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)

Calnexin (CNX) is a membrane protein of the endoplasmic reticulum that has been defined primarily as a lectin, yet is capable of functioning as a molecular chaperone with non-glycosylated proteins in vitro. Here, we assess the relative contributions of the oligosaccharide- and polypeptide-binding sites of CNX to its in vitro chaperone functions by comparing it with the Hsp70 chaperone of the endoplasmic reticulum, BiP. Both proteins were equally effective in preventing the aggregation of non-glycosylated citrate synthase, indicating that the polypeptide-binding site of CNX is capable of functioning at a level similar to that of Hsp70. However, when confronted with glycoprotein substrates, the lectin site of CNX provided a significant advantage over BiP in suppressing aggregation. CNX also cooperated with BiP and the J domain of Sec63p in the ATP-dependent refolding of glycoprotein and non-glycosylated substrates. The lectin site of CNX was essential for refolding of the glycoprotein. These findings reinforce the function of CNX as a bona fide chaperone and illustrate how its lectin site confers advantages relative to other chaperones when confronted with glycoprotein substrates.
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PMID:Relationship between calnexin and BiP in suppressing aggregation and promoting refolding of protein and glycoprotein substrates. 1151 79

Calnexin is a membrane-bound lectin of the endoplasmic reticulum (ER) that binds transiently to newly synthesized glycoproteins. By interacting with oligosaccharides of the form Glc(1)Man(9)GlcNAc(2), calnexin enhances the folding of glycoprotein substrates, retains misfolded variants in the ER, and in some cases participates in their degradation. Calnexin has also been shown to bind polypeptides in vivo that do not possess a glycan of this form and to function in vitro as a molecular chaperone for nonglycosylated proteins. To test the relative importance of the lectin site compared with the polypeptide-binding site, we have generated six calnexin mutants defective in oligosaccharide binding using site-directed mutagenesis. Expressed as glutathione S-transferase fusions, these mutants were still capable of binding ERp57, a thiol oxidoreductase, and preventing the aggregation of a nonglycosylated substrate, citrate synthase. They were, however, unable to bind Glc(1) Man(9)GlcNAc(2) oligosaccharide and were compromised in preventing the aggregation of the monoglucosylated substrate jack bean alpha-mannosidase. Two of these mutants were then engineered into full-length calnexin for heterologous expression in Drosophila cells along with the murine class I histocompatibility molecules K(b) and D(b) as model glycoproteins. In this system, lectin site-defective calnexin was able to replace wild type calnexin in forming a complex with K(b) and D(b) heavy chains and preventing their degradation. Thus, at least for class I molecules, the lectin site of calnexin is dispensable for some of its chaperone functions.
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PMID:Lectin-deficient calnexin is capable of binding class I histocompatibility molecules in vivo and preventing their degradation. 1469 98

The calnexin homologue (Cne1p) of Saccharomyces cerevisiae was expressed in Escherichia coli to evaluate its chaperone function. The chaperone function was examined as to the effects on the suppression of thermal denaturation and the enhancement of refolding, using citrate synthase (CS) as a nonspecific chaperone substrate. Cne1p effectively suppressed the thermal denaturation of CS and enhanced the refolding of thermally or chemically denatured CS in a concentration-dependent manner. In addition, the chaperone function of Cne1p was greatly affected in the presence of monoglucosylated oligosaccharides (G1M9) that specifically bind to the lectin site. These results indicated that Cne1p functions as a molecular chaperone in Saccharomyces cerevisiae.
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PMID:Expression and characterization of Saccharomyces cerevisiae Cne1p, a calnexin homologue. 1517

Cne1p, a calnexin homologue from Saccharomyces cerevisiae, has been shown to possess a conserved P-domain and lectin site as mammalian calnexin. The effect of P-domain and lectin site on the function of Cne1p was investigated in vitro using recombinant P-domain, P-domain deletion mutant of Cne1p, and lectin site mutant of Cne1ps (E181A and E398A) The binding of monoglucosylated oligosaccharide (G1M9) with Cne1p was clearly demonstrated using lectin site mutants. The P-domain deletion mutant and the letcin site mutants partially decreased the ability to suppress the aggregation of citrate synthase (CS) and chicken egg yolk immunoglobulin at levels different from Cne1p. Furthermore, the P-domain deletion mutant and the lectin site mutants decreased the ability to enhance the refolding of CS. These results suggest that the cooperation between the P-domain and the lectin site are important for the complete function of Cne1p. Thus, we conclude that P-domain in cooperation with the lectin site of Cne1p functions as a chaperone.
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PMID:P-domain and lectin site are involved in the chaperone function of Saccharomyces cerevisiae calnexin homologue. 1525 57

Recently, it became clear that aminoglycoside antibiotics affect protein-protein interactions involving protein disulfide isomerase as well as protein synthesis in the endoplasmic reticulum. In this study, we used affinity column chromatography to screen gentamicin-binding proteins in microsomes derived from bovine kidney in order to learn about the possible mechanisms of gentamicin-associated nephrotoxicity. One of the gentamicin-binding proteins was identified as calreticulin (CRT) by N-terminal amino acid sequence analysis. Interestingly, gentamicin inhibited the chaperone and oxidative refolding activities of CRT when N-glycosylated substrates such as alpha1-antitrypsin and alpha-mannosidase were used as substrates, but it did not inhibit the chaperone activity of CRT when unglycosylated citrate synthase was used. Moreover, CRT suppressed the aggregation of deglycosylated and denatured alpha-mannosidase, but gentamicin did not inhibit its chaperone activity. Experiments with domain mutants suggest that the lectin site of CRT is the main target for gentamicin binding and that binding of gentamicin to this site inhibits the chaperone activity of CRT.
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PMID:Gentamicin binds to the lectin site of calreticulin and inhibits its chaperone activity. 1535 34

Calreticulin (CRT) is a soluble molecular chaperone of the endoplasmic reticulum that functions to promote protein folding as well as to retain misfolded proteins. Similar to its membrane-bound paralog calnexin (CNX), CRT is a lectin that preferentially interacts with glycoproteins bearing Glc1Man5-9GlcNAc2 oligosaccharides. Although the lectin site of CNX has been delineated through X-ray crystallographic and mutagenic studies, the corresponding site for CRT has not been as well characterized. To address this issue, we attempted to construct lectin-deficient CRT mutants, using the structure of CNX as a guide to identify potential oligosaccharide-binding residues. Mutation of 4 such CRT residues (Y109, K111, Y128, D317) completely abrogated oligosaccharide binding. In contrast, mutation of CRT residues M131 and D160, which correspond to important residues in the lectin site of CNX, had no effect on oligosaccharide binding. These findings suggest that the organization of the lectin site in CRT largely resembles that of CNX but is not identical. The deficiency in oligosaccharide binding by the mutants was not due to misfolding because they exhibited wild-type protease digestion patterns, were capable of binding the thiol oxidoreductase ERp57, and functioned just as efficiently as wild-type CRT in suppressing the aggregation of the nonglycosylated substrate citrate synthase. However, they were impaired in their ability to suppress the aggregation of the glycosylated substrate jack bean alpha-mannosidase. This provides the first direct demonstration of the importance of CRT's lectin site in suppressing the aggregation of nonnative glycoproteins.
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PMID:Delineation of the lectin site of the molecular chaperone calreticulin. 1618 69

Clusterin is the first well characterized, constitutively secreted extracellular chaperone that binds to exposed regions of hydrophobicity on non-native proteins. It may help control the folding state of extracellular proteins by targeting them for receptor-mediated endocytosis and intracellular lysosomal degradation. A notable feature of secreted clusterin is its heavy glycosylation. Although carbohydrate comprises approximately 20-25% of the total mass of the mature molecule, its function is unknown. Results from the current study demonstrate that deglycosylation of human serum clusterin had little effect on its overall secondary structure content but produced a small increase in solvent-exposed hydrophobicity and enhanced the propensity of the molecule to aggregate in solution. These changes were associated with increased binding to a variety of ligands but did not substantially impact the ability of clusterin to inhibit heat-induced precipitation of citrate synthase. Evidence suggesting that the normally conjugated sugars are important in the interaction of secreted clusterin with a lectin-type receptor on liver cells is also presented. Bulk expression of fully processed, glycosylated clusterin in mammalian cells is difficult, often producing inappropriately disulfide-bonded high molecular weight aggregates; this has hampered previous studies aimed at identifying those regions of the molecule important in its chaperone action. The current results suggest that it may be possible in the future to study the structure and chaperone function of clusterin using recombinant protein (lacking sugars) conveniently bulk-expressed in bacteria.
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PMID:Effects of glycosylation on the structure and function of the extracellular chaperone clusterin. 1726 Sep 71

Animals native to high altitude must overcome the constraining effects of hypoxia on tissue O₂ supply to support routine metabolism, thermoregulation in the cold, and exercise. Deer mice (Peromyscus maniculatus) native to high altitude have evolved an enhanced aerobic capacity in hypoxia, along with increased capillarity and oxidative capacity of locomotory muscle. Here, we examined whether exposure to chronic hypoxia during development or adulthood affects muscle phenotype. Deer mice from a highland population were bred in captivity at sea level, and exposed to normoxia or one of four treatments of hypobaric hypoxia (12kPa O₂, simulating hypoxia at ~4300m): adult hypoxia (6-8weeks), post-natal hypoxia (birth to adulthood), pre-natal hypoxia (before conception to adulthood), and parental hypoxia (in which mice were conceived and raised in normoxia, but their parents were previously exposed to hypoxia). Litter size was similar across treatments, and pups survived the hypoxia exposures and grew to similar body masses at ~6-8months of age. Hypoxia had no effect on the masses of gastrocnemius and soleus muscles. There was a strong concordance between two distinct histological methods for staining capillaries in the gastrocnemius - alkaline phosphatase activity and binding of Griffonia simplicifolia lectin I - each of which showed that capillarity and muscle fibre size were largely unaffected by hypoxia. Maximal activities of several metabolic enzymes (cytochrome c oxidase, citrate synthase, isocitrate dehydrogenase, and lactate dehydrogenase) in the gastrocnemius were also largely unaffected by hypoxia. Therefore, the evolved muscle phenotype of high-altitude deer mice is relatively insensitive to hypoxia across life stages.
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PMID:Effects of hypoxia at different life stages on locomotory muscle phenotype in deer mice native to high altitudes. 2917 84

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