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, produced by the bacterium Bacillus anthracis, is the major cause of death in animals infected with anthrax. One component of this toxin, lethal factor (LF), is suspected to be a metalloprotease, but no physiological substrates have been identified. Here it is shown that LF is a protease that cleaves the amino terminus of mitogen-activated protein kinase kinases 1 and 2 (MAPKK1 and MAPKK2) and that this cleavage inactivates MAPKK1 and inhibits the MAPK signal transduction pathway. The identification of a cleavage site for LF may facilitate the development of LF inhibitors.
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PMID:Proteolytic inactivation of MAP-kinase-kinase by anthrax lethal factor. 966 Jul

The lethal toxin of Bacillus anthracis consists of two proteins, PA and LF, which together induce lethal effects in some animal species and cause macrophage lysis. LF is a zinc-binding protein with metalloprotease activity. With a two-hybrid system approach we identified MAP kinase kinases (MAPKKs) Mekl and Mek2 as proteins interacting with LF. LF was shown to cleave Mek1 and Mek2 and an additional MAPKK family member MKK3, within their N-terminal region. We examined macrophage cell lines and primary peritoneal cells with different sensitivities to LF but did not find a direct correlation between MAPKKs cleavage and cell death. On the other hand, sublytic doses of LF cleave MAPKKs and cause a reduction in the LPS/IFNgamma-induced production of proinflammatory mediators. These findings are discussed with respect to the possible role of LF in the initial phase of infection.
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PMID:Lethal factor of Bacillus anthracis cleaves the N-terminus of MAPKKs: analysis of the intracellular consequences in macrophages. 1111 21

Lethal factor (LF) is a protein (relative molecular mass 90,000) that is critical in the pathogenesis of anthrax. It is a highly specific protease that cleaves members of the mitogen-activated protein kinase kinase (MAPKK) family near to their amino termini, leading to the inhibition of one or more signalling pathways. Here we describe the crystal structure of LF and its complex with the N terminus of MAPKK-2. LF comprises four domains: domain I binds the membrane-translocating component of anthrax toxin, the protective antigen (PA); domains II, III and IV together create a long deep groove that holds the 16-residue N-terminal tail of MAPKK-2 before cleavage. Domain II resembles the ADP-ribosylating toxin from Bacillus cereus, but the active site has been mutated and recruited to augment substrate recognition. Domain III is inserted into domain II, and seems to have arisen from a repeated duplication of a structural element of domain II. Domain IV is distantly related to the zinc metalloprotease family, and contains the catalytic centre; it also resembles domain I. The structure thus reveals a protein that has evolved through a process of gene duplication, mutation and fusion, into an enzyme with high and unusual specificity.
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PMID:Crystal structure of the anthrax lethal factor. 1170 May 39

Anthrax is a severe bacterial infection that occurs when Bacillus anthracis spores gain access into the body and germinate in macrophages, causing septicemia and toxemia. Anthrax toxin is a binary A-B toxin composed of protective antigen (PA), lethal factor (LF), and edema factor (EF). PA mediates the entry of either LF or EF into the cytosol of host cells. LF is a zinc metalloprotease that inactivates mitogen-activated protein kinase kinase inducing cell death, and EF is an adenylyl cyclase impairing host defences. Inhibitors targeting different steps of toxin activity have recently been developed. Anthrax toxin has also been exploited as a therapeutic agent against cancer.
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PMID:Anthrax toxin: a tripartite lethal combination. 1243 80

The lethal factor (LF) of anthrax toxin is the toxic component of the exotoxin (lethal toxin) secreted by toxic strains of Bacillus anthracis. The lethal factor is a zinc-dependent metalloprotease that specifically cleaves the mitogen-activated protein kinase kinase (MAPKK) family of enzymes. We took advantage of this substrate specificity to develop an electrochemiluminescence (ECL) peptide cleavage assay. The ECL assay uses the stable ruthenium (Ru) metal chelate that, in the presence of tripropylamine, generates a light reaction triggered by the application of an electric potential. The Ru label is specifically incorporated into the C-terminal CYS residue of a synthetic peptide (23mer) containing the MAPKK2 cleavage sequence of LF. Streptavidin-coated paramagnetic beads were the solid phase and facilitated separation and characterization of the enzymatic reaction products based upon N-terminal biotinylation of the peptide substrate. Intact peptide bound via the biotin moiety generated high signal due to the Ru label, whereas binding of the cleaved peptide fragment devoid of Ru label reduced the ECL signal. The proposed assay provides a novel opportunity for the screening of potential therapeutics against anthrax.
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PMID:An enzymatic electrochemiluminescence assay for the lethal factor of anthrax. 1296 63

Anthrax lethal toxin is the major cause of death in systemic anthrax. Lethal toxin consists of two proteins: protective antigen and LF (lethal factor). Protective antigen binds to a cell-surface receptor and transports LF into the cytosol. LF is a metalloprotease that targets MKKs [MAPK (mitogen-activated protein kinase) kinases]/MEKs [MAPK/ERK (extracellular-signal-regulated kinase) kinases], cleaving them to remove a small N-terminal stretch but leaving the bulk of the protein, including the protein kinase domain, intact. LF-mediated cleavage of MEK1 and MKK6 has been shown to inhibit signalling through their cognate MAPK pathways. However, the precise mechanism by which this proteolytic cleavage inhibits signal transmission has been unclear. Here we show that the C-terminal LF-cleavage products of MEK1, MEK2, MKK3, MKK4, MKK6 and MKK7 are impaired in their ability to bind to their MAPK substrates, suggesting a common mechanism for the LF-induced inhibition of signalling.
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PMID:Anthrax lethal factor-cleavage products of MAPK (mitogen-activated protein kinase) kinases exhibit reduced binding to their cognate MAPKs. 1461 89

In addition to their inhibitory effects, cannabinoids also exert stimulatory activity which can be detected at the cellular level. In a previous study, we demonstrated a stimulatory effect of the synthetic cannabinoid receptor agonist desacetyllevonantradol (DALN) on Ca(2+) flux into N18TG2 neuroblastoma cells, and suggested a dual mechanism: one pathway mediated by PKA and the other one by protein kinase C (PKC). Here we studied the PKC-mediated effect of DALN on Ca(2+) influx. The stimulatory effect of DALN on Ca(2+) influx was partially blocked by the PKC inhibitor chelerythrine, by the metalloprotease inhibitor o-phenanthroline and by the MEK (mitogen-activated protein-kinase kinase, MAPK kinase) inhibitor PD98059. Immunobloting of ERK1/2 MAPK demonstrated phosphorylation by DALN, and indicated the involvement of vascular endothelial growth factor (VEGF) receptor tyrosin kinases (RTKs) in MAPK activation as it was blocked by oxindole-1. Transactivation of the VEGFR-MAPK cascade by DALN involved CB1 cannabinoid receptors coupled to Gi/Go GTP-binding proteins as it was blocked by SR141716A and by pertussis toxin (PTX). The pharmacological implications of this novel mechanism of cannabinoid activity are discussed.
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PMID:The involvement of VEGF receptors and MAPK in the cannabinoid potentiation of Ca2+ flux into N18TG2 neuroblastoma cells. 1474 3

Anthrax lethal factor (LF) is a Zn2+ -metalloprotease that cleaves and inactivates mitogen-activated protein kinase kinases (MEKs). We have used site-directed mutagenesis to identify a cluster of residues in domain II of LF that lie outside the active site and are required for cellular proteolytic activity toward MEKs. Alanine substituted for Leu293, Lys294, Leu514, Asn516, or Arg491 caused a 10-50-fold reduction in LF toxicity. Further, whereas pairwise substitution of alanine for Leu514 and either Leu293, Lys294, or Arg491 completely abrogated LF toxicity, pairwise mutation of Leu514 and Asn516 resulted in toxicity comparable with N516A alone. The introduction of these mutations reduced LF-mediated cleavage of MEK2 in cell-based assays but altered neither the ability of LF to bind protective antigen nor its ability to translocate across a membrane. Interestingly, direct in vitro measurement of LF activity indicated that decreased toxicity was not always accompanied by reduced proteolytic activity. However, mutations in this region significantly reduced the ability of LF to competitively inhibit B-Raf phosphorylation of MEK. These results provide evidence that elements of domain II are involved in the association of LF into productive complex with MEKs.
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PMID:Involvement of domain II in toxicity of anthrax lethal factor. 1546 30

Interleukin-8 (IL-8) has been reported to promote tumor cell growth in colon cancer cells after binding to its receptors, which are members of the G-protein coupled receptor (GPCR) family. Recent studies demonstrated that stimulation of GPCR can induce shedding of epidermal growth factor (EGF) ligands via activation of a disintegrin and metalloprotease (ADAM), with subsequent transactivation of the EGF receptor (EGFR). In this study, we investigated mechanisms of cell proliferation and migration stimulated by IL-8 in a human colon carcinoma cell line (Caco2). IL-8 increased DNA synthesis of Caco2 in a dose dependent manner and this was inhibited by ADAM, EGFR kinase, and MEK inhibitors. IL-8 transiently induced EGFR tyrosine phosphorylation after 5-90 min and this was completely inhibited by ADAM inhibitor. Neutralizing antibody against HB-EGF as a key ligand for EGFR also blocked transactivation of EGFR and cell proliferation by IL-8. Since IL-8-induced cell migration was further suppressed by the ADAM inhibitor and the HB-EGF neutralizing antibody, our data indicate that IL-8 induces cell proliferation and migration by an ADAM-dependent pathway, and that HB-EGF plays an important role as the major ligand for this pathway.
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PMID:IL-8 promotes cell proliferation and migration through metalloproteinase-cleavage proHB-EGF in human colon carcinoma cells. 1574 28

The design of inhibitors for anthrax lethal factor (LF) is currently of interest as an approach for the treatment of anthrax because LF plays a major role in the cytotoxicity of target cells. LF is a zinc-dependent metalloprotease that specifically cleaves the mitogen-activated protein kinase kinase (MKK) family. Current assay systems for the screening of LF inhibitor use the optimized synthetic peptide coupled with various kinds of fluorophores, enabling fast, sensitive, and robust assays suited to high-throughput screening. However, evidence suggests that the regions beside the cleavage site are also involved in specificity and proteolytic activity of LF. In the current study, we tried to develop a high-throughput assay for LF activity based on native substrate, mitogen-activated ERK kinase 1 (MEK1). The assay system relies on the enhanced chemiluminescence signal resulting from a specific antibody against the C-terminal region of native substrate. A glutathione-coated multiwell plate was used as a solid support to immobilize the native substrate by its N-terminal glutathione-S-transferase moiety. Immobilized substrate increases the specificity and sensitivity of LF-catalyzed substrate hydrolysis compared with the solution phase assay. This assay system might be used to discover a wide spectrum of anthrax inhibitors.
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PMID:Development of high-throughput assay of lethal factor using native substrate. 1586 25


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