Exposed Thiols Confer Localization in the Endoplasmic Reticulum by Retention Rather than Retrieval

Isidoro, Ciro; Maggioni, C.; Démoz, Marina; Pizzagalli, Antonella; Fra, Annamaria; Sitia, Roberto

doi:10.1074/jbc.271.42.26138

Cited by 42 publications

(36 citation statements)

References 43 publications

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“…This observation is consistent with the effect of free thiols in ER retention (33). Our results indicate that retention of mutant PrP is mediated through the formation of stable complexes with the ER resident chaperones PDI and calnexin.…”

Section: Discussionsupporting

confidence: 79%

Prion Protein Glycosylation Is Sensitive to Redox Change

Capellari

Zaidi

Urig

et al. 1999

Journal of Biological Chemistry

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The conversion of soluble prion protein into an insoluble, pathogenic, protease-resistant isoform is a key event in the development of prion diseases. Although the mechanism by which the conversion engenders a pathogenic event is unclear, there is increasing evidence to suggest that this may depend on the function of the prion protein in preventing oxidative damage. Therefore, in this study, we assessed the interrelationship between redox-sensitive cysteine, glycosylation, and prion metabolism. Cells were treated with a thioreductant, dithiothreitol, to assess the effect of the cellular oxidation state on the synthesis of the prion protein. This change in redox balance affected the glycosylation of the prion protein, resulting in the sole production of glycosylated forms. The role of the single disulfide bridge in mediating this effect within the prion protein was confirmed by mutating the cysteine residues involved in its formation. These data suggest that conditions that increase the rate of formation of the disulfide bridge favor formation of the unglycosylated prion protein. Thus, since the presence of glycans on the prion protein is protective against its pathogenic conversion, a change in the redox status of the cell would increase the risk of developing a prion disease by favoring the production of the unglycosylated form.

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Section: Discussionsupporting

confidence: 79%

Prion Protein Glycosylation Is Sensitive to Redox Change

Capellari

Zaidi

Urig

et al. 1999

Journal of Biological Chemistry

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“…These results were confirmed by direct immunofluorescence using human CtsWspecific antisera as well as subcellular fractionation of HeLa cells transfected with untagged CtsW (3). ER localization usually requires retention signals such as a KDEL motif (9), KDEL-like motifs (10), C-terminal cysteine residues (11,12), basic dipeptides within KKXX motifs (13), or internal signal sequences (14), all of which are missing from the CtsW cDNA sequence. Whether the 21-aa insertion, the 8-aa C-terminal extension, or other mechanisms participate in the active retention of CtsW in the ER will require further elucidation.…”

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confidence: 50%

Characterization of Murine Cathepsin W and Its Role in Cell-mediated Cytotoxicity

Ondr

Pham

2004

Journal of Biological Chemistry

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Cathepsin W is a member of the papain-like family of cysteine proteases. In this report, we have isolated the cDNA for murine CtsW (mCtsW) from a splenocyte library. The deduced 371-amino-acid sequence shares 68% identity with human CtsW and includes the conserved catalytic triad cysteine, histidine, and asparagine found in all members of this family. In addition to the fulllength form of mCtsW, we have isolated an alternatively spliced form of the mRNA that lacks a complete catalytic triad. An S1 nuclease protection assay and a Western blot analysis showed that mCtsW is mainly restricted to the CD8 ؉ T cell and natural killer cell compartments. In addition, we confirmed that, like its human homologue, mCtsW is localized mainly to the endoplasmic reticulum and its expression is up-regulated upon activation. We also characterized the mCtsW locus using bacterial artificial chromosome clones. The gene consists of 10 coding exons and 9 introns spanning 3.2 kb. To elucidate the physiologic role of this protease, we generated mice deficient in mCtsW. Our data establish that mCtsW is not required for cytotoxic lymphocyte-induced target cell death in vitro. In addition, mCtsW deficiency does not alter the susceptibility of cytotoxic lymphocytes to suicide or fratricide after degranulation. Thus, mCtsW does not have a unique role in target cell apoptosis or cytotoxic cell survival in vitro.

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“…One plausible explanation for this eect is that mutations of Cys609, Cys618 or Cys620 and not of Cys630 or Cys634 would disrupt the intracellular tracking of RET. For instance, it is known that exposed thiol groups in immunoglobulins result in a cytoplasmic retention of unassembled molecules through the formation of disul®de-bonds with proteins of the endoplasmic reticulum (see references in Isidoro et al, 1996). A similar argument could be brought forward if mutations of Cys609, Cys618 or Cys620 would free a cysteine more accessible to the reticulum environment than the partners of Cys630 or Cys634.…”

Section: Discussionmentioning

confidence: 92%

Dual effect on the RET receptor of MEN 2 mutations affecting specific extracytoplasmic cysteines

Chappuis-Flament

Pasini

et al. 1998

Oncogene

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The RET gene encodes a receptor tyrosine kinase whose function is essential during the development of kidney and the intestinal nervous system. Germline mutations aecting one of ®ve cysteines (Cys609, 611, 618, 620 and 634) located in the juxtamembrane domain of the RET receptor are responsible for the vast majority of two cancer-prone disorders, multiple endocrine neoplasia type 2A (MEN 2A) and familial medullary thyroid carcinoma (FMTC). These mutations lead to the replacement of a cysteine by an alternate amino acid. Mutations of the RET gene are also the underlying genetic cause of Hirschsprung disease (HSCR), a congenital aganglionosis of the hindgut. In a fraction of kindreds, MEN 2A cosegregate with HSCR and aected individuals carry a single mutation at codons 609, 618 or 620. To examine the consequences of cysteine substitution on RET function, we have introduced a Cys to Arg mutation into the wild-type RET at either codons 609, 618, 620, 630 or 634. We now report that each mutation induces a constitutive catalytic activity due to the aberrant disul®de homodimerization of RET. However, mutations 630 and 634 activate RET more strongly than mutations 609, 618 or 620 as demonstrated by quantitative assays in rodent ®broblasts and pheochromocytoma PC12 cells. Biochemical analysis revealed that mutations 618 and 620, and to a lesser extent mutation 609, result in a marked reduction of the level of RET at the cell surface and as a consequence decrease the amount of RET covalent dimer. These ®ndings provide a molecular basis explaining the range of phenotype engendered by alterations of RET cysteines and suggest a novel mechanism whereby mutations of cysteines 609, 618 and 620 exert both activating and inactivating eects.Keywords: MEN 2; Hirschsprung disease; RET receptor; tyrosine kinase IntroductionMutations in the RET proto-oncogene, which encodes a transmembrane protein tyrosine kinase, cause two inherited neural crest disorders: multiple endocrine neoplasia type 2 (MEN 2) and Hirschsprung disease (HSCR). Recent studies have provided evidence that RET is a component of a multi-subunit complex which acts as a receptor for the Glial cell-line Derived Neurotrophic Factor (GDNF) and for neurturin, two homologous members of the transforming growth factor b (TGF-b) family (reviewed in Lindsay and Yancopoulos, 1996. For references see Milbrandt et al., 1998). GDNF and neurturin-binding require accessory receptors called GFRa-1 and GFRa-2 that associate with RET and are anchored to the plasma membrane via a glycosyl ± phosphatidylinositol linkage (reviewed in Lindsay and Yancopoulos, 1996. For references see Milbrandt et al., 1998). Mice homozygous for a disruptive mutation either in the RET or the GDNF gene show renal agenesis or dysgenesis of the kidneys and lack the intestinal nervous system (reviewed in Lindsay and Yancopoulos, 1996). These results indicate that RET and GDNF play a critical role during renal organogenesis and are required for the development of a subset of vagal neural crest cells.MEN 2 is a f...

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Exposed Thiols Confer Localization in the Endoplasmic Reticulum by Retention Rather than Retrieval

Cited by 42 publications

References 43 publications

Prion Protein Glycosylation Is Sensitive to Redox Change

Prion Protein Glycosylation Is Sensitive to Redox Change

Characterization of Murine Cathepsin W and Its Role in Cell-mediated Cytotoxicity

Dual effect on the RET receptor of MEN 2 mutations affecting specific extracytoplasmic cysteines

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