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:1.8.1.4 (diaphorase)
2,754 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The aceEF-lpd operon of Escherichia coli encodes the pyruvate dehydrogenase (E1p), dihydrolipoamide acetyltransferase (E2p) and dihydrolipoamide dehydrogenase (E3) subunits of the pyruvate dehydrogenase multienzyme complex (PDH complex). An isopropyl beta-D-thiogalactopyranoside-inducible expression system was developed for amplifying fully lipoylated wild-type and mutant PDH complexes to over 30% of soluble protein. The extent of lipoylation was related to the degree of aeration during amplification. The specific activities of the isolated PDH complexes and the E1p component were 50-75% of the values normally observed for the unamplified complex. This could be due to altered stoichiometries of the overproduced complexes (higher E3 and lower E1p contents) or inactivation of E1p. The chaperonin, GroEL, was identified as a contaminant which copurifies with the complex. Site-directed substitutions of an invariant glycine residue (G231A, G231S and G231M) in the putative thiamine pyrophosphate-binding fold of the E1p component had no effect on the production of high-molecular-mass PDH complexes but their E1p and PDH complex activities were very low or undetectable, indicating that G231 is essential for the structural or catalytic integrity of E1p. A minor correction to the nucleotide sequence, which leads to the insertion of an isoleucine residue immediately after residue 273, was made. Substitution of the conserved histidine and arginine residues (H602 and R603) in the putative active-site motif of the E2p subunit confirmed that H602 of the E. coli E2p is essential, whereas R603 could be replaced without inactivating E2p. Deletions affecting putative secondary structural elements at the boundary of the E2p catalytic domain inhibited catalytic activity without affecting the assembly of the E2p core or its ability to bind E1p, indicating that the latter functions are determined elsewhere in the domain. The results further consolidate the view that chloramphenicol acetyltransferase serves as a useful structural and functional model for the catalytic domain of the lipoate acyltransferases.
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PMID:Overproduction of the pyruvate dehydrogenase multienzyme complex of Escherichia coli and site-directed substitutions in the E1p and E2p subunits. 144 21

The interaction between the pyruvate decarboxylase (E1) component and a di-domain (lipoyl domain plus peripheral subunit-binding domain) from the dihydrolipoyl acetyltransferase (E2) component of the Bacillus stearothermophilus pyruvate dehydrogenase multienzyme complex was investigated. Only 1 mol of di-domain (binding domain) was bound to 1 mol of heterotetrameric E1 (alpha 2 beta 2) and the binding was without effect on the kinetic activity of E1. Similarly, the di-domain bound to separate E1 beta subunits at a maximal polypeptide chain ratio of 1:2, but no detectable interaction was found with the E1 alpha subunit. However, addition of the monomeric E1 alpha subunit to an E1 beta-di-domain complex generated a fully functional E1 (alpha 2 beta 2)-di-domain complex, indicating that the E1 beta subunit plays the critical part in binding the E1 component to the di-domain and suggesting that no chaperonin is needed in vitro to promote the assembly of the three separate proteins. Mixing the E1 and dihydrolipoyl dehydrogenase (E3) components in the presence of di-domain revealed that E1 and E3 cannot bind simultaneously to the same molecule of di-domain, a new feature of the assembly pathway and an important factor in determining the ultimate structure of the assembled enzyme complex.
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PMID:Interaction of component enzymes with the peripheral subunit-binding domain of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus: stoichiometry and specificity in self-assembly. 770 67

The mammalian renal toxicant tetrafluoroethylcysteine (TFEC) is metabolized to a reactive intermediate that covalently modifies the lysine residues of a select group of mitochondrial proteins, forming difluorothioamidyl lysine protein adducts. Cellular damage is initiated by this process and cell death ensues. NH2-terminal sequence analysis of purified mitochondrial proteins containing difluorothioamidyl lysine adducts identified the lipoamide succinyltransferase and dihydrolipoamide dehydrogenase subunits of the alpha-ketoglutarate dehydrogenase complex (alphaKGDH), a key regulatory component of oxidative metabolism, as targets for TFEC action. Adduct formation resulted in marked inhibition of alphaKGDH enzymatic activity, whereas the related pyruvate dehydrogenase complex was unmodified by TFEC and its activity was not inhibited in vivo. Covalent modification of alphaKGDH subunits also resulted in interactions with mitochondrial chaperonin HSP60 in vivo and with HSP60 and mitochondrial HSP70 in vitro. These observations confirm the role of mammalian stress proteins in the recognition of abnormal proteins and provide supporting evidence for reactive metabolite-induced cell death by modification of critical protein targets.
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PMID:Mitochondrial stress protein recognition of inactivated dehydrogenases during mammalian cell death. 981 14

Duchenne muscular dystrophy is the most commonly inherited neuromuscular disorder in humans. Although the primary genetic deficiency of dystrophin in X-linked muscular dystrophy is established, it is not well-known how pathophysiological events trigger the actual fibre degeneration. We have therefore performed a DIGE analysis of normal diaphragm muscle versus the severely affected x-linked muscular dystrophy (MDX) diaphragm, which represents an established animal model of dystrophinopathy. Out of 2398 detectable 2-D protein spots, 35 proteins showed a drastic differential expression pattern, with 21 proteins being decreased, including Fbxo11-protein, adenylate kinase, beta-haemoglobin and dihydrolipoamide dehydrogenase, and 14 proteins being increased, including cvHSP, aldehyde reductase, desmin, vimentin, chaperonin, cardiac and muscle myosin heavy chain. This suggests that lack of sarcolemmal integrity triggers a generally perturbed protein expression pattern in dystrophin-deficient fibres. However, the most significant finding was the dramatic increase in the small heat shock protein cvHSP, which was confirmed by 2-D immunoblotting. Confocal fluorescence microscopy revealed elevated levels of cvHSP in MDX fibres. An immunoblotting survey of other key heat shock proteins showed a differential expression pattern in MDX diaphragm. Stress response appears to be an important cellular mechanism in dystrophic muscle and may be exploitable as a new approach to counteract muscle degeneration.
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PMID:Proteome analysis of the dystrophin-deficient MDX diaphragm reveals a drastic increase in the heat shock protein cvHSP. 1683 51

Differentially expressed and immunogenic spore proteins of the Bacillus cereus group of bacteria, which includes Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis, were identified. Comparative proteomic profiling of their spore proteins distinguished the three species from each other as well as the virulent from the avirulent strains. A total of 458 proteins encoded by 232 open reading frames were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis for all the species. A number of highly expressed proteins, including elongation factor Tu (EF-Tu), elongation factor G, 60-kDa chaperonin, enolase, pyruvate dehydrogenase complex, and others exist as charge variants on two-dimensional gels. These charge variants have similar masses but different isoelectric points. The majority of identified proteins have cellular roles associated with energy production, carbohydrate transport and metabolism, amino acid transport and metabolism, posttranslational modifications, and translation. Novel vaccine candidate proteins were identified using B. anthracis polyclonal antisera from humans postinfected with cutaneous anthrax. Fifteen immunoreactive proteins were identified in B. anthracis spores, whereas 7, 14, and 7 immunoreactive proteins were identified for B. cereus and in the virulent and avirulent strains of B. thuringiensis spores, respectively. Some of the immunodominant antigens include charge variants of EF-Tu, glyceraldehyde-3-phosphate dehydrogenase, dihydrolipoamide acetyltransferase, Delta-1-pyrroline-5-carboxylate dehydrogenase, and a dihydrolipoamide dehydrogenase. Alanine racemase and neutral protease were uniquely immunogenic to B. anthracis. Comparative analysis of the spore immunome will be of significance for further nucleic acid- and immuno-based detection systems as well as next-generation vaccine development.
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PMID:Proteomic profiling and identification of immunodominant spore antigens of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis. 1695 62

Leishmania infantum is the etiologic agent of zoonotic visceral leishmaniasis (VL) in countries in the Mediterranean basin, where dogs are the domestic reservoirs and represent important elements in the transmission of the disease. Since the major focal areas of human VL exhibit a high prevalence of seropositive dogs, the control of canine VL could reduce the infection rate in humans. Efforts toward this have focused on the improvement of diagnostic tools, as well as on vaccine development. The identification of parasite antigens including suitable major histocompatibility complex (MHC) class I- and/or II-restricted epitopes is very important since disease protection is characterized by strong and long-lasting CD8+ T and CD4+ Th1 cell-dominated immunity. In the present study, total protein extract from late-log phase L. infantum promastigotes was analyzed by two-dimensional western blots and probed with sera from asymptomatic and symptomatic dogs. A total of 42 protein spots were found to differentially react with IgG from asymptomatic dogs, while 17 of these identified by Coommasie stain were extracted and analyzed. Of these, 21 proteins were identified by mass spectrometry; they were mainly involved in metabolism and stress responses. An in silico analysis predicted that the chaperonin HSP60, dihydrolipoamide dehydrogenase, enolase, cyclophilin 2, cyclophilin 40, and one hypothetical protein contain promiscuous MHCI and/or MHCII epitopes. Our results suggest that the combination of immunoproteomics and bioinformatics analyses is a promising method for the identification of novel candidate antigens for vaccine development or with potential use in the development of sensitive diagnostic tests.
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PMID:Identification of Immunoreactive Leishmania infantum Protein Antigens to Asymptomatic Dog Sera through Combined Immunoproteomics and Bioinformatics Analysis. 2690 26

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