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)

Small heat shock proteins (sHSPs) are widely distributed, and their function and diversity of structure have been much studied in the field of molecular chaperones. In plants, which frequently have to cope with hostile environments, sHSPs are much more abundant and diverse than in other forms of life. In response to high temperature stress, sHSPs of more than twenty kinds can make up more than 1% of soluble plant proteins. We isolated a genomic clone, NtHSP18.3, from Nicotiana tabacum that encodes the complete open reading frame of a cytosolic class I small heat shock protein. To investigate the function of NtHSP18.3 in vitro, it was overproduced in Escherichia coli and purified. The purified NtHSP18.3 had typical molecular chaperone activity as it protected citrate synthase and luciferase from high temperature-induced aggregation. When E. coli celluar proteins were incubated with NtHSP18.3, a large proportion of the proteins remained soluble at temperatures as high as 70 degrees . Native gel analysis suggested that NtHSP18.3 is a dodecameric oligomer as the form present and showing molecular chaperone activity at the condition tested. Binding of bis-ANS to the oligomers of NtHSP18.3 indicated that exposure of their hydrophobic surfaces increased as the temperature was raised. Taken together, our data suggested that NtHSP18.3 is a molecular chaperone that functions as a dodecameric complex and possibly in a temperature-induced manner.
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PMID:Biochemical analysis of a cytosolic small heat shock protein, NtHSP18.3, from Nicotiana tabacum. 1599 48

E7 oncoprotein is the major transforming activity in human papillomavirus and shares sequence and functional properties with adenovirus E1A and SV40 T-antigen, in particular by targeting the pRb tumor suppressor. HPV 16 E7 forms spherical oligomers that display chaperone activity in thermal denaturation and chemical refolding assays of two model polypeptide substrates, citrate synthase and luciferase, and it does so at substoichiometric concentrations. We show that the E7 chaperone can stably bind model polypeptides and hold them in a state with significant tertiary structure, but does not bind the fully native proteins. The E7 oligomers bind native in vitro translated pRb without the requirement of it being unfolded, since the N-terminal domain of E7 containing the LXCXE binding motif is exposed. The N-terminal domain of E7 can interfere with pRb binding but not with the chaperone activity, which requires the C-terminal domain, as in most reported E7 activities. The ability to bind up to approximately 72 molecules of pRb by the oligomeric E7 form could be important either for sequestering pRb from Rb-E2F complexes or for targeting it for proteasome degradation. Thus, both the dimeric and oligomeric chaperone forms of E7 can bind Rb and various potential targets. We do not know at present if the chaperone activity of E7 plays an essential role in the viral life cycle; however, a chaperone activity may explain the large number of cellular targets reported for this oncoprotein.
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PMID:Chaperone holdase activity of human papillomavirus E7 oncoprotein. 1641 41

The Drosophila melanogaster family of small heat shock proteins (sHsps) is composed of 4 main members (Hsp22, Hsp23, Hsp26, and Hsp27) that display distinct intracellular localization and specific developmental patterns of expression in the absence of stress. In an attempt to determine their function, we have examined whether these 4 proteins have chaperone-like activity using various chaperone assays. Heat-induced aggregation of citrate synthase was decreased from 100 to 17 arbitrary units in the presence of Hsp22 and Hsp27 at a 1:1 molar ratio of sHsp to citrate synthase. A 5 M excess of Hsp23 and Hsp26 was required to obtain the same efficiency with either citrate synthase or luciferase as substrate. In an in vitro refolding assay with reticulocyte lysate, more than 50% of luciferase activity was recovered when heat denaturation was performed in the presence of Hsp22, 40% with Hsp27, and 30% with Hsp23 or Hsp26. These differences in luciferase reactivation efficiency seemed related to the ability of sHsps to bind their substrate at 42 degrees C, as revealed by sedimentation analysis of sHsp and luciferase on sucrose gradients. Therefore, the 4 main sHsps of Drosophila share the ability to prevent heat-induced protein aggregation and are able to maintain proteins in a refoldable state, although with different efficiencies. The functional reasons for their distinctive cell-specific pattern of expression could reflect the existence of defined substrates for each sHsp within the different intracellular compartments.
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PMID:Differences in the chaperone-like activities of the four main small heat shock proteins of Drosophila melanogaster. 1657 29

Small heat shock proteins (sHSPs) are a ubiquitous class of molecular chaperones that interacts with substrates to prevent their irreversible insolubilization during denaturation. How sHSPs interact with substrates remains poorly defined. To investigate the role of the conserved C-terminal alpha-crystallin domain versus the variable N-terminal arm in substrate interactions, we compared two closely related dodecameric plant sHSPs, Hsp18.1 and Hsp16.9, and four chimeras of these two sHSPs, in which all or part of the N-terminal arm was switched. The efficiency of substrate protection and formation of sHSP-substrate complexes by these sHSPs with three different model substrates, firefly luciferase, citrate synthase, and malate dehydrogenase (MDH) provide new insights into sHSP/substrate interactions. Results indicate that different substrates have varying affinities for different domains of the sHSP. For luciferase and citrate synthase, the efficiency of substrate protection was determined by the identity of the N-terminal arm in the chimeric proteins. In contrast, for MDH, efficient protection clearly required interactions with the alpha-crystallin domain in addition to the N-terminal arm. Furthermore, we show that sHSP-substrate complexes with varying stability and composition can protect substrate equally, and substrate protection is not correlated with sHSP oligomeric stability for all substrates. Protection of MDH by the dimeric chimera composed of the Hsp16.9 N-terminal arm and Hsp18.1 alpha-crystallin domain supports the model that a dimeric form of the sHSP can bind and protect substrate. In total, results demonstrate that sHSP-substrate interactions are complex, likely involve multiple sites on the sHSP, and vary depending on substrate.
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PMID:The N-terminal arm of small heat shock proteins is important for both chaperone activity and substrate specificity. 1709 May 42

In previous studies, the only small HSPs that have been studied in Xenopus laevis are members of the HSP30 family. We now report the analysis of Xenopus HSP27, a homolog of the human small HSP, HSP27. To date the presence of both hsp30 and hsp27 genes has been demonstrated only in minnow and chicken. Xenopus HSP27 cDNA encodes a 213 aa protein that contains an alpha-crystallin domain as well as a polar C-terminal extension. Xenopus HSP27 shares 71% identity with chicken HSP24 but only 19% identity with Xenopus HSP30C. Northern blot analysis revealed that Xenopus HSP27 gene expression was developmentally regulated. Constitutive and heat shock-induced hsp27 mRNA accumulation was first detectable at the early tailbud stage while HSP27 protein was detected at the tadpole stage. Furthermore, hsp27 mRNA was enriched in selected tissues under both control and heat shock conditions. Whole mount in situ hybridization analysis detected the presence of this message in the lens vesicle, heart, head, somites, and tail region. Purified recombinant HSP27 protein displayed molecular chaperone properties since it had the ability to inhibit heat-induced aggregation of target proteins including citrate synthase, malate dehydrogenase and luciferase. Thus, Xenopus HSP27, like HSP30, is a developmentally-regulated heat-inducible molecular chaperone.
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PMID:Analysis of the expression and function of the small heat shock protein gene, hsp27, in Xenopus laevis embryos. 1726 55

When cells are submitted to an increase in temperature, heat shock proteins (Hsp) are synthesized to help heat stress resistance. Small Hsps, which are diverse and abundant in plants, have the major function of preventing irreversible protein aggregation. The diversity of small Hsps in plants is intriguing and characterization of their chaperone activity is important to understand plant tolerance to heat stress. A previous study showed that small Hsps, mainly represented by class I (cytosolic), correspond to about 5% of all sugarcane Expressed Sequencing Tags belonging to the molecular chaperone category. Here, we present biochemical and biophysical characterization of two sugarcane small Hsps from class I, which were named SsHsp17.2 and SsHsp17.9 according to their monomer molecular mass of 17.2 and 17.9 kDa, respectively. The recombinant proteins have identity of about 75% to each other and similar structural characteristics. However, their stability and their chaperone activity were not equivalent: SsHsp17.9 was more efficient in protecting citrate synthase and malate dehydrogenase from aggregation whereas SsHsp17.2 was more efficient in protecting luciferase from aggregation. There is only one region, which is located at the N-terminus, of low homology between these two proteins. Based on that and on previous works pointing to multiple sites, mainly at the N-terminus, involved with substrate specificity in small Hsps, we suggest that this specific region is one of these sites. In addition, this is the first report on the chaperone activity of sugarcane small Hsps.
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PMID:Biochemical and biophysical characterization of small heat shock proteins from sugarcane. Involvement of a specific region located at the N-terminus with substrate specificity. 1733 76

The Sgt1 protein is a binding partner of heat shock proteins such as Hsp90, Hsp70 or Hsc70. In this work we show that the level of Sgt1 is increased in HEp-2 cells exposed to heat shock or radicicol. The citrate synthase aggregation assay shows that Sgt1 attenuates aggregation of the enzyme induced by increased temperature as efficiently as p23, a known co-chaperone of Hsp90. We have cloned two fragments of the human Sgt1 gene promoter (-708/+98 and -351/+98) into pGL3-luciferase vector and found that both fragments generated a 2-fold increase in luciferase activity upon heat shock. Furthermore, electrophoretic mobility shift assay revealed binding of the HSF-1 transcription factor to the heat shock element in the proximal (-42/-2) Sgt1 gene promoter fragment. These results indicate that Sgt1 is a co-chaperone protein with an expression pattern matching that of the well known heat shock proteins.
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PMID:Sgt1 has co-chaperone properties and is up-regulated by heat shock. 1835 34

Regulation of cell division requires the concerted function of proteins and protein complexes that properly mediate cytoskeletal dynamics. NudC is an evolutionarily conserved protein of undetermined function that associates with microtubules and interacts with several key regulators of mitosis, such as polo-kinase 1 (Plk1) and dynein. NudC is essential for proper mitotic progression, and homologs have been identified in species ranging from fungi to humans. In this paper, we report the characterization of the Caenorhabditis elegans NudC homolog, NUD-1, as a protein exhibiting molecular chaperone activity. All NudC/NUD-1 proteins share a conserved p23/HSP20 domain predicted by three-dimensional modeling [Garcia-Ranea, Mirey, Camonis, Valencia, FEBS Lett 529(2-3):162-167, 2002]. We demonstrate that nematode NUD-1 is able to prevent the aggregation of two substrate proteins, citrate synthase (CS) and luciferase, at stoichiometric concentrations. Further, NUD-1 also protects the native state of CS from thermal inactivation by significantly reducing the inactivation rate of this enzyme. To further determine if NUD-1/substrate complexes were productive or simply "dead-end" unfolding intermediates, a luciferase refolding assay was utilized. Following thermal denaturation, rabbit reticulocyte lysate and ATP were added and luciferase activity measured. In the presence of NUD-1, nearly all of the luciferase activity was regained, indicating that unfolded intermediates complexed with NUD-1 could be refolded. These studies represent the first functional evidence for a member of this mitotically essential protein family as having chaperone activity and facilitates elucidation of the role such proteins play in chaperone complexes utilized in cell division. C. elegans NUD-1 is a member of an evolutionary conserved protein family of unknown function involved in the regulation of cytoskeletal dynamics. NUD-1 and its mammalian homolog, NudC, function with the dynein motor complex to ensure proper cell division, and knockdown or overexpression of these proteins leads to disruption of mitosis. In this paper, we show that NUD-1 possesses ATP-independent chaperone activity comparable to that of small heat shock proteins and cochaperones and that changes in phosphorylation state functionally alter chaperone activity in a phosphomimetic NUD-1 mutant.
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PMID:The microtubule-associated protein, NUD-1, exhibits chaperone activity in vitro. 1862 91

Partially or completely unfolded polypeptides are highly prone to aggregation due to nonspecific interactions between their exposed hydrophobic surfaces. Extracellular proteins are continuously subjected to stresses conditions, but the existence of extracellular chaperones remains largely unexplored. The results presented here demonstrate that one of the most abundant extracellular proteins, fibrinogen has chaperone-like activity. Fibrinogen can specifically bind to nonnative form of citrate synthase and inhibit its thermal aggregation and inactivation in an ATP-independent manner. Interestingly, fibrinogen maintains thermal-denatured luciferase in a refolding competent state allowing luciferase to be refolded in cooperation with rabbit reticulocyte lysate. Fibrinogen also inhibits fibril formation of yeast prion protein Sup35 (NM). Furthermore, fibrinogen rescues thermal-induced protein aggregation in the plasma of fibrinogen-deficient mice. Our studies demonstrate the chaperone-like activity of fibrinogen, which not only provides new insights into the extracellular chaperone protein system, but also suggests potential diagnostic and therapeutic approaches to fibrinogen-related pathological conditions.
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PMID:Fibrinogen has chaperone-like activity. 1905 6

Human fibrinogen is an important coagulation factor as well as an acute phase protein in the circulatory system. Fibrinogen-420 is distinguished from the conventional alpha chain of fibrinogen-340 by the presence of an additional 236-residue carboxyl terminus globular domain (alpha(E)C). The alpha(E)C domain of human fibrinogen-420 is a stable and early proteolytic cleavage product in the circulation. A genuine physiological function for alpha(E)C has not yet been established. Our study aims to characterize the novel chaperone-like activity of alpha(E)C. alpha(E)C efficiently protects a series of model proteins from thermally induced aggregation. Furthermore, alpha(E)C specifically recognizes the partially denatured form instead of the native form of citrate synthase (CS) and potentially protects it from thermally induced inactivation. The protective effect may result from formation of soluble complexes between alpha(E)C and partially denatured CS as tested by size exclusion column and electron microscope. In addition, alpha(E)C can keep the partially denatured luciferase in a folding competent state and help it refold in cooperation with rabbit reticulocyte lysate (RRL). Furthermore, alpha(E)C can also form complexes with thermally stressed plasma proteins. Our findings reveal the novel function of alpha(E)C as a chaperone-like protein, which not only provides new insights into the extracellular chaperone system but also has implications on the physiological and pathological relevance of fibrinogen.
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PMID:Alpha(E)C, the C-terminal extension of fibrinogen, has chaperone-like activity. 1928 87


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