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Query: UNIPROT:P06889 (Mol)
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ER-associated degradation (ERAD) removes defective and mis-folded proteins from the eukaryotic secretory pathway, but mutations in the ER lumenal Hsp70, BiP/Kar2p, compromise ERAD efficiency in yeast. Because attenuation of ERAD activates the UPR, we screened for kar2 mutants in which the unfolded protein response (UPR) was induced in order to better define how BiP facilitates ERAD. Among the kar2 mutants isolated we identified the ERAD-specific kar2-1 allele (Brodsky et al. J. Biol. Chem. 274, 3453-3460). The kar2-1 mutation resides in the peptide-binding domain of BiP and decreases BiP's affinity for a peptide substrate. Peptide-stimulated ATPase activity was also reduced, suggesting that the interdomain coupling in Kar2-1p is partially compromised. In contrast, Hsp40 cochaperone-activation of Kar2-1p's ATPase activity was unaffected. Consistent with UPR induction in kar2-1 yeast, an ERAD substrate aggregated in microsomes prepared from this strain but not from wild-type yeast. Overexpression of wild-type BiP increased substrate solubility in microsomes obtained from the mutant, but the ERAD defect was exacerbated, suggesting that simply retaining ERAD substrates in a soluble, retro-translocation-competent conformation is insufficient to support polypeptide transit to the cytoplasm.
Mol Biol Cell 2003 Aug
PMID:Dependence of endoplasmic reticulum-associated degradation on the peptide binding domain and concentration of BiP. 1292 75

Lectin (calreticulin [CRT])-N-glycan-mediated quality control of glycoprotein folding is operative in trypanosomatid protozoa but protein-linked monoglucosylated N-glycans are exclusively formed in these microorganisms by UDP-Glc:glycoprotein glucosyltransferase (GT)-dependent glucosylation. The gene coding for this enzyme in the human pathogen Trypanosoma cruzi was identified and sequenced. Even though several of this parasite glycoproteins have been identified as essential components of differentiation and mammalian cell invasion processes, disruption of both GT-encoding alleles did not affect cell growth rate of epimastigote form parasites and only partially affected differentiation and mammalian cell invasion. The cellular content of one of the already identified T. cruzi glycoprotein virulence factors (cruzipain, a lysosomal proteinase) only showed a partial (5-20%) decrease in GT null mutants in spite of the fact that >90% of all cruzipain molecules interacted with CRT during their folding process in wild-type cells. Although extremely mild cell lysis and immunoprecipitation procedures were used, no CRT-cruzipain interaction was detected in GT null mutants but secretion of the proteinase was nevertheless delayed because of a lengthened interaction with Grp78/BiP probably caused by the detected induction of this chaperone in GT null mutants. This result provides a rationale for the absence of a more drastic consequence of GT absence. It was concluded that T. cruzi endoplasmic reticulum folding machinery presents an exquisite plasticity that allows the parasite to surmount the absence of the glycoprotein-specific folding facilitation mechanism.
Mol Biol Cell 2003 Sep
PMID:The interplay between folding-facilitating mechanisms in Trypanosoma cruzi endoplasmic reticulum. 1297 44

The mammalian unfolded protein response (UPR) protects the cell against the stress of misfolded proteins in the endoplasmic reticulum (ER). We have investigated here the contribution of the UPR transcription factors XBP-1, ATF6alpha, and ATF6beta to UPR target gene expression. Gene profiling of cell lines lacking these factors yielded several XBP-1-dependent UPR target genes, all of which appear to act in the ER. These included the DnaJ/Hsp40-like genes, p58(IPK), ERdj4, and HEDJ, as well as EDEM, protein disulfide isomerase-P5, and ribosome-associated membrane protein 4 (RAMP4), whereas expression of BiP was only modestly dependent on XBP-1. Surprisingly, given previous reports that enforced expression of ATF6alpha induced a subset of UPR target genes, cells deficient in ATF6alpha, ATF6beta, or both had minimal defects in upregulating UPR target genes by gene profiling analysis, suggesting the presence of compensatory mechanism(s) for ATF6 in the UPR. Since cells lacking both XBP-1 and ATF6alpha had significantly impaired induction of select UPR target genes and ERSE reporter activation, XBP-1 and ATF6alpha may serve partially redundant functions. No UPR target genes that required ATF6beta were identified, nor, in contrast to XBP-1 and ATF6alpha, did the activity of the UPRE or ERSE promoters require ATF6beta, suggesting a minor role for it during the UPR. Collectively, these results suggest that the IRE1/XBP-1 pathway is required for efficient protein folding, maturation, and degradation in the ER and imply the existence of subsets of UPR target genes as defined by their dependence on XBP-1. Further, our observations suggest the existence of additional, as-yet-unknown, key regulators of the UPR.
Mol Cell Biol 2003 Nov
PMID:XBP-1 regulates a subset of endoplasmic reticulum resident chaperone genes in the unfolded protein response. 1455 94

mda-7/IL-24 (HGMW-approved symbol IL24) is a tumor suppressor gene whose expression is lost during tumor progression. Gene transfer using adenoviral mda-7/IL-24 (Ad-mda7) exhibits minimal toxicity on normal cells while inducing potent apoptosis in a variety of cancer cell lines. Ad-mda7-transduced cells express high levels of MDA-7 protein intracellularly and also secrete a soluble form of MDA-7 protein. In this study, we sought to determine whether the intracellular or secreted MDA-7 protein was responsible for anti-tumor activity in H1299 lung tumor cells. Ad-mda7 transduction of lung tumor cells increased expression of stress-related proteins, including BiP, GADD34, PP2A, caspases 7 and 12, and XBP-1, consistent with activation of the UPR pathway, a key sensor of endoplasmic reticulum (ER)-mediated stress. Blocking secretion of MDA-7 did not inhibit apoptosis, demonstrating that intracellular MDA-7 was responsible for cytotoxicity. Consistent with this result, when applied directly to lung cancer cells, soluble MDA-7 protein exhibited minimal cytotoxic effect. We then generated mda-7 expression constructs using vectors that target the expressed protein to various subcellular compartments, including cytoplasm, nucleus, and ER. Only full-length and ER-targeted MDA-7 elicited cell death in tumor cells. Thus in lung cancer cells, Ad-mda7 activates the UPR stress pathway and induces apoptosis via intracellular MDA-7 expression in the secretory pathway.
Mol Ther 2004 Mar
PMID:The tumor suppressor activity of MDA-7/IL-24 is mediated by intracellular protein expression in NSCLC cells. 1500 2

Anticancer drugs often show complex mechanisms of action, including effects on multiple cellular targets. Detailed understanding of these intricate effects is important for the understanding of cytotoxicity. In this study, we examined apoptosis induction by ellipticines, a class of cytotoxic plant alkaloids known to inhibit topoisomerase II. The potent ellipticine derivative 6-propanamine ellipticine (6-PA-ELL) induced rapid apoptosis in MDA-MB-231 breast cancer cells, preceded by a conformational change in Bak and cytochrome c release. Experiments using knock-out mouse embryo fibroblasts established that Bak was of particular importance for cytotoxicity. 6-PA-ELL increased the expression of the endoplasmic reticulum chaperones GRP78/BiP and GRP94, suggesting induction of endoplasmic reticulum stress. Induction of GRP78 expression was dependent on the endoplasmic reticulum stress response element (ERSE) of the GRP78 promoter. Examination of different ellipticine derivatives revealed a correlation between pro-apoptotic activity and the ability to induce GRP78 expression. Furthermore, 6-PA-ELL was found to induce splicing of the mRNA encoding the XBP1 transcription factor, characteristic of endoplasmic reticulum stress, and to induce activation of the endoplasmic reticulum-specific caspase-12 in mouse colon cancer cells. We finally demonstrate that 6-PA-ELL induces apoptotic signaling also in enucleated cells, consistent with the existence of a cytoplasmic target for this compound. Our data suggest that induction of endoplasmic reticulum stress may contribute to the cytotoxicity of ellipticines.
Mol Cancer Ther 2004 Apr
PMID:Induction of endoplasmic reticulum stress by ellipticine plant alkaloids. 1507 93

Most loss-of-function mutations of the glycoprotein hormone receptors have been found to be due to the misfolding of the receptor, resulting in its intracellular retention and, therefore, decreased cell surface expression. Chaperone proteins within the endoplasmic reticulum play an essential role in facilitating the folding of newly synthesized proteins and in recognizing and segregating misfolded proteins, thereby preventing their transit to the Golgi. The present study was conducted to begin to elucidate the role of chaperone proteins in the folding of the glycoprotein hormone receptors and misfolded mutants thereof. Toward this end, we examined the potential associations of calnexin, calreticulin, Grp94, BiP, ERp57, and protein disulfide-isomerase with each of the three glycoprotein hormone receptors. Calnexin, calreticulin, and protein disulfide-isomerase were found to associate with the immature forms of all three wild-type (wt) glycoprotein hormone receptors. As examples of misfolded glycoprotein hormone receptors, we studied two human LH receptor (hLHR) loss-of-function mutants that we show to be expressed predominantly as immature forms that are retained intracellularly. Significantly, the patterns of chaperone protein associations with the misfolded hLHR mutants differ from that observed with the wt hLHR. Furthermore, and unexpectedly, the chaperone protein associations were found to differ between the two misfolded hLHR mutants. Altogether, our studies show that although the same chaperone proteins are used by the three wt glycoprotein hormone receptors, different chaperone proteins associate with misfolded mutants thereof, and the specificity of interactions can vary between mutants, most likely reflecting the different stages of folding they achieve before being targeted for degradation.
Mol Endocrinol 2004 Jul
PMID:Intracellularly located misfolded glycoprotein hormone receptors associate with different chaperone proteins than their cognate wild-type receptors. 1510 36

Oxidation and folding of secretory proteins in the endoplasmic reticulum (ER) depends on the presence of chaperones and oxidoreductases. Two of the oxidoreductases present in the ER of mammalian cells are protein disulfide isomerase (PDI) and ERp57. In this study, we investigated the influence of ERp57 on the in vitro reoxidation and refolding of an antibody Fab fragment. Our results show that ERp57 shares functional properties with PDI and that both are clearly different from other oxidoreductases. The reactivation of the denatured and reduced Fab fragment was enhanced significantly in the presence of ERp57 with kinetics and redox dependence of the reactivation reaction comparable to those obtained for PDI. These properties were not influenced by the presence of calnexin. Furthermore, whereas PDI cooperates with the immunoglobulin heavy chain binding protein (BiP), no synergistic effect could be observed for BiP and ERp57. These results indicate that the cooperation of the two oxidoreductases with different partner proteins may explain their different roles in the folding of proteins in the ER.
J Mol Biol 2004 Aug 20
PMID:Influence of the oxidoreductase ER57 on the folding of an antibody fab fragment. 1532 18

GM1-ganglioside (GM1) is a major sialoglycolipid of neuronal membranes that, among other functions, modulates calcium homeostasis. Excessive accumulation of GM1 due to deficiency of lysosomal beta-galactosidase (beta-gal) characterizes the neurodegenerative disease GM1-gangliosidosis, but whether the accumulation of GM1 is directly responsible for CNS pathogenesis was unknown. Here we demonstrate that activation of an unfolded protein response (UPR) associated with the upregulation of BiP and CHOP and the activation of JNK2 and caspase-12 leads to neuronal apoptosis in the mouse model of GM1-gangliosidosis. GM1 loading of wild-type neurospheres recapitulated the phenotype of beta-gal-/- cells and activated this pathway by depleting ER calcium stores, which ultimately culminated in apoptosis. Activation of UPR pathways did not occur in mice double deficient for beta-gal and ganglioside synthase, beta-gal-/-/GalNAcT-/-, which do not accumulate GM1. These findings suggest that the UPR can be induced by accumulation of the sialoglycolipid GM1 and this causes a novel mechanism of neuronal apoptosis.
Mol Cell 2004 Sep 10
PMID:GM1-ganglioside-mediated activation of the unfolded protein response causes neuronal death in a neurodegenerative gangliosidosis. 1535 Feb 19

We recently identified ERdj3 as a component of unassembled immunoglobulin (Ig) heavy chain:BiP complexes. ERdj3 also associates with a number of other protein substrates, including unfolded light chains, a nonsecreted Ig light chain mutant, and the VSV-G ts045 mutant at the nonpermissive temperature. We produced an ERdj3 mutant that was unable to stimulate BiP's ATPase activity in vitro or to bind BiP in vivo. This mutant retained the ability to interact with unfolded protein substrates, suggesting that ERdj3 binds directly to proteins instead of via interactions with BiP. BiP remained bound to unfolded light chains longer than ERdj3, which interacted with unfolded light chains initially, but quickly disassociated before protein folding was completed. This suggests that ERdj3 may bind first to substrates and serve to inhibit protein aggregation until BiP joins the complex, whereas BiP remains bound until folding is complete. Moreover, our findings support a model where interactions with BiP help trigger the release of ERdj3 from the substrate:BiP complex.
Mol Biol Cell 2005 Jan
PMID:ERdj3, a stress-inducible endoplasmic reticulum DnaJ homologue, serves as a cofactor for BiP's interactions with unfolded substrates. 1552 76

Human ADAM33 is a multiple-domain, type-I transmembrane zinc metalloprotease recently implicated in asthma susceptibility [Nature 418 (2002) 426]. To provide an active protease for functional studies, expression of a recombinant ADAM33 zymogen (pro-catalytic domains, pro-CAT) was attempted in several insect cells. The pro-CAT was cloned into baculovirus under the regulation of the polyhedron promoter and using either the honeybee mellitin or ADAM33 signal sequence. Sf9 or Hi5 cells infected with these recombinant viruses expressed the majority of the protein unprocessed and as inclusion bodies ( approximately 10 mg/L). On the other hand, similar constructs could be expressed, processed, and secreted by Drosophila S2 cells using a variety of constitutive (actin, pAc5.1) or inducible (metallothionein, PMT) promoters and leader sequences (e.g., native and BiP). Higher expression level of 10-fold was observed for the inducible system resulting in an average yield of 20 mg/L after purification. The majority of the catalytic domain purified from the Drosophila conditioned media remained associated with the pro-domain after several chromatography steps. An induction cocktail containing cadmium chloride and zinc chloride was subsequently developed for the PMT system as an alternative to using cupric sulfate or cadmium chloride as single inducers. The novel induction cocktail resulted in an increased ratio of secreted catalytic to pro-domain, and yielded milligram amounts of highly purified protease. The availability of this modified expression system facilitated purification of the wild type and several glycosylation mutants, one of which (N231Q) crystallized recently for X-ray structure determination [J. Mol. Biol. 335 (2003) 129].
 ...
PMID:Protease domain of human ADAM33 produced by Drosophila S2 cells. 1555 45


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