Analyzing folding and degradation of metabolically labelled polypeptides by conventional and diagonal sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Soldà, Tatiana; Olivari, Silvia; Molinari, Maurizio

doi:10.1251/bpo111

Cited by 4 publications

(5 citation statements)

References 23 publications

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“…With this technique, proteins lacking disulphide bonds will migrate on the diagonal because of the identical mobility in both dimensions. Proteins that contain intrachain disulphide bonds will be more compact and will run above the diagonal; proteins that form interchain disulphide-linked complexes will run below the diagonal . Our results indicate that the ERp57/TUBB3 complex is not modulated by disulphide bridges, since the only signal obtained using the anti-TUBB3 antibody was localized in the diagonal (Figure C).…”

Section: Resultsmentioning

confidence: 66%

Comparative Proteomic Analysis of Paclitaxel Sensitive A2780 Epithelial Ovarian Cancer Cell Line and Its Resistant Counterpart A2780TC1 by 2D-DIGE: The Role of ERp57

Cicchillitti

Urbani

et al. 2009

J. Proteome Res.

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Epithelial ovarian cancer is the leading cause of gynecological cancer mortality. Despite good response to surgery and initial chemotherapy, chemoresistance occurrence represents a major obstacle to a successful therapy. To better understand biological mechanisms at the basis of paclitaxel resistance, a comparative proteomic approach based on DIGE coupled with mass spectrometry (MALDI-TOF and LC-MS/MS) was applied to the human epithelial ovarian cancer cell lines A2780 and its paclitaxel resistant counterpart A2780TC1. Most of the differentially expressed proteins between the two cell lines belong to the class of stress response (29%), metabolism (21%), and cell cycle and apoptosis (17%). We focused on proteins which were most strongly modulated by paclitaxel resistance and in particular on the disulphide isomerase ERp57, which may represent a chemoresistance biomarker. ERp57 was found to interact with class III beta-tubulin (TUBB3), involved in paclitaxel resistance in ovarian and other cancers. Moreover, we demonstrated a novel localization of this protein in cytoskeleton and described that ERp57/TUBB3 interaction occurs also in the nuclear compartment and in association with a multimeric complex formed by nucleolin, nucleophosmin, hnRNPK, and mortalin. Our data suggest that ERp57 plays an important role in chemoresistance mechanisms in ovarian cancer by modulating the attachment of microtubules to chromosomes following paclitaxel treatment through its interaction with TUBB3.

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Section: Resultsmentioning

confidence: 66%

Comparative Proteomic Analysis of Paclitaxel Sensitive A2780 Epithelial Ovarian Cancer Cell Line and Its Resistant Counterpart A2780TC1 by 2D-DIGE: The Role of ERp57

Cicchillitti

Urbani

et al. 2009

J. Proteome Res.

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“…It is followed by a post-translational phase, where disulfides are rearranged into the native set that fulfills quality control requirements. The first phase is completed in about 2 min, the time required to synthesize and translocate in the ER lumen the few hundred residues of the nascent chains (514 for HA, 438 for E1, and 482 for p62, respectively, for the model proteins used in this study) and was investigated in this work with a sophisticated technical approach based on metabolic labeling of nascent chains and their analysis by diagonal gel electrophoresis (20,21). The second phase lasts for about 15-20 min in wt cells for HA and a bit longer for E1 and p62.…”

Section: Discussionmentioning

confidence: 99%

“…Analysis of co-translational formation of intra-and intermolecular disulfide bonds by diagonal gel electrophoresis is described in Ref. 20.…”

Section: Analysis Of Transcripts By Reverse Transcription-pcr-mentioning

confidence: 99%

Consequences of ERp57 Deletion on Oxidative Folding of Obligate and Facultative Clients of the Calnexin Cycle

Soldà¹,

Garbi

Hämmerling

et al. 2006

Journal of Biological Chemistry

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105

115

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Members of the protein-disulfide isomerase superfamily catalyze the formation of intra-and intermolecular disulfide bonds, a ratelimiting step of protein folding in the endoplasmic reticulum (ER).Here we compared maturation of one obligate and two facultative calnexin substrates in cells with and without ERp57, the calnexinassociated, glycoprotein-specific oxidoreductase. ERp57 deletion did not prevent the formation of disulfide bonds during co-translational translocation of nascent glycopolypeptides in the ER. It affected, however, the post-translational phases of oxidative influenza virus hemagglutinin (HA) folding, resulting in significant loss of folding efficiency for this obligate calnexin substrate. Without ERp57, HA also showed reduced capacity to recover from an artificially induced aberrant conformation, thus revealing a crucial role of ERp57 during post-translational reshuffling to the native set of HA disulfides. ERp57 deletion did not affect maturation of the model facultative calnexin substrates E1 and p62 (and of most cellular proteins, as shown by lack of induction of ER stress). ERp72 was identified as one of the ER-resident oxidoreductases associating with the orphan ERp57 substrates to maintain their folding competence.Polypeptide asparagines emerging in the ER 4 lumen as part of an Asn-X-Ser/Thr motif are covalently modified with preassembled glycans containing 2 N-acetylglucosamine, 9 mannose, and 3 glucose residues (1). N-Glycosylation prepares nascent chains for association with the two lectin-like molecular chaperones calnexin and calreticulin and the glycoprotein-dedicated oxidoreductase ERp57 (2-5).Deletion of individual members of the calnexin chaperone system, namely calreticulin (6), UGT1 (7), and ERp57 (8), is embryonic lethal in mice. Deletion of calnexin does not result in embryonic lethality but causes a severe progressive pathology leading to premature death (9). The suboptimal glycoprotein folding efficiency upon chaperone deletion might be detrimental for organism viability, but all of these deletions are well tolerated at the cellular level. In fact, only a restricted number of endogenous, recombinant, or virus-encoded glycoproteins (e.g. the major histocompatibility complex class I peptide loading complex (8, 10), the influenza virus HA (11), and the ADAM1/ADAM2 fertilin complex (12)) have so far been shown to strongly depend on the calnexin chaperone system for maturation. Consistently, and as previously shown for calnexin, calreticulin, and UGT1 deletions, cells lacking ERp57 show normal viability and proliferation rates and unperturbed maturation and transport of several surface glycoproteins containing disulfide bonds (8). Conditional deletion of ERp57 in B lymphocytes/ plasma cells revealed no defect in the production of immunoglobulin chains, the most abundant N-glycosylated/disulfide-bonded products of these cells (8). The thorough analysis by Garbi et al. showed that ERp57 deletion specifically affected assembly and stability of the major histocompatibility class I p...

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“…Chaperone proteins were immunoprecipitated using a protocol modified from one previously described (44). Cells were lysed on ice for 30 min in 2% CHAPS/50 mM Hepes, pH 6.8 or 7.4/200 mM NaCl/1 mM CaCl 2 containing the following protease inhibitors: 1 mM PMSF and 10 μg/ml each of Antipain, Aprotinin, Chymostatin, Leupeptin and Pepstatin A (Sigma-Aldrich).…”

Section: Methodsmentioning

confidence: 99%

Interactions of NK Cell Receptor KIR3DL1*004 with Chaperones and Conformation-Specific Antibody Reveal a Functional Folded State As Well As Predominant Intracellular Retention

Taner

Pando

Roberts

et al. 2011

The Journal of Immunology

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Variable interaction between the Bw4 epitope of HLA-B and the polymorphic KIR3DL1/S1 system of inhibitory and activating NK cell receptors diversifies the development, repertoire formation and response of human NK cells. KIR3DL1*004, a common KIR3DL1 allotype, in combination with Bw4+, HLA-B slows progression of HIV infection to AIDS. Analysis here of KIR3DL1*004 membrane traffic in NK cells shows this allotype is largely misfolded but stably retained in the endoplasmic reticulum, where it binds to the chaperone calreticulin and does not induce the unfolded protein response. A small fraction of KIR3DL1*004 folds correctly and leaves the endoplasmic reticulum to be expressed on the surface of primary NK and transfected NKL cells, in a form that can be triggered to inhibit NK cell activation and secretion of interferon-γ. Consistent with this small proportion of correctly-folded molecules, trace amounts of MHC Class I co-immunoprecipitated with KIR3DL1*004. There was no indication of any extensive intracellular interaction between unfolded KIR3DL1*004 and cognate Bw4+ HLA-B. A similarly limited interaction of Bw4 with KIR3DL1*002, when both were expressed by the same cell, was observed despite the efficient folding of KIR3DL1*002 and its abundance on the NK cell surface. Several positions of polymorphism modulate KIR3DL1 abundance at the cell surface, differences that do not necessarily correlate with the potency of allotype function. In this context our results suggest the possibility that the effect of Bw4+ HLA-B and KIR3DL1*004 in slowing progression to AIDS is mediated by interaction of Bw4+ HLA-B with the small fraction of cell surface KIR3DL1*004.

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Analyzing folding and degradation of metabolically labelled polypeptides by conventional and diagonal sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Cited by 4 publications

References 23 publications

Comparative Proteomic Analysis of Paclitaxel Sensitive A2780 Epithelial Ovarian Cancer Cell Line and Its Resistant Counterpart A2780TC1 by 2D-DIGE: The Role of ERp57

Comparative Proteomic Analysis of Paclitaxel Sensitive A2780 Epithelial Ovarian Cancer Cell Line and Its Resistant Counterpart A2780TC1 by 2D-DIGE: The Role of ERp57

Consequences of ERp57 Deletion on Oxidative Folding of Obligate and Facultative Clients of the Calnexin Cycle

Interactions of NK Cell Receptor KIR3DL1*004 with Chaperones and Conformation-Specific Antibody Reveal a Functional Folded State As Well As Predominant Intracellular Retention

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