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:2.3.3.1 (citrate synthase)
4,488 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The 90-kDa heat shock protein (Hsp90) is the most abundant molecular chaperone of the eukaryotic cytoplasm. Its cysteine groups participate in the interactions of Hsp90 with the heme-regulated eIF-2alpha kinase and molybdate, a stabilizer of Hsp90-protein complexes. In our present studies we investigated the reactivity of the sulfhydryl groups of Hsp90. Our data indicate that Hsp90 as well as two Hsp90 peptides containing Cys-521 and Cys-589/590 are able to reduce cytochrome c. The effect of Hsp90 can be blocked by sulfhydryl reagents including arsenite and cadmium, which indicates the involvement of the vicinal cysteines Cys589/590 in the reduction of cytochrome c. Hsp90 neither reduces the disulfide bonds of insulin nor possesses a NADPH:quinone oxidoreductase activity. Oxidizing conditions impair the chaperone activity of Hsp90 toward citrate synthase. The high and specific reactivity of Hsp90 cysteine groups toward cytochrome c may indicate a role of this chaperone in modulation of the redox status of the cytosol in resting and apoptotic cells.
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PMID:Reactive cysteines of the 90-kDa heat shock protein, Hsp90. 1114 36

Small heat shock proteins protect cells from stress presumably by acting as molecular chaperones. Here we report on the functional characterization of a developmentally regulated, heat-inducible member of the Xenopus small heat shock protein family, Hsp30C. An expression vector containing the open reading frame of the Hsp30C gene was expressed in Escherichia coli. These bacterial cells displayed greater thermoresistance than wild type or plasmid-containing cells. Purified recombinant protein, 30C, was recovered as multimeric complexes which inhibited heat-induced aggregation of either citrate synthase or luciferase as determined by light scattering assays. Additionally, 30C attenuated but did not reverse heat-induced inactivation of enzyme activity. In contrast to an N-terminal deletion mutant, removal of the last 25 amino acids from the C-terminal end of 30C severely impaired its chaperone activity. Furthermore, heat-treated concentrated solutions of the C-terminal mutant formed nonfunctional complexes and precipitated from solution. Immunoblot and gel filtration analysis indicated that 30C binds with and maintains the solubility of luciferase preventing it from forming heat-induced aggregates. Coimmunoprecipitation experiments suggested that the carboxyl region is necessary for 30C to interact with target proteins. These results clearly indicate a molecular chaperone role for Xenopus Hsp30C and provide evidence that its activity requires the carboxyl terminal region.
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PMID:Functional characterization of Xenopus small heat shock protein, Hsp30C: the carboxyl end is required for stability and chaperone activity. 1114 66

In the present study we investigated the substrate-binding characteristics of three members of the 90 kDa heat shock protein (HSP90) family, namely the alpha isoform of human HSP90 (HSP90alpha), human GRP94 (94 kDa glucose-regulated protein, a form of HSP90 from endoplasmic reticulum), and HtpG (the Escherichia coli homologue of HSP90) and the domain responsible for these characteristics. The recombinant forms of HSP90alpha, GRP94 and HtpG existed as dimers and became oligomerized at higher temperatures. Among the three family members, HtpG required the highest temperature (65 degrees C) for its transition to oligomeric forms. The precipitation of the substrate protein, glutathione S-transferase, which occurred at 55 degrees C, was efficiently prevented by the simultaneous presence of a sufficient amount of HSP90alpha or GRP94, but not by HtpG, which was still present as a dimer at that temperature. However, precipitation was stopped completely at 65-70 degrees C, at which temperature HtpG was oligomerized. Thus the transition of HSP90-family proteins to a state with self-oligomerization ability is essential for preventing the precipitation of substrate proteins. We then investigated the domain responsible for the substrate binding of HtpG on the basis of the three domain structures. The self-oligomerizing and substrate-binding activities towards glutathione S-transferase and citrate synthase were both located in a single domain, the N-terminal domain (residues 1-336) of HtpG. We therefore propose that the primary peptide-binding site is located in the N-terminal domain of HSP90-family proteins.
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PMID:Substrate-binding characteristics of proteins in the 90 kDa heat shock protein family. 1123 71

The small heat shock protein (smHSP) family has been extensively studied in eukaryotic cells. SmHSP assemble into large multimeric structures and possess chaperone activity that can prevent protein aggregation in vitro. Few studies on prokaryotic smHSP are actually available and no smHSP from lactic acid bacteria has been characterized at a biochemical level to date. Here we report on the Lo18 membrane-associated smHSP from the lactic acid bacterium Oenococcus oeni. Using size exclusion chromatography, nondenaturing pore-exclusion PAGE and in vitro and in vivo cross-linking experiments, the multimeric structure of Lol8 from O. oeni or expressed in Escherichia coli was investigated. In vitro, Lo18 is able to form a trimer and a higher oligomer which could be a dodecamer. Experiments strongly suggest that the same structures exist in vivo. First, Lo18 prevented thermal aggregation of citrate synthase and lactate dehydrogenase even at 60degreesC. These findings showed that the prokaryotic smHSP Lo18 can function as a molecular chaperone in vitro. Second, Lo18 did not protect lactate dehydrogenase from thermal inactivation and did not assist in enzymatic activity restoration after thermal aggregation, suggesting that other components may be needed for the refolding of the enzyme in an active conformation. Third, we showed that membrane association of Lo18 depends on the temperature upshift. Moreover, expression of this smHSP was induced by administration of a membrane fluidiser, the benzyl alcohol, suggesting that Lo18 expression could be regulated by the level of membrane fluidity.
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PMID:Biochemical and physiological studies of the small heat shock protein Lo18 from the lactic acid bacterium Oenococcus oeni. 1154 77

Rice (Oryza sativa) class I low-molecular mass (LMM) heat shock protein (HSP), Oshsp16.9, has been shown to be able to confer thermotolerance in Escherichia coli. To define the regions for this intriguing property, deletion mutants of this hsp have been constructed and overexpressed in E. coli XL1-blue cells after isopropyl beta-D-thioglactopyranoside induction. The deletion of amino acid residues 30 through 36 (PATSDND) in the N-terminal domain or 73 through 78 (EEGNVL) in the consensus II domain of Oshsp16.9 led to the loss of chaperone activities and also rendered the E. coli incapable of surviving at 47.5 degrees C. To further investigate the function of these two domains, we determined the light scattering changes of Oshsp16.9 mutant proteins at 320 nm under heat treatment either by themselves or in the presence of a thermosensitive enzyme, citrate synthase. It was observed that regions of amino acid residues 30 through 36 and 73 through 78 were responsible for stability of Oshsp16.9 and its interactions with other unfolded protein substrates, such as citrate synthase. Studies of two-point mutants of Oshsp16.9, GST-N74E73K and GST-N74E74K, indicate that amino acid residues 73 and 74 are an important part of the substrate-binding site of Oshsp16.9. Non-denaturing gel analysis of purified Oshsp16.9 revealed that oligomerization of Oshsp16.9 was necessary but not sufficient for its chaperone activity.
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PMID:Functional regions of rice heat shock protein, Oshsp16.9, required for conferring thermotolerance in Escherichia coli. 1184 69

Mycobacterium tuberculosis heat shock protein 16.3 (MTB HSP 16.3) accumulates as the dominant protein in the latent stationary phase of tuberculosis infection. MTB HSP 16.3 displays several characteristics of small heat shock proteins (sHsps): its expression is increased in response to stress, it protects against protein aggregation in vitro, and it contains the core 'alpha-crystallin' domain found in all sHsps. In this study we characterized the chaperone activity of recombinant MTB HSP 16.3 in several different assays and compared the results to those obtained with recombinant human alphaB-crystallin, a well characterized member of the sHsp family. Recombinant MTB HSP 16.3 was expressed in Escherichia coli and purified to apparent homogeneity. Similar to alphaB-crystallin, MTB HSP16.3 suppressed citrate synthase aggregation and in the presence of 3.5 mm ATP the chaperone activity was enhanced by twofold. ATP stabilized MTB HSP 16.3 against proteolysis by chymotrypsin, and no effect was observed with ATPgammaS, a nonhydrolyzable analog of ATP. Increased expression of MTB HSP 16.3 resulted in protection against thermal killing in E. coli at 48 degrees C. While the sequence similarity between human alphaB-crystallin and MTB HSP 16.3 is only 18%, these results suggest that the functional similarities between these proteins containing the core 'alpha-crystallin' domain are much closer.
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PMID:Functional similarities between the small heat shock proteins Mycobacterium tuberculosis HSP 16.3 and human alphaB-crystallin. 1195 82

A free-roaming dog in Okinawa island showed Anaplasma (Ehrlichia) platys-like inclusions within the platelets of peripheral blood samples. The inclusions were positive in indirect fluorescence test with anti-A. phagocytophila serum. The platelet count of the dog was 170,000 microl(-1). The sequence analysis of the 16S rRNA, citrate synthase and heat shock protein genes of DNA from the infected platelets confirmed that the inclusions were A. platys. This is the first detection of A. platys inclusions in dogs in Japan.
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PMID:Demonstration of Anaplasma (Ehrlichia) platys inclusions in peripheral blood platelets of a dog in Japan. 1244

The alpha isoform of human 90-kDa heat shock protein (HSP90alpha) is composed of three domains: the N-terminal (residues 1-400); middle (residues 401-615) and C-terminal (residues 621-732). The middle domain is simultaneously associated with the N- and C-terminal domains, and the interaction with the latter mediates the dimeric configuration of HSP90. Besides one in the N-terminal domain, an additional client-binding site exists in the C-terminal domain of HSP90. The aim of the present study is to elucidate the regions within the C-terminal domain responsible for the bindings to the middle domain and to a client protein, and to define the relationship between the two functions. A bacterial two-hybrid system revealed that residues 650-697 of HSP90alpha were essential for the binding to the middle domain. An almost identical region (residues 657-720) was required for the suppression of heat-induced aggregation of citrate synthase, a model client protein. Replacement of either Leu665-Leu666 or Leu671-Leu672 to Ser-Ser within the hydrophobic segment (residues 662-678) of the C-terminal domain caused the loss of bindings to both the middle domain and the client protein. The interaction between the middle and C-terminal domains was also found in human 94-kDa glucose-regulated protein. Moreover, Escherichia coli HtpG, a bacterial HSP90 homologue, formed heterodimeric complexes with HSP90alpha and the 94-kDa glucose-regulated protein through their middle-C-terminal domains. Taken together, it is concluded that the identical region including the hydrophobic segment of the C-terminal domain is essential for both the client binding and dimer formation of the HSP90-family molecular chaperone and that the dimeric configuration appears to be similar in the HSP90-family proteins.
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PMID:A hydrophobic segment within the C-terminal domain is essential for both client-binding and dimer formation of the HSP90-family molecular chaperone. 1249 85

The most likely animal source of a human case of cardiac disease in Washoe County, Nev., was identified by comparison of DNA sequences of three genes (citrate synthase gltA, 60-kDa heat shock protein gene groEL, and 16S rRNA gene) of Bartonella washoensis cultured from the human patient in question and of Bartonella isolates obtained from the following Nevada rodents: Peromyscus maniculatus (17 isolates), Tamias minimus (11 isolates), Spermophilus lateralis (3 isolates), and Spermophilus beecheyi (7 isolates). Sequence analyses of gltA amplicons obtained from Bartonella from the rodents demonstrated considerable heterogeneity and resulted in the identification of 16 genetic variants that were clustered within three groups in phylogenetic analysis. Each of the three groups was associated with a rodent genus, Peromyscus, Tamias, or Spermophilus: The gltA, 16S rRNA gene, and groEL sequences of a Bartonella isolate obtained from a California ground squirrel (S. beecheyi) were completely identical to homologous sequences of B. washoensis, strongly suggesting that these animals were the source of infection in the human case.
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PMID:Bartonella strains from ground squirrels are identical to Bartonella washoensis isolated from a human patient. 1257 61

To examine whether genes associated with cellular defense against oxidative stress are associated with insulin sensitivity, patients with type 2 diabetes (n = 7) and age-matched (n = 5) and young (n = 9) control subjects underwent a euglycemic-hyperinsulinemic clamp for 120 min. Muscle samples were obtained before and after the clamp and analyzed for heat shock protein (HSP)72 and heme oxygenase (HO)-1 mRNA, intramuscular triglyceride content, and the maximal activities of beta-hydroxyacyl-CoA dehydrogenase (beta-HAD) and citrate synthase (CS). Basal expression of both HSP72 and HO-1 mRNA were lower (P < 0.05) by 33 and 55%, respectively, when comparing diabetic patients with age-matched and young control subjects, with no differences between the latter groups. Both basal HSP72 (r = 0.75, P < 0.001) and HO-1 (r = 0.50, P < 0.05) mRNA expression correlated with the glucose infusion rate during the clamp. Significant correlations were also observed between HSP72 mRNA and both beta-HAD (r = 0.61, P < 0.01) and CS (r = 0.65, P < 0.01). HSP72 mRNA was induced (P < 0.05) by the clamp in all groups. Although HO-1 mRNA was unaffected by the clamp in both the young and age-matched control subjects, it was increased (P < 0.05) approximately 70-fold in the diabetic patients after the clamp. These data demonstrate that genes involved in providing cellular protection against oxidative stress are defective in patients with type 2 diabetes and correlate with insulin-stimulated glucose disposal and markers of muscle oxidative capacity. The data provide new evidence that the pathogenesis of type 2 diabetes involves perturbations to the antioxidant defense mechanism within skeletal muscle.
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PMID:Intramuscular heat shock protein 72 and heme oxygenase-1 mRNA are reduced in patients with type 2 diabetes: evidence that insulin resistance is associated with a disturbed antioxidant defense mechanism. 1294 74


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