jul 75


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