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)

Although immunoglobulin light chains are usually secreted in association with heavy chains, free light chains can be secreted by lymphocytes. To identify the structural features of light chains that are essential for their secretion, we mutated a conserved sequence in the variable domain of a lambda I light chain. The effects of the mutations on secretion were assayed by transient expression in COS-1 cells. One mutant (AV60), which replaced Ala-60 with Val, was secreted as efficiently as wild-type lambda I by transfected COS-1 cells. This result was not surprising because secreted lambda II chains contain valine in this position. However, a second lambda I mutant (AV60FS62), which replaced Phe-62 with Ser as well as Ala-60 with Val, was not secreted. This mutant was arrested in the endoplasmic reticulum, as judged by immunofluorescence and by its association with a lumenal endoplasmic reticulum protein, immunoglobulin heavy chain binding protein (BiP). The defect in secretion was not due to gross misfolding of the lambda I chain, since cells cotransfected with AV60FS62 and an immunoglobulin heavy chain gene produced functional antigen-binding antibodies. These assembled IgM molecules were still not secreted. Hence, the replacement of Phe-62 with Ser specifically affects a determinant on the lambda I light chain that is necessary for the intracellular transport of this molecule.
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PMID:A single amino acid substitution in the variable region of the light chain specifically blocks immunoglobulin secretion. 212 54

A G273D mutation immediately proximal to the first calcium binding domain of platelet GPIIb impairs the export of GPIIb-IIIa heterodimers to the platelet surface. To examine how this mutation might alter the structure of GPIIb, G273 was replaced by other amino acids and the resulting mutants were coexpressed with GPIIIa in COS-1 cells. Although replacement with Ala or Val had no effect on GPIIb-IIIa expression, replacement with Glu, Lys, Pro, or Asn caused intracellular retention of GPIIb-IIIa. Concurrently, the consequences of these replacements were examined by comparative modeling by introducing them into the analogous position of the first helix-loop-helix (HLH) motif of calmodulin, based on homology between the calcium binding domains of GPIIb and the calcium binding loops of HLH-containing proteins. The modeling revealed that as the side chain of the introduced amino acid increased in size, it progressively interfered with hydrophobic interactions between the incoming and outgoing helices of the motif. To test whether this observation also applies to GPIIb, V286, located immediately distal to the first GPIIb calcium binding domain, was replaced by Asp and Phe. Expression of these mutants in COS-1 cells also resulted in the intracellular retention of GPIIb-IIIa, suggesting that interactions between sequences that flank the first calcium binding domain of GPIIb affect its folding. Finally, the endoplasmic reticulum chaperone BiP was detected in immunoprecipitates of GPIIb-IIIa containing GPIIb with Ala, Val, Lys, or Pro, but not Gly, at position 273. This suggests that although BiP binding is a sensitive indication of the fidelity of GPIIb-IIIa folding, it is not sufficient to account for the intracellular retention of the heterodimer.
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PMID:Effect of mutagenesis of GPIIb amino acid 273 on the expression and conformation of the platelet integrin GPIIb-IIIa. 891 16

Transformed yeasts producing a mutant form of bovine beta-lactoglobulin (beta-LG), W19Y, in which Trp(19) was replaced with Tyr, were shown to secrete 6 times more than those producing wild type beta-LG. Northern blot analysis suggested that the enhanced level of secretion was not the result of upregulated transcription of W19Y. The ratio of the amount of W19Y secreted into the supernatant to the amount of W19Y remaining inside the cells was much larger than that in the case of wild type beta-LG as shown by immunoblot analysis. A pulse/chase experiment revealed that the speed of secretion of W19Y was significantly accelerated, compared to wild type beta-LG. These results indicated that W19Y was more efficiently and rapidly transported in the course of secretion than wild type beta-LG. Our previous study showed that the DeltaG of unfolding of W19Y in water is 6.9 kcal/mol smaller than that of wild type beta-LG. Furthermore, immunoblot analysis of intracellular beta-LG under non-reducing conditions indicated that W19Y as well as wild type beta-LG maintained a specific folded structure inside the yeast cells, whereas other non-secretable mutant beta-LGs with Phe or Ala at position 19 (W19F and W19A, respectively) did not. These data suggest that low molecular stability and the maintenance of a specific folded structure inside the yeast cells are prerequisites for efficient and rapid secretion. W19Y was more efficiently secreted than wild type beta-LG also in transformed ern1 mutant yeast cells expressing only a basal level of BiP which is considered to function in quality control in the endoplasmic reticulum (ER) by playing an important role in determining the secretion efficiency of secretory proteins. Thus, the reason for the enhanced secretion of W19Y is considered to be that the improved folding ability of W19Y can allow the half-life of the W19Y-BiP complex to become shorter than that of the wild type beta-LG-BiP complex, leading to faster translocation of W19Y into transport vesicles, or that W19Y can fold in a BiP-independent manner in the ER of the yeast cells. Our findings demonstrate that the amount of protein secreted can be improved by alteration of a single amino acid residue crucial for its structure.
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PMID:Accelerated secretion of mutant beta-lactoglobulin in Saccharomyces cerevisiae resulting from a single amino acid substitution. 1040 52

Surfactant protein B (SP-B) is essential to the function of pulmonary surfactant and to alveolar type 2 cell phenotype. Human SP-B is the 79-amino acid product of extensive post-translational processing of a 381-amino acid preproprotein. Processing involves modification of the primary translation product from 39 to 42 kDa and at least 3 subsequent proteolytic cleavages to produce the mature 8-kDa SP-B. To examine the intracellular sites of SP-B processing, we carried out immunofluorescence cytochemistry and inhibitor studies on human fetal lung in explant culture and isolated type 2 cells in monolayer culture using polyclonal antibodies to human SP-B(8) (Phe(201)-Met(279)) and specific epitopes within the N- (NFProx, Ser(145)-Leu(160); NFlank Gln(186)-Gln(200)) and C-terminal (CFlank, Gly(284)-Ser(304)) propeptides of pro-SP-B. Fluorescence immunocytochemistry using epitope-specific antisera showed colocalization of pro-SP-B with the endoplasmic reticulum resident protein BiP. The 25-kDa intermediate was partially endo H-sensitive, colocalized with the medial Golgi resident protein MG160, and shifted into the endoplasmic reticulum in the presence of brefeldin A, which interferes with anterograde transport from endoplasmic reticulum to Golgi. The 9-kDa intermediate colocalized in part with MG160 but not with Lamp-1, a transmembrane protein resident in late endosomes and lamellar bodies. Brefeldin A induced a loss of colocalization between MG160 and NFlank, shifting NFlank immunostaining to a juxtanuclear tubular array. In pulse-chase studies, brefeldin A blocked all processing of 42-kDa pro-SP-B whereas similar studies using monensin blocked the final N-terminal processing event of 9 to 8 kDa SP-B. We conclude that: 1) the first enzymatic cleavage of pro-SP-B to the 25-kDa intermediate is in the brefeldin A-sensitive, medial Golgi; 2) cleavage of the 25-kDa intermediate to a 9-kDa form is a trans-Golgi event that is slowed but not blocked by monensin; 3) the final cleavage of 9 to 8 kDa SP-B is a monensin-sensitive, post-Golgi event occurring prior to transfer of SP-B to lamellar bodies.
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PMID:Intracellular localization of processing events in human surfactant protein B biosynthesis. 1072 8

The C-terminal tail of the calcium receptor (CaR) regulates the affinity of the receptor for ligand, desensitization, and membrane localization. To determine the role of specific amino acids in the bovine parathyroid CaR in mediating signal transduction and cell-surface expression, we transfected truncated and mutated CaR cDNAs into HEK-293 cells. The ability of high extracellular [Ca(2+)] ([Ca(2+)](o)) to increase total inositol phosphate (InsP) production, an index of phospholipase C (PLC) activation, was determined. Receptor expression was assessed by immunoblotting and immunocytochemistry. In cells transiently or stably expressing receptors with the C-terminal tail truncated after residue 895 (CaR-(1-895)) or 929 (CaR-(1-929)), raising [Ca(2+)](o) increased InsPs to levels comparable with those of cells expressing wild-type CaRs. There were no PLC responses to high [Ca(2+)](o) (up to 30 mm) in cells expressing CaRs with C-terminal tails of only 3 residues (CaR-(1-866)), even though these receptors were expressed in the membrane. We scanned the residues between Ser(866) and Val(895) using tandem-Ala and single-site mutagenesis. Two point mutants (His(880) --> Ala and Phe(882) --> Ala CaR) showed 50-70% reductions in high [Ca(2+)](o)-induced InsP production. The levels of expression and glycosylation of these mutants were comparable with wild-type CaRs, but both receptors were profoundly retained in intracellular organelles and co-localized with the endoplasmic reticulum marker BiP. This suggested that the signaling defects of these receptors were likely because of defective trafficking of receptors to the cell surface. Modeling of the C-terminal domain of the CaR indicated that His(880) and Phe(882) are situated in a putative alpha-helical structure of 15 amino acids between residues 877 and 891 in the C-terminal tail. Our studies support the idea that specific amino acids, and possibly a unique secondary structure in the C-terminal tail, are required for the efficient targeting of the CaR to the cell surface required for PLC activation.
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PMID:Amino acids in the cytoplasmic C terminus of the parathyroid Ca2+-sensing receptor mediate efficient cell-surface expression and phospholipase C activation. 1153 93

ERdj3 was identified as a soluble, lumenal DnaJ family member that binds to unassembled immunoglobulin heavy chains along with the BiP chaperone complex in the endoplasmic reticulum of mammalian cells. Here we demonstrated that ERdj3 binds directly to unfolded substrates. Secondary structure predictions suggested that the substrate binding domain of ERdj3 was likely to closely resemble Ydj1, a yeast cytosolic DnaJ family member, which was previously crystallized with a peptide bound to the C-terminal fragment composed of domains I, II, and III. Mutation of conserved residues in domain I, which formed the peptide binding site in Ydj1, affected ERdj3's substrate binding ability in mammalian cells and in vitro binding studies. Somewhat unexpectedly, we found that domain II, which is highly conserved among ERdj3 homologues, but very different from domain II of Ydj1, was also critical for substrate binding. In addition, we demonstrated that ERdj3 forms multimers in cells and found that the conserved carboxy-terminal residue phenylalanine 326 played a critical role in self-assembly. In vitro binding assays revealed that mutation of this residue to alanine diminished ERdj3's substrate binding ability, arguing that multimerization is important for substrate binding. Together, these studies demonstrate that the Ydj1 structure is conserved in another family member and reveal that among this group of DnaJ proteins domain II, which is not present in the closely related type II family members, also plays an essential role in substrate binding.
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PMID:ERdj3, a luminal ER DnaJ homologue, binds directly to unfolded proteins in the mammalian ER: identification of critical residues. 1909 Jun 75

Among the amino acids, cysteine stands apart based on its highly reactive sulfur group. In general, cysteine is underrepresented in proteins. Yet, when present, the features of cysteine often afford unique function. We have shown previously that a cysteine within the ATPase domain of yeast BiP (Kar2) serves as a sensor of the endoplasmic reticulum (ER) redox environment [1, 2]. Under conditions of increased oxidant (oxidative stress), this cysteine becomes oxidized, changing Kar2 from an ATP-dependent foldase to an ATP-independent holdase. We were struck by the high degree of conservation for this cysteine between BiP orthologs, and we sought to determine how cysteine substitution impacts Kar2 function. We observed that no single amino acid replacement is capable of recreating the range of functions that can be achieved by wild-type Kar2 with its cysteine in either unmodified or oxidized states. However, we were able to generate mutants that could selectively replicate the distinct activities exhibited by either unmodified or oxidized Kar2. We found that the ATPase activity displayed by unmodified Kar2 is fully maintained when Cys63 is replaced with Ala or Val. Conversely, we demonstrate that several amino acid substitutions (including His, Phe, Pro, Trp, and Tyr) support an enhanced viability during oxidative stress associated with oxidized Kar2, although these alleles are compromised as an ATPase. We reveal that the range of activity demonstrated by wild-type Kar2 can be replicated by co-expression of Kar2 mutants that mimic either the unmodified or oxidized Kar2 state, allowing for growth during standard and oxidative stress conditions.
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PMID:A Conserved Cysteine within the ATPase Domain of the Endoplasmic Reticulum Chaperone BiP is Necessary for a Complete Complement of BiP Activities. 2754 5

In macroautophagy/autophagy, cargoes are collected by specific receptors, such as SQSTM1/p62 (sequestosome 1), and delivered to phagophores for lysosomal degradation. To date, little is known about how cells modulate SQSTM1 activity and autophagosome biogenesis in response to accumulating cargoes. In this study, we show that SQSTM1 is an N-recognin whose ZZ domain binds N-terminal arginine (Nt-Arg) and other N-degrons (Nt-Lys, Nt-His, Nt-Trp, Nt-Phe, and Nt-Tyr) of the N-end rule pathway. The substrates of SQSTM1 include the endoplasmic reticulum (ER)-residing chaperone HSPA5/GRP78/BiP. Upon N-end rule interaction with the Nt-Arg of arginylated HSPA5 (R-HSPA5), SQSTM1 undergoes self-polymerization via disulfide bonds of Cys residues including Cys113, facilitating cargo collection. In parallel, Nt-Arg-bound SQSTM1 acts as an inducer of autophagosome biogenesis and autophagic flux. Through this dual regulatory mechanism, SQSTM1 plays a key role in the crosstalk between the ubiquitin (Ub)-proteasome system (UPS) and autophagy. Based on these results, we employed 3D-modeling of SQSTM1 and a virtual chemical library to develop small molecule ligands to the ZZ domain of SQSTM1. These autophagy inducers accelerated the autophagic removal of mutant HTT (huntingtin) aggregates. We suggest that SQSTM1 can be exploited as a novel drug target to modulate autophagic processes in pathophysiological conditions.
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PMID:Regulation of autophagic proteolysis by the N-recognin SQSTM1/p62 of the N-end rule pathway. 2926 Oct 1