Disulfide recognition in an optimized threading potential.
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An energy potential is constructed and trained to succeed in fold recognition for the general population of proteins as well as an important class which has previously been problematic: small, disulfide-bearing proteins. The potential is modeled on solvation, with the energy a function of side chain burial and the number of disulfide bonds. An accurate disulfide recognition algorithm identifies cysteine pairs which have the appropriate orientation to form a disulfide bridge. The potential has 22 energy parameters which are optimized so the Protein Data Bank (PDB) structure for each sequence in a training set is the lowest in energy out of thousands of alternative structures. One parameter per amino acid type reflects burial preference and a single parameter is used in an overpacking term. Additionally, one optimized parameter provides a favorable contribution for each disulfide identified in a given protein structure. With little training, the potential is >80% accurate in ungapped threading tests using a variety of proteins. The same level of accuracy is observed in a threading test of small proteins which have disulfide bonds. Importantly, the energy potential is also successful with proteins having uncrosslinked cysteines. (+info)
Structure, function, and regulation of human cystine/glutamate transporter in retinal pigment epithelial cells.
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PURPOSE: The purpose of this investigation was to provide evidence for the expression of the cystine/glutamate transporter (x(c)(-)) in the human retinal pigment epithelial cell line ARPE-19, clone the light chain of the transporter from an ARPE-19 cell cDNA library and study its function, and investigate the regulation of this transporter by nitric oxide (NO) in ARPE-19 cells. METHODS: Uptake of radiolabeled cystine and glutamate was measured in ARPE-19 cells. The functional identity of x(c)(-) in these cells was established by substrate specificity and Na(+)-independence of the uptake process. The human x(c)(-) light chain (human xCT) was cloned from an ARPE-19 cell cDNA library. The functional identity of the cloned human xCT was investigated by heterologous coexpression of the light chain with the heavy chain (human 4F2hc) in HeLa cells. ARPE-19 cells were treated with or without the NO donor 3-nitroso-N:-acetylpenicillamine (SNAP) and the expression of x(c)(-) was studied at the functional and molecular levels. RESULTS: ARPE-19 cells take up cystine as well as glutamate in the absence of Na(+). Substrate specificity studies indicate that although the uptake of cystine in the absence of Na(+) is mediated by multiple amino acid transport systems including x(c)(-), the uptake of glutamate in the absence of Na(+) occurs exclusively via x(c)(-). The human xCT cloned from ARPE-19 cells is a protein of 501 amino acids. These cells express the heavy chain 4F2hc as evidenced from RT-PCR analysis. Coexpression of human xCT with 4F2hc in HeLa cells leads to the induction of cystine and glutamate uptake with characteristics similar to that of x(c)(-). The activity of x(c)(-) in ARPE-19 cells is upregulated by SNAP, and the process is associated with an increase in the expression of xCT with no detectable change in the expression of 4F2hc. CONCLUSIONS: ARPE-19 cells express the cystine/glutamate transporter x(c)(-) (the light chain xCT and the heavy chain 4F2hc) as is evident from functional and molecular studies. NO upregulates this transport system and the process is associated with an increase in xCT mRNA but with no change in 4F2hc mRNA. (+info)
Functional contributions of noncysteine residues within the cystine knots of human chorionic gonadotropin subunits.
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Human chorionic gonadotropin (hCG) is a heterodimeric member of a family of cystine knot-containing proteins that contain the consensus sequences Cys-X(1)-Gly-X(2)-Cys and Cys-X(3)-Cys. Previously, we characterized the contributions that cystine residues of the hCG subunit cystine knots make in folding, assembly, and bioactivity. Here, we determined the contributions that noncysteine residues make in hCG folding, secretion, and assembly. When the X(1), X(2), and X(3) residues of hCG-alpha and -beta were substituted by swapping their respective cystine knot motifs, the resulting chimeras appeared to fold correctly and were efficiently secreted. However, assembly of the chimeras with their wild type partner was almost completely abrogated. No single amino acid substitution completely accounted for the assembly inhibition, although the X(2) residue made the greatest individual contribution. Analysis by tryptic mapping, high performance liquid chromatography, and SDS-polyacrylamide gel electrophoresis revealed that substitution of the central Gly in the Cys-X(1)-Gly-X(2)-Cys sequence of either the alpha- or beta-subunit cystine knot resulted in non-native disulfide bond formation and subunit misfolding. This occurred even when the most conservative change possible (Gly --> Ala) was made. From these studies we conclude that all three "X" residues within the hCG cystine knots are collectively, but not individually, required for the formation of assembly-competent hCG subunits and that the invariant Gly residue is required for efficient cystine knot formation and subunit folding. (+info)
Diet-induced hyperhomocysteinemia exacerbates neointima formation in rat carotid arteries after balloon injury.
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BACKGROUND: Increasing evidence indicates that elevated plasma homocysteine levels are associated with an increased risk of atherosclerosis and endothelial dysfunction, although little specific information on the mechanisms responsible for the atherogenic effects of homocysteine or on the in vivo contribution made by hyperhomocysteinemia to atherosclerosis is currently available. Because homocysteine is known to exert a direct inhibitory effect on endothelial cell growth in vitro, we hypothesized that this effect contributes to the progression of atherosclerotic lesions initiated by endothelial damage caused by mechanical injury. METHODS AND RESULTS: We prepared diet-induced hyperhomocysteinemic rats in which neointima formation after balloon injury to the common carotid artery was assessed. Moderate hyperhomocysteinemia (plasma homocysteine levels 3- to 4-fold higher than control) significantly exacerbated neointima formation. Oral administration of folate, which had a homocysteine-lowering effect, diminished neointima formation induced by moderate hyperhomocysteinemia. Furthermore, the attenuation of reendothelialization was shown in diet-induced hyperhomocysteinemic rats with Evans blue staining. CONCLUSIONS: Diet-induced hyperhomocysteinemia, even mild to moderate, exacerbates neointima formation after denuding injury, making hyperhomocysteinemia a likely risk factor for postangioplasty restenosis. It may be mediated through an inhibitory effect of homocysteine on reendothelialization. Homocysteine lowering with folate supplementation can effectively ameliorate the detrimental effects of moderate hyperhomocysteinemia. Clinical trials would seem to be warranted. (+info)
Effect of oxygen on induction of the cystine transporter by bacterial lipopolysaccharide in mouse peritoneal macrophages.
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Amino acid transport in mouse peritoneal macrophages is mediated by several membrane carriers with different substrate specificity and sensitivity to environmental stimuli. We reported previously that transport activities of cystine and arginine in the macrophages were induced markedly by low concentrations of bacterial lipopolysaccharide (LPS). It is known that a variety of macrophage functions are affected by ambient oxygen tension. In this study, we have investigated the effects of oxygen on the induction of amino acid transport activity by LPS and found that the induction of cystine, but not arginine, transport activity was dependent on the ambient oxygen tension. When the macrophages were cultured with 2% O(2) in the presence of 1 ng/ml LPS, induction of cystine transport activity was reduced by approximately 70% compared with cells cultured under normoxic conditions. In macrophages, transport of cystine is mediated by a Na(+)-independent anionic amino acid transporter named system x(c)(-). System x(c)(-) is composed of two protein components, xCT and 4F2hc, and the expression of xCT was closely correlated with system x(c)(-) activity. A putative NF-kappaB binding site was found in the 5'-flanking region of the xCT gene, but the enhanced expression of xCT by LPS and oxygen was not mediated by NF-kappaB binding. An increase in intracellular GSH in macrophages paralleled induction of xCT, but not gamma-glutamylcysteine synthetase. These results suggest the importance of system x(c)(-) in antioxidant defense in macrophages exposed to LPS and oxidative stress. (+info)
Identification of a novel thioredoxin-related transmembrane protein.
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We recently identified a series of transforming growth factor-beta-responsive genes in A549 human adenocarcinoma cell line by a gene trap screening method. Here we report the molecular cloning and characterization of one of these genes, designated TMX, that encodes a novel protein of 280 amino acid residues. The TMX protein possesses an N-terminal signal peptide followed by one thioredoxin (Trx)-like domain with a unique active site sequence, Cys-Pro-Ala-Cys, and a potential transmembrane domain. There are putative TMX homologs with identical active site sequences in the Caenorhabditis elegans and Drosophila genomes. Using recombinant proteins expressed in Escherichia coli, we demonstrated the activity of the Trx domain of TMX to cleave the interchain disulfide bridges in insulin in vitro. The TMX transcript is widely expressed in normal human tissues, and subcellular fractionation and immunostaining for an epitope-tagged TMX protein suggest that TMX is predominantly localized in the endoplasmic reticulum (ER). When TMX was expressed in HEK293 cells, it significantly suppressed the apoptosis induced by brefeldin A, an inhibitor of ER-Golgi transport. This activity was abolished when two Cys residues in the active site sequence were mutated to Ser, suggesting that the Trx-like activity of TMX may help relieve ER stress caused by brefeldin A. (+info)
The moroccan food snail, Helix aspersa, as a source of Salmonella.
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A total of 270 samples, nine lots of 30 samples each, of imported Moroccan food snails was examined for the presence of Salmonella. Eighty-four samples (an overall incidence of 31.11%) and all nine lots contained Salmonella. No significant difference (P greater than 0.25) in the number of positive samples was observed by using either selenite cystine both or tetrathionate broth when the samples had been pre-enriched in lactose broth. When used as direct selective enrichments with samples not pre-enriched in lactose broth, tetrathionate broth was significantly (P less than 0.05) more productive than selenite cystine broth. The overall detection of Salmonella-positive samples by direct enrichment was significantly greater (P less than 0.001) than by pre-enrichment. A variety of uncommon serotypes occurrence and incidence, and the concomitant human health potential, of Salmonella in one species of live, imported food snails. (+info)
Bone dysplasia sclerosteosis results from loss of the SOST gene product, a novel cystine knot-containing protein.
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Sclerosteosis is an autosomal recessive sclerosing bone dysplasia characterized by progressive skeletal overgrowth. The majority of affected individuals have been reported in the Afrikaner population of South Africa, where a high incidence of the disorder occurs as a result of a founder effect. Homozygosity mapping in Afrikaner families along with analysis of historical recombinants localized sclerosteosis to an interval of approximately 2 cM between the loci D17S1787 and D17S930 on chromosome 17q12-q21. Here we report two independent mutations in a novel gene, termed "SOST." Affected Afrikaners carry a nonsense mutation near the amino terminus of the encoded protein, whereas an unrelated affected person of Senegalese origin carries a splicing mutation within the single intron of the gene. The SOST gene encodes a protein that shares similarity with a class of cystine knot-containing factors including dan, cerberus, gremlin, prdc, and caronte. The specific and progressive effect on bone formation observed in individuals affected with sclerosteosis, along with the data presented in this study, together suggest that the SOST gene encodes an important new regulator of bone homeostasis. (+info)