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)

We have cloned, purified to homogeneity, and characterized as a molecular chaperone the Escherichia coli YedU protein. The purified protein shows a single band at 31 kDa on SDS-polyacrylamide gels and forms dimers in solution. Like other chaperones, YedU interacts with unfolded and denatured proteins. It promotes the functional folding of citrate synthase and alpha-glucosidase after urea denaturation and prevents the aggregation of citrate synthase under heat shock conditions. YedU forms complexes with the permanently unfolded protein, reduced carboxymethyl alpha-lactalbumin. In contrast to DnaK/Hsp70, ATP does not stimulate YedU-dependent citrate synthase renaturation and does not affect the interaction between YedU and unfolded proteins, and YedU does not display any peptide-stimulated ATPase activity. We conclude that YedU is a novel chaperone which functions independently of an ATP/ADP cycle.
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PMID:Characterization of the Escherichia coli YedU protein as a molecular chaperone. 1256 79

We initiated a proteomic study as part of a programme aimed at discovering novel functions of the plant cell wall. Cell-wall fragments isolated from cell-suspension cultures of Arabidopsis thaliana were stripped of protein sequentially using CaCl2 and a urea-based buffer. The protein fractions were separated by two-dimensional gel electrophoresis and individual proteins were identified by MS. We identified a number of proteins considered to be resident in other organelles but not the cell wall on the basis of their classical biological function. These included citrate synthase, which is known to be targeted to mitochondria, peroxisomes and glyoxysomes, and luminal binding protein, which is an ER (endoplasmic reticulum)-resident protein. Searches of the Arabidopsis database revealed that there are several genes encoding putative citrate synthase and luminal binding protein. We have also performed detailed analyses of the protein sequences and this paper shows how each one contains encrypted targeting information that results in the export of the protein to the extracellular matrix. We discuss the presence of alternative non-classical secretory pathways in plants.
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PMID:Proteomic analysis of the Arabidopsis cell wall reveals unexpected proteins with new cellular locations. 1515 77

UK114, the goat liver tumour antigen, is a member of a widely distributed family of conserved low-molecular-mass proteins (YER057c/YjgF/UK114), the function of which is ill understood. To the various orthologues diverse functions have been ascribed, such as translation inhibition, regulation of purine repressor or calpain activation. Owing to a limited sequence similarity to Hsp90 (heat-shock protein 90), they have also been proposed to be molecular chaperones; however, this has never been tested. In the present paper, we report the cloning and characterization of the Drosophila orthologue, DUK114. In brief, DUK114 had no effect that would have qualified it as a calpain activator. In contrast, it proved to be a very potent molecular chaperone in in vitro assays. In a heat-aggregation test, it significantly decelerated the formation of citrate synthase aggregates. In a reverse assay, the recovery of the enzyme from urea- and heat-induced denatured states was accelerated almost 3-fold. On a molar basis, the chaperone activity of the 15-kDa DUK114 is comparable with that of Hsp90, the almost 6-times-larger archetypal molecular chaperone. In similar assays, DUK114 was ineffective with Drosophila calpain A or calpain B. To test for its chaperone activity in vivo, DUK114 was transfected into Schneider (S2) cells; after heat shock, the number of viable non-transfected cells started to increase after a lag time; in the presence of DUK114, cell proliferation started at once. Our work is the first experimental evidence that DUK114, and possibly other members of this family, are molecular chaperones.
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PMID:DUK114, the Drosophila orthologue of bovine brain calpain activator protein, is a molecular chaperone. 1525 Aug 25

Since, like other osmolytes, proline can act as a protein stabilizer, we investigated the thermoprotectant properties of proline in vitro and in vivo. In vivo, elevated proline pools in Escherichia coli (obtained by altering the feedback inhibition by proline of gamma-glutamylkinase, the first enzyme of the proline biosynthesis pathway) restore the viability of a dnaK-deficient mutant at 42 degrees C, suggesting that proline can act as a thermoprotectant for E. coli cells. Furthermore, analysis of aggregated proteins in the dnaK-deficient strain at 42 degrees C by two-dimensional gel electrophoresis shows that high proline pools reduce the protein aggregation defect of the dnaK-deficient strain. In vitro, like other "chemical chaperones," and like the DnaK chaperone, proline protects citrate synthase against thermodenaturation and stimulates citrate synthase renaturation after urea denaturation. These results show that a protein aggregation defect can be compensated for by a single mutation in an amino acid biosynthetic pathway and that an ubiquitously producible chemical chaperone can compensate for a defect in one of the major chaperones involved in protein folding and aggregation.
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PMID:The chemical chaperone proline relieves the thermosensitivity of a dnaK deletion mutant at 42 degrees C. 1554 89

Two small heat shock proteins (sHsps), Hsp17.8 and Hsp17.1, were identified in the cyanobacterium Anabaena sp. PCC 7120. Recombinant Hsp17.8 and Hsp17.1 were overexpressed in Escherichia coli and characterized here. Hsp17.8 was purified by sequential chromatography on DEAE-Sepharose and Superose 6 10/300 column, and Hsp17.1 was purified by Superose 6 10/300 column in 4M urea. Size exclusion chromatography demonstrated that both purified proteins form large oligomers approximately 420kDa and 410kDa, respectively. Both Hsp17.8 and Hsp17.1 showed chaperone-like activity to protect citrate synthase (CS) from thermal aggregation at 43 degrees C. Furthermore, both proteins were found to form complexes with denatured CS at 45 degrees C. Our study also demonstrated that despite a high degree of sequence homology and similar subunit size, Hsp17.1 showed higher hydrophobicity indicated by 8-anilino-1-naphthalene sulfonate fluorescence and thus greater chaperone-like activity. This is the first report of characterization and comparison of an sHsp system containing two chaperones in cyanobacteria.
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PMID:Purification and characterization of two small heat shock proteins from Anabaena sp. PCC 7120. 1601 54

1. The activities of enzymes of the urea cycle, carbamoyl phosphate synthetase, ornithine transcarbamoylase, argininosuccinate synthetase, argininosuccinase (the last two comprising the arginine synthetase system) and arginase, were measured in the liver during development of the rat. All five enzymes exhibited relatively low activities in foetal liver and a rapid postnatal increase was found. The rate-limiting enzyme of urea synthesis in the rat, the condensing enzyme of the arginine synthetase system, showed the lowest activity at birth and the most rapid postnatal increase, a fivefold increase within 24hr. after birth. A second increase of activity was noted after the tenth day. These results suggest that the postnatal increase of arginine synthetase activity initiates the ability for urea synthesis in the rat. 2. Some factors influencing the development of the rate-limiting arginine synthetase system were studied in more detail. (a) Intraperitoneal administration of puromycin inhibited the postnatal increaseof the enzyme activity. (b) Starvation of newborn animals for 24hr. after birth had no effect on the postnatal development of the enzyme. (c) Bilateral adrenalectomy at birth caused a marked diminution in the postnatal increase of the enzyme activity and injections of triamcinolone were effective in preventing the effect of adrenalectomy. (d) Administration of triamcinolone alone had a marked stimulatory effect on the postnatal development of this enzyme. (e) Premature and postmature birth had virtually no effect on the developmental pattern of the arginine synthetase activity, suggesting that the increase of this enzyme activity after birth is not initiated by the birth process.
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PMID:Factors influencing the development of urea-synthesizing enzymes in rat liver. 1674 4

Sodium fluoroacetate was introduced as a rodenticide in the US in 1946. However, its considerable efficacy against target species is offset by comparable toxicity to other mammals and, to a lesser extent, birds and its use as a general rodenticide was therefore severely curtailed by 1990. Currently, sodium fluoroacetate is licensed in the US for use against coyotes, which prey on sheep and goats, and in Australia and New Zealand to kill unwanted introduced species. The extreme toxicity of fluoroacetate to mammals and insects stems from its similarity to acetate, which has a pivotal role in cellular metabolism. Fluoroacetate combines with coenzyme A (CoA-SH) to form fluoroacetyl CoA, which can substitute for acetyl CoA in the tricarboxylic acid cycle and reacts with citrate synthase to produce fluorocitrate, a metabolite of which then binds very tightly to aconitase, thereby halting the cycle. Many of the features of fluoroacetate poisoning are, therefore, largely direct and indirect consequences of impaired oxidative metabolism. Energy production is reduced and intermediates of the tricarboxylic acid cycle subsequent to citrate are depleted. Among these is oxoglutarate, a precursor of glutamate, which is not only an excitatory neurotransmitter in the CNS but is also required for efficient removal of ammonia via the urea cycle. Increased ammonia concentrations may contribute to the incidence of seizures. Glutamate is also required for glutamine synthesis and glutamine depletion has been observed in the brain of fluoroacetate-poisoned rodents. Reduced cellular oxidative metabolism contributes to a lactic acidosis. Inability to oxidise fatty acids via the tricarboxylic acid cycle leads to ketone body accumulation and worsening acidosis. Adenosine triphosphate (ATP) depletion results in inhibition of high energy-consuming reactions such as gluconeogenesis. Fluoroacetate poisoning is associated with citrate accumulation in several tissues, including the brain. Fluoride liberated from fluoroacetate, citrate and fluorocitrate are calcium chelators and there are both animal and clinical data to support hypocalcaemia as a mechanism of fluoroacetate toxicity. However, the available evidence suggests the fluoride component does not contribute. Acute poisoning with sodium fluoroacetate is uncommon. Ingestion is the major route by which poisoning occurs. Nausea, vomiting and abdominal pain are common within 1 hour of ingestion. Sweating, apprehension, confusion and agitation follow. Both supraventricular and ventricular arrhythmias have been reported and nonspecific ST- and T-wave changes are common, the QTc may be prolonged and hypotension may develop. Seizures are the main neurological feature. Coma may persist for several days. Although several possible antidotes have been investigated, they are of unproven value in humans. The immediate, and probably only, management of fluoroacetate poisoning is therefore supportive, including the correction of hypocalcaemia.
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PMID:Sodium fluoroacetate poisoning. 1728 93

Phosphorylation appears to be one of the modulators of chaperone functions of small heat shock proteins. However, the role of phosphorylation is not completely understood. We have investigated the structural and functional consequences of a phosphorylation-mimicking mutation in alpha B-crystallin, a small heat shock protein with chaperone activity. We have used a phosphorylation-mimicking mutant, 3D alpha B-crystallin, in which all the three phosphorylatable serine residues are replaced with aspartic acid. 3D alpha B-Crystallin showed enhanced chaperone-like activity towards DTT-induced aggregation of insulin, heat-induced aggregation of citrate synthase and SDS-induced amyloid fibril formation of alpha-synuclein. Fluorescence and circular dichroism spectroscopic studies showed that 3D alpha B-crystallin exhibits lower stability towards urea-induced denaturation compared to alpha B-crystallin. Subunit exchange studies using fluorescence resonance energy transfer showed that 3D alpha B-crystallin exhibits an observable increase in subunit exchange compared to alpha B-crystallin. Since only part of alpha B-crystallin is phosphorylated in vivo, our subunit exchange studies indicate that formation of mixed oligomers between the unphosphorylated and phosphorylated subunits are likely to play a role in vivo. Our study shows that mixed-oligomer formation modulates the chaperone-like activity. We propose that the degree of phosphorylation of the alpha B-crystallin oligomers and temperature are key modulators to achieve a wide range of chaperone capabilities of the small heat shock protein, alpha -crystallin.
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PMID:Effect of phosphorylation on alpha B-crystallin: differences in stability, subunit exchange and chaperone activity of homo and mixed oligomers of alpha B-crystallin and its phosphorylation-mimicking mutant. 1806 12

Experimental metabolic alkalosis is known to stimulate whole-animal urea production and active ion secretion by the rectal gland in the dogfish shark. Furthermore, recent evidence indicates that a marked alkaline tide (systemic metabolic alkalosis) follows feeding in this species and that the activities of the enzymes of the ornithine-urea cycle (OUC) for urea synthesis in skeletal muscle and liver and of energy metabolism and ion transport in the rectal gland are increased at this time. We therefore evaluated whether alkalosis and/or NaCl/volume loading (which also occurs with feeding) could serve as a signal for activation of these enzymes independent of nutrient loading. Fasted dogfish were infused for 20 h with either 500 mmol L(-1) NaHCO3 (alkalosis + volume expansion) or 500 mmol L(-1) NaCl (volume expansion alone), both isosmotic to dogfish plasma, at a rate of 3 mL kg(-1) h(-1). NaHCO3 infusion progressively raised arterial pH to 8.28 (control = 7.85) and plasma [HCO3-] to 20.8 mmol L(-1) (control = 4.5 mmol L(-1)) at 20 h, with unchanged arterial P(CO2), whereas NaCl/volume loading had no effect on blood acid-base status. Rectal gland Na+,K+-ATPase activity was increased 50% by NaCl loading and more than 100% by NaHCO3 loading, indicating stimulatory effects of both volume expansion and alkalosis. Rectal gland lactate dehydrogenase activity was elevated 25% by both treatments, indicating volume expansion effects only, whereas neither treatment increased the activities of the aerobic enzymes citrate synthase, NADP-isocitrate dehydrogenase, or the ketone body-utilizing enzyme beta-hydroxybutyrate dehydrogenase in the rectal gland or liver. The activity of ornithine-citrulline transcarbamoylase in skeletal muscle was doubled by NaHCO3 infusion, but neither treatment altered the activities of other OUC-related enzymes (glutamine synthetase, carbamoylphosphate synthetase III). We conclude that both the alkaline tide and salt loading/volume expansion act as signals to activate some but not all of the elevated metabolic pathways and ionoregulatory mechanisms needed during processing of a meal.
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PMID:Is the alkaline tide a signal to activate metabolic or ionoregulatory enzymes in the dogfish shark (Squalus acanthias)? 1841 54

YajL is the closest prokaryotic homolog of the parkinsonism-associated protein DJ-1 (40% sequence identity and similar three-dimensional structure), a protein of unknown function involved in the cellular response to oxidative stress. We report here that a yajL mutant of Escherichia coli displays an increased sensitivity to oxidative stress. It also exhibits a protein aggregation phenotype in aerobiosis, but not in anaerobiosis or in aerobic cells overexpressing superoxide dismutase, suggesting that protein aggregation depends on the presence of reactive oxygen species produced by respiratory chains. The protein aggregation phenotype of the yajL mutant, which can be rescued by the wild-type yajL gene, but not by the corresponding cysteine 106 mutant allele, is similar to that of multiple mutants deficient in superoxide dismutases and catalases, although intracellular hydrogen peroxide levels were not increased in the yajL mutant, suggesting that protein aggregation in this strain does not result from a hydrogen peroxide detoxification defect. Aggregation-prone proteins included 17 ribosomal proteins, the ATP synthase beta subunit, flagellin, and the outer membrane proteins OmpA and PAL; all of them are part of multiprotein complexes, suggesting that YajL might be involved in optimal expression of these complexes, especially during oxidative stress. YajL stimulated the renaturation of urea-unfolded citrate synthase and the solubilization of the urea-unfolded ribosomal proteins S1 and L3 and was more efficient as a chaperone in its oxidized form than in its reduced form. The mRNA levels of several aggregated proteins of the yajL mutant were severely affected, suggesting that YajL also acts at the level of gene expression. These two functions of YajL might explain the protein aggregation phenotype of the yajL mutant.
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PMID:Protein aggregation in a mutant deficient in yajL, the bacterial homolog of the Parkinsonism-associated protein DJ-1. 2012 4


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