Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P11021 (BiP)
 2,049 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To study the importance of individual sulfhydryl residues during the folding and assembly in vivo of influenza virus hemagglutinin (HA), we have constructed and expressed a series of mutant HA proteins in which cysteines involved in three disulfide bonds have been substituted by serine residues. Investigations of the structure and intracellular transport of the mutant proteins indicate that (a) cysteine residues in the ectodomain are essential both for efficient folding of HA and for stabilization of the folded molecule; (b) cysteine residues in the globular portion of the ectodomain are likely to form native disulfide bonds rapidly and directly, without involvement of intermediate, nonnative linkages; and (c) cysteine residues in the stalk portion of the ectodomain also appear not to form intermediate disulfide bonds, even though they have the opportunity to do so, being separated from their correct partners by hundreds of amino acids including two or more other sulfhydryl residues. We propose a role for the cellular protein BiP in shielding the cysteine residues of the stalk domain during the folding process, thus preventing them from forming intermediate, nonnative disulfide bonds.
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PMID:Disulfide bond formation during the folding of influenza virus hemagglutinin. 132 Nov 56

The mouse Wnt-1 gene, a target for insertional activation in mouse mammary tumor virus-induced mammary tumors, encodes poorly secreted, cysteine-rich glycoproteins required for proper central nervous system development. We have been analyzing the biosynthesis of Wnt-1 proteins in several cell lines that express Wnt-1 cDNA from heterologous promoters. A protein of 78 kDa was found to be associated with the intracellular forms of Wnt-1 proteins in mammalian and avian cells by using multiple antisera against Wnt-1 proteins. We have identified p78 as the binding protein BiP with anti-BiP antisera and by its release from Wnt-1 immunoprecipitates upon incubation with MgCl2 and ATP. Experiments with a Wnt-1 mutant that lacks the sequence encoding the signal peptide indicates that Wnt-1 proteins must enter the secretory pathway in order to interact with BiP. We demonstrate that Wnt-1 proteins are associated with BiP in cells in which active Wnt-1 proteins are produced, such as a cultured mammary epithelial cell line and Wnt-1 transgenic mouse mammary tumor cells. The association of Wnt-1 proteins with BiP may be a factor in determining the efficiency of secretion of Wnt-1 gene products.
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PMID:Interaction of Wnt-1 proteins with the binding protein BiP. 153 Oct 88

A heterozygous single base change in exon 49 of COL1A1, which converted the codon for pro alpha 1(I) carboxyl-terminal propeptide residue 94 from tryptophan (TGG) to cysteine (TGT) was identified in a baby with lethal osteogenesis imperfecta (OI64). The C-propeptide mutations in OI64 and in another lethal osteogenesis imperfecta cell strain (OI26), which has a frameshift mutation altering the sequence of the carboxyl-terminal half of the propeptide (Bateman, J. F., Lamande, S. R., Dahl, H.-H. M., Chan, D., Mascara, T. and Cole, W. G. (1989) J. Biol. Chem. 264, 10960-10964), disturbed procollagen folding and retarded the formation of disulfide-linked trimers. Although assembly was delayed, the presence of slowly migrating, overmodified alpha 1(I) and alpha 2(I) chains indicated that mutant pro alpha 1(I) could associate with normal pro alpha 1(I) and pro alpha 2(I) to form pepsin-resistant triple-helical molecules, a proportion of which were secreted. Further evidence of the aberrant folding of mutant procollagen in OI64 and OI26 was provided by experiments demonstrating that the endoplasmic reticulum resident molecular chaperone BiP, which binds to malfolded proteins, was specifically bound to type I procollagen and was coimmunoprecipitated in the osteogenesis imperfecta cells but not control cells. Experiments with brefeldin A, which inhibits protein export from the endoplasmic reticulum, demonstrated that unassembled mutant pro alpha 1(I) chains were selectively degraded within the endoplasmic reticulum resulting in reduced collagen production by the osteogenesis imperfecta cells. This biosynthetic deficiency was reflected in the inability of OI64 and OI26 cells to produce a substantial in vitro collagenous matrix when grown in the continuous presence of ascorbic acid to allow collagen matrix formation. Both these carboxyl-terminal propeptide mutants showed a marked reduction in collagen accumulation to 20% (or less) of control cultures, comparable to the reduced collagen content of tissues from OI26.
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PMID:Endoplasmic reticulum-mediated quality control of type I collagen production by cells from osteogenesis imperfecta patients with mutations in the pro alpha 1 (I) chain carboxyl-terminal propeptide which impair subunit assembly. 772 66

We have used four glycoproteins as markers to study how disulfide bond formation and protein folding effect the intracellular transport of proteins in yeast. Under normal conditions, the vacuolar enzyme carboxypeptidase Y (CPY) and the secretory stress-protein hsp150 acquired disulfide bonds in the endoplasmic reticulum (ER). Treatment of living cells with the reducing agent dithiothreitol (DTT) prevented disulfide formation of newly synthesized CPY and hsp150, resulting in retention of the proteins in the ER. When DTT was removed, the sulfhydryls were reoxidized, and the transport of the proteins to their correct destinations was resumed. Even mature CPY, located in the vacuole, could be reduced with DTT, and reoxidized after removal of the drug. DTT treatment blocked intracellular transport of hsp150 only when present during the synthesis and translocation of the protein. Reduction of folded hsp150, accumulated in the ER due to a sec block prior to DTT treatment, did not inhibit its secretion. The Kar2p/BiP protein, a component of the ER lumen, was found to be associated with fully translocated reduced hsp150, but not with native hsp150, suggesting that Kar2p/BiP may be involved in the putative retention mechanism. The cysteine-free pro-alpha-factor, and invertase which was shown to have free sulfhydryls, were secreted and modified similarly in the presence and absence of DTT, showing that the secretory pathway of yeast functioned under reducing conditions.
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PMID:Selective retention of secretory proteins in the yeast endoplasmic reticulum by treatment of cells with a reducing agent. 801 5

hGHDAF28 is a chimaeric protein consisting of human growth hormone fused to a crippled signal sequence for glycosylphosphatidylinositol (GPI)-anchor addition from decay-accelerating factor, and serves as a model for quality control of GPI-anchor addition. hGHDAF28 is retained in a pre-Golgi compartment and degraded intracellularly by a mechanism with similarity to that for other endoplasmic reticulum (ER)-retained proteins (Field, Moran, Lee, Keller and Caras (1994) J. Biol. Chem. 269, 10830-10837). We have studied the specific pathway of degradation for hGHDAF28 using a number of compounds which affect protein folding and trafficking pathways in eukaryotic cells. We found that high concentrations of dithiothreitol (DTT) accelerated loss of hGHDAF28 by degradation from cell lysates, without promoting secretion or alteration of disulphide-bond distribution, in contrast to a number of other examples of ER-retained proteins where DTT alters disulphide-bond formation. Additionally, degradation of hGHDAF28 was sensitive to pH, being promoted at pH 6.0 and inhibited at pH 8.0; however, the latter effect was transient, indicating incomplete blockade. Degradation was also partially enhanced by depletion of ER calcium with thapsigargin, but this was again a partial and transient effect. Furthermore, degradation was temperature sensitive, with a gradual decrease in rate observed at lower temperatures. However, a sharp decrease in turnover between 15 degrees C and 20 degrees C, indicative of a requirement for transport to a post-ER compartment, was not observed. Degradation of hGHDAF28 was insensitive to treatment with nocodozole or compounds preventing cytoplasmic autophagy, suggesting that ER degradation is independent of classical autophagy and microtubule-dependent processes. In addition, disruption of N-glycosylation with tunicamycin, or inhibition of processing of immature N-glycan chains with castanospermine or deoxynojirimycin, had little effect on the stability of hGHDAF28, suggesting that disruption of the BiP/calnexin quality-control system by bulk cellular secretory proteins does not influence the ER-degradation pathway of hGHDAF28. Intermolecular hGHDAF28 cysteine bonds result in the formation of aggregates which are probably important in the retention of the molecule. The insensitivity of this structure to reduction in vivo, together with the enhanced degradation rate, indicates that DTT mediates its effect on stability via a molecule involved in degradation of hGHDAF28, possibly a thiol-sensitive protease.
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PMID:Quality control of glycosylphosphatidylinositol anchor attachment in mammalian cells: a biochemical study. 903 50

The accumulation of misfolded proteins in the cytosol leads to increased expression of heat-shock proteins, while accumulation of such proteins in the endoplasmic reticulum (ER) stimulates the expression of many ER resident proteins, most of which function as molecular chaperones. Recently, inhibitors of the proteasome have been identified that can block the rapid degradation of abnormal cytosolic and ER-associated proteins. We therefore tested whether these agents, by causing the accumulation of abnormal proteins, might stimulate the expression of cytosolic heat-shock proteins and/or ER molecular chaperones and thereby induce thermotolerance. Exposure of Madin-Darby canine kidney cells to various proteasome inhibitors, including the peptide aldehydes (MG132, MG115, N-acetyl-leucyl-leucyl-norleucinal) and lactacystin, inhibited the degradation of short-lived proteins and increased markedly the levels of mRNAs encoding cytosolic heat-shock proteins (Hsp70, polyubiquitin) and ER chaperones (BiP, Grp94, ERp72), as shown by Northern blot analysis. However, inhibitors of cysteine proteases (E64), serine proteases (leupeptin), or metalloproteases (1, 10-phenanthroline) had no effect on the levels of these mRNAs. The relative efficacies of the peptide aldehyde inhibitors in inducing these mRNAs correlated with their potencies against the proteasome. Furthermore, reduction of the aldehyde group of MG132 decreased its inhibitory effect on proteolysis and largely prevented the induction of these mRNAs. Although treatment with the proteasome inhibitors caused rapid increases in mRNA levels (as early as 2 h after treatment with MG132), the inhibitors did not detectably affect total protein synthesis, total protein secretion, ER morphology, or the retention of ER-lumenal proteins, even after 18 h of treatment. Together, the findings suggest that inhibition of proteasome function induces heat-shock proteins and ER chaperones due to the accumulation of sufficient amounts of abnormal proteins and/or the inhibition of degradation of a key regulatory factor (e.g. heat-shock factor). Since expression of heat-shock proteins can protect cells from thermal injury, we tested whether the proteasome inhibitors might also confer thermotolerance. Treatment of cells with MG132 for as little as 2 h, markedly increased the survival of cells subjected to high temperatures (up to 46 degrees C). Thus, these agents may have applications in protecting against cell injury.
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PMID:Proteasome inhibition leads to a heat-shock response, induction of endoplasmic reticulum chaperones, and thermotolerance. 908 35

Polymerization caused by defective folding of heat-denatured ovalbumin was examined. A compactly misfolded ovalbumin that was produced by cooling heat-denatured protein rapidly tended to aggregate in the presence of salt. Two different forms of aggregates were observed as the concentration of salt was varied: a linear polymer at a physiological concentration and a massive agglomerate at a higher concentration. Salt-induced polymerization depended on the species of anion and the order of effectiveness followed the lyotropic series of Hofmeister. Defective folding of heat-denatured ovalbumin induced the exposure of cysteine residues in sequences located in the interior of the native protein. The misfolded ovalbumin, but not the native protein, bound to bovine BiP and stimulated its ATPase activity with the K(m) of 64 microM and the V(max) of 0.5 nmol/min per milligram. Measurement of surface plasmon resonance revealed that only the misfolded ovalbumin was recognized with the K(d) of 4.12 X 10(-8) M by the Fab fragment of a monoclonal antibody raised against hen ovalbumin, and its epitope was determined to be a hydrophobic segment in the beta-strand of central sheet A. Transmission electron microscopy showed that the linear polymerization was inhibited by the addition of bovine BiP and the Fab fragment. These results demonstrated that the compactly misfolded ovalbumin polymerized through hydrophobic interaction occurring among the areas exposed as a result of defective folding of the heat-denatured protein. Exposure of the region of, or adjacent to, the central beta-sheet A was required for axial contact among the misfolded molecules, suggesting that this process may be explained by reference to the mechanism proposed for loop-sheet polymerization in the Z type variant of a serpin alpha1-antitrypsin.
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PMID:Linear polymerization caused by the defective folding of a non-inhibitory serpin ovalbumin. 916 32

The mouse autosomal dominant mutation Mody develops hyperglycemia with notable pancreatic beta-cell dysfunction. This study demonstrates that one of the alleles of the gene for insulin 2 in Mody mice encodes a protein product that substitutes tyrosine for cysteine at the seventh amino acid of the A chain in its mature form. This mutation disrupts a disulfide bond between the A and B chains and can induce a drastic conformational change of this molecule. Although there was no gross defect in the transcription from the wild-type insulin 2 allele or two alleles of insulin 1, levels of proinsulin and insulin were profoundly diminished in the beta cells of Mody mice, suggesting that the number of wild-type (pro)insulin molecules was also decreased. Electron microscopy revealed a dramatic reduction of secretory granules and a remarkably enlarged lumen of the endoplasmic reticulum. Little proinsulin was processed to insulin, but high molecular weight forms of proinsulin existed with concomitant overexpression of BiP, a molecular chaperone in the endoplasmic reticulum. Furthermore, mutant proinsulin expressed in Chinese hamster ovary cells was inefficiently secreted, and its intracellular fraction formed complexes with BiP and was eventually degraded. These findings indicate that mutant proinsulin was trapped and accumulated in the endoplasmic reticulum, which could induce beta-cell dysfunction and account for the dominant phenotype of this mutation.
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PMID:A mutation in the insulin 2 gene induces diabetes with severe pancreatic beta-cell dysfunction in the Mody mouse. 988 31

Entamoeba histolytica, the protozoan parasite that phagocytoses bacteria and host cells, has a vesicle/vacuole-filled cytosol like that of macrophages. In contrast, the infectious cyst form has four nuclei and a chitin wall. Here, anti-chitinase antibodies identified hundreds of small secretory vesicles in encysting E. invadens parasites and in E. histolytica trophozoites overexpressing chitinase under an actin gene promoter. Abundant small secretory vesicles were also identified with antibodies to the surface antigen Ariel and with a fluorescent substrate of cysteine proteinases. Removal of an N-terminal signal sequence directed chitinase to the cytosol. Addition of a C-terminal KDEL peptide, identified on amebic BiP, retained chitinase in a putative endoplasmic reticulum, which was composed of a few vesicles of mixed sizes. A putative Golgi apparatus, which was Brefeldin A sensitive and composed of a few large, perinuclear vesicles, was identified with antibodies to ADP-ribosylating factor and to epsilon-COP. We conclude that the amebic secretory pathway is similar to those of other eukaryotic cells, even if its appearance is somewhat different.
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PMID:Chitinase secretion by encysting Entamoeba invadens and transfected Entamoeba histolytica trophozoites: localization of secretory vesicles, endoplasmic reticulum, and Golgi apparatus. 1033 23

Protein disulfide isomerase (PDI) interacts with secretory proteins, irrespective of their thiol content, late during translocation into the ER; thus, PDI may be part of the quality control machinery in the ER. We used yeast pdi1 mutants with deletions in the putative peptide binding region of the molecule to investigate its role in the recognition of misfolded secretory proteins in the ER and their export to the cytosol for degradation. Our pdi1 deletion mutants are deficient in the export of a misfolded cysteine-free secretory protein across the ER membrane to the cytosol for degradation, but ER-to-Golgi complex transport of properly folded secretory proteins is only marginally affected. We demonstrate by chemical cross-linking that PDI specifically interacts with the misfolded secretory protein and that mutant forms of PDI have a lower affinity for this protein. In the ER of the pdi1 mutants, a higher proportion of the misfolded secretory protein remains associated with BiP, and in export-deficient sec61 mutants, the misfolded secretory protein remain bounds to PDI. We conclude that the chaperone PDI is part of the quality control machinery in the ER that recognizes terminally misfolded secretory proteins and targets them to the export channel in the ER membrane.
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PMID:Export of a cysteine-free misfolded secretory protein from the endoplasmic reticulum for degradation requires interaction with protein disulfide isomerase. 1061 3


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