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)

Encysted embryos of the primitive crustacean Artemia franciscana are among the most resistant of all multicellular eukaryotes to environmental stress, in part due to massive amounts of a small heat shock/alpha-crystallin protein (p26) that acts as a molecular chaperone. These embryos also contain very large amounts of the disaccharide trehalose, well known for its ability to protect macromolecules and membranes against damage due to water removal and temperature extremes. Therefore, we looked for potential interactions between trehalose and p26 in the protection of a model substrate, citrate synthase (CS), against heat denaturation and aggregation and in the restoration of activity after heating in vitro. Both trehalose and p26 decreased the aggregation and irreversible inactivation of CS at 43 degrees C. At approximate physiological concentrations (0.4 M), trehalose did not interfere with the ability of p26 to assist in the reactivation of CS after heating, but higher concentrations (0.8 M) were inhibitory. We also showed that CS and p26 interact physically during heating and that trehalose interferes with complex formation and disrupts CS-p26 complexes that form at high temperatures. We suggest from these results that trehalose may act as a "release factor," freeing folding intermediates of CS that p26 can chaperone to the native state. Trehalose and p26 can act synergistically in vitro, during and after thermal stress, suggesting that these interactions also occur in vivo.
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PMID:Influence of trehalose on the molecular chaperone activity of p26, a small heat shock/alpha-crystallin protein. 1159 74

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

Oligomerization into multimeric complexes is a prerequisite for the chaperone function of almost all alpha-crystallin type heat shock proteins (alpha-Hsp), but the molecular details of complex assembly are poorly understood. The alpha-Hsp proteins from Bradyrhizobium japonicum are suitable bacterial models for structure-function studies of these ubiquitous stress proteins. They fall into two distinct classes, A and B, display chaperone activity in vitro and form oligomers of approximately 24 subunits. We constructed 19 derivatives containing truncations or point mutations within the N- and C-terminal regions and analyzed them by gel filtration, citrate synthase assay and coaffinity purification. Truncation of more than the initial few amino acids of the N-terminal region led to the formation of distinct dimeric to octameric structures devoid of chaperone activity. In the C-terminal extension, integrity of an isoleucine-X-isoleucine (I-X-I) motif was imperative for alpha-Hsp functionality. This I-X-I motif is one of the characteristic consensus motifs of the alpha-Hsp family, and here we provide experimental evidence of its structural and functional importance. alpha-Hsp proteins lacking the C-terminal extension were inactive, but still able to form dimers. Here, we demonstrate that the central alpha-crystallin domain alone is not sufficient for dimerization. Additional residues at the end of the N-terminal region were required for the assembly of two subunits.
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PMID:A critical motif for oligomerization and chaperone activity of bacterial alpha-heat shock proteins. 1213 98

HSV-2 R1, the R1 subunit of herpes simplex virus (HSV) ribonucleotide reductase, protects cells against apoptosis. Here, we report the presence in HSV-2 R1 of a stretch exhibiting similarity to the alpha-crystallin domain of the small heat shock proteins, a domain known to be important for oligomerization and cytoprotective activities of these proteins. Also, the HSV-2 R1 protein, which forms multimeric structures in the absence of nucleotide, displayed chaperone ability as good as Hsp27 in a thermal denaturation assay using citrate synthase. In contrast, mammalian R1, which does not contain an alpha-crystallin domain, has neither chaperone nor anti-apoptotic activity. Thus, we propose that the chaperone activity of HSV-2 R1 could play an important role in viral pathogenesis.
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PMID:The R1 subunit of herpes simplex virus ribonucleotide reductase has chaperone-like activity similar to Hsp27. 1280 78

A newly identified 22 kDa protein that interacts with Hsp27 (heat-shock protein 27) was shown to possess the characteristic alpha-crystallin domain, hence named Hsp22, and categorized as a member of the sHsp (small Hsp) family. Independent studies from different laboratories reported the protein with different names such as Hsp22, H11 kinase, E2IG1 and HspB8. We have identified, on the basis of the nucleotide sequence analysis, putative heat-shock factor 1 binding sites upstream of the Hsp22 translation start site. We demonstrate that indeed Hsp22 is heat-inducible. We show, in vitro, chaperone-like activity of Hsp22 in preventing dithiothreitol-induced aggregation of insulin and thermal aggregation of citrate synthase. We have cloned rat Hsp22, overexpressed and purified the protein to homogeneity and studied its structural and functional aspects. We find that Hsp22 fragments on storage. MS analysis of fragments suggests that the fragmentation might be due to the presence of labile peptide bonds. We have established conditions to improve its stability. Far-UV CD indicates a randomly coiled structure for Hsp22. Quaternary structure analyses by glycerol density-gradient centrifugation and gel filtration chromatography show that Hsp22 exists as a monomer in vitro, unlike other members of the sHsp family. Hsp22 exhibits significantly exposed hydrophobic surfaces as reported by bis-8-anilinonaphthalene-l-sulphonic acid fluorescence. We find that the chaperone-like activity is temperature dependent. Thus Hsp22 appears to be a true member of the sHsp family, which exists as a monomer in vitro and exhibits chaperone-like activity.
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PMID:Mammalian Hsp22 is a heat-inducible small heat-shock protein with chaperone-like activity. 1503 Mar 16

A hallmark of alpha-crystallin-type small heat shock proteins (sHsps) is their highly dynamic oligomeric structure which promotes intermolecular interactions involved in subunit exchange and substrate binding (chaperone-like activity). We studied the oligomeric features of two classes of bacterial sHsps by size exclusion chromatography and nanoelectrospray mass spectrometry. Proteins of both classes formed large complexes that rapidly dissociated upon dilution and at physiologically relevant heat shock temperatures. As the secondary structure was not perturbed, temperature- and concentration-dependent dissociations were fully reversible. Complexes formed between sHsps and the model substrate citrate synthase were stable and exceeded the size of sHsp oligomers. Small Hsps, mutated in a highly conserved glycine residue at the C-terminal end of the alpha-crystallin domain, formed labile complexes that disassembled more readily than the corresponding wild-type proteins. Reduced complex stability coincided with reduced chaperone activity.
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PMID:Temperature and concentration-controlled dynamics of rhizobial small heat shock proteins. 1518 65

Artemia franciscana embryos undergo encystment, developmental arrest and diapause, the last characterized by profound metabolic dormancy and extreme stress resistance. Encysted embryos contain an abundant small heat shock protein termed p26, a molecular chaperone that undoubtedly has an important role in development. To understand better the role of p26 in Artemia embryos, the structural and functional characteristics of full-length and truncated p26 expressed in Escherichia coli and COS-1 cells were determined. p26 chaperone activity declined with increasing truncation of the protein, and those deletions with the greatest adverse effect on protection of citrate synthase during thermal stress had the most influence on oligomerization. When produced in either prokaryotic or eukaryotic cells the p26 alpha-crystallin domain consisting of amino acid residues 61-152 existed predominantly as monomers, and p26 variants lacking the amino-terminal domain but with intact carboxyl-terminal extensions were mainly monomers and dimers. The amino terminus was, therefore, required for efficient dimer formation. Assembly of higher order oligomers was enhanced by the carboxyl-terminal extension, although removing the 10 carboxyl-terminal residues had relatively little effect on oligomerization and chaperoning. Full-length and carboxyl-terminal truncated p26 resided in the cytoplasm of transfected COS-1 cells; however, variants missing the complete amino-terminal domain and existing predominantly as monomers/dimers entered the nuclei. A mechanism whereby oligomer disassembly assisted entry of p26 into nuclei was suggested, this of importance because p26 translocates into Artemia embryo nuclei during development and stress. However, when examined in Artemia, the p26 oligomer size was unchanged under conditions that allowed movement into nuclei, suggesting a process more complex than just oligomer dissociation.
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PMID:Oligomerization, chaperone activity, and nuclear localization of p26, a small heat shock protein from Artemia franciscana. 1525 52

Small heat shock proteins (sHsps) form large oligomers that are characterised by their dynamic behaviour, e.g., complex disassembly/reassembly and extensive subunit exchange. These processes are interrelated with sHsp/substrate interaction. sHsps bind a broad spectrum of unrelated substrate proteins under denaturing conditions. Detailed knowledge about the binding process and regions critical for sHsp/substrate interaction is missing. In this study, we screened cellulose-bound peptide spot libraries derived from a bacterial sHsp and the model-substrate citrate synthase to detect oligomerisation and substrate interaction sites, respectively. In line with previous results, it was demonstrated that multiple contacts involving the N- and C-terminal extensions and the central alpha-crystallin domain are required for oligomerisation. Incubation of the citrate synthase membrane with sHsps revealed a putative substrate interaction site. A soluble peptide with the sequence RTKYWELIYEDCMDL (CS(191-205)) corresponding to that site inhibited chaperone activity of sHsps, presumably by blocking their substrate-binding sites.
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PMID:Detection of oligomerisation and substrate recognition sites of small heat shock proteins by peptide arrays. 1553 Apr 6

Protein pin arrays identified seven interactive sequences for chaperone activity in human alphaB crystallin using natural lens proteins, beta(H) crystallin and gammaD crystallin, and in vitro chaperone target proteins, alcohol dehydrogenase and citrate synthase. The N-terminal domain contained two interactive sequences, (9)WIRRPFFPFHSP(20) and (43)SLSPFYLRPPSFLRAP(58). The alpha crystallin core domain contained four interactive sequences, (75)FSVNLDVK(82) (beta3), (113)FISREFHR(120), (131)LTITSSLS(138) (beta8), and (141)GVLTVNGP(148) (beta9). The C-terminal domain contained one interactive sequence, (157)RTIPITRE(164), that included the highly conserved I-X-I/V motif. Two interactive sequences, (73)DRFSVNLDVKHFS(85) and (131)LTITSSLSDGV(141), belonging to the alpha crystallin core domain were synthesized as peptides and assayed for chaperone activity in vitro. Both synthesized peptides inhibited the thermal aggregation of beta(H) crystallin, alcohol dehydrogenase, and citrate synthase in vitro. Five of the seven chaperone sequences identified by the pin arrays overlapped with sequences identified previously as sequences for subunit-subunit interactions in human alphaB crystallin. The results suggested that interactive sequences in human alphaB crystallin have dual roles in subunit-subunit assembly and chaperone activity.
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PMID:Interactive domains for chaperone activity in the small heat shock protein, human alphaB crystallin. 1627 33

Knowledge of the interactive domains on the surface of small heat shock proteins (sHSPs) is necessary for understanding the assembly of complexes and the activity as molecular chaperones. The primary sequences of 26 sHSP molecular chaperones were aligned and compared. In the interactive beta3 sequence, 73DRFSVNLDVKHFS85 of human alphaB crystallin, Ser-76, Asn-78, Lys-82, and His-83 were identified as nonconserved residues on the exposed surface of the alpha crystallin core domain. Site-directed mutagenesis produced the mutant alphaB crystallins: S76E, N78G, K82Q, and H83F. Domain swapping with homologous beta3 sequences, 32EKFEVGLDVQFFT44 from Caenorhabditis elegans sHSP12.2 or 69DKFVIFLDVKHFS81 from alphaA crystallin, resulted in the mutant alphaB crystallins, CE1 and alphaA1, respectively. Decreased chaperone activity was observed with the point mutants N78G, K82Q, and H83F and with the mutant, CE1, in aggregation assays using betaL crystallin, alcohol dehydrogenase (ADH), or citrate synthase (CS). The S76E mutant had minimal effect on chaperone activity, and domain swapping with alphaA crystallin had no effect on chaperone activity. The mutations that resulted in altered chaperone activity, produced minimal modification to the secondary, tertiary, and quaternary structure of human alphaB crystallin as determined by ultraviolet circular dichroism spectroscopy, chymotrypsin proteolysis, and size exclusion chromatography. Chaperone activity was influenced by the amount of unfolding of the target proteins and independent of complex size. The results characterized the importance of the exposed side chains of Glu-78, Lys-82, and His-83 in the interactive beta3 sequence of the alpha crystallin core domain in alphaB crystallin for chaperone function.
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PMID:The function of the beta3 interactive domain in the small heat shock protein and molecular chaperone, human alphaB crystallin. 1681 25


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