Protein disulfide isomerase–like proteins play opposing roles during retrotranslocation

Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
The Journal of Cell Biology (Impact Factor: 9.69). 07/2006; 173(6):853-9. DOI: 10.1083/jcb.200602046
Source: PubMed

ABSTRACT Misfolded proteins in the endoplasmic reticulum (ER) are retained in the organelle or retrotranslocated to the cytosol for proteasomal degradation. ER chaperones that guide these opposing processes are largely unknown. We developed a semipermeabilized cell system to study the retrotranslocation of cholera toxin (CT), a toxic agent that crosses the ER membrane to reach the cytosol during intoxication. We found that protein disulfide isomerase (PDI) facilitates CT retrotranslocation, whereas ERp72, a PDI-like protein, mediates its ER retention. In vitro analysis revealed that PDI and ERp72 alter CT's conformation in a manner consistent with their roles in retrotranslocation and ER retention. Moreover, we found that PDI's and ERp72's opposing functions operate on endogenous ER misfolded proteins. Thus, our data identify PDI family proteins that play opposing roles in ER quality control and establish an assay to further delineate the mechanism of CT retrotranslocation.

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Available from: Wayne I Lencer, Aug 17, 2015
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    • "Once the toxin is released from BiP, CTA is reduced to generate CTA1 via the action of an unidentified reductase. PDI then unfolds CTA1 (Tsai et al., 2001; Forster et al., 2006), priming the toxin for retrotranslocation across the Hrd1 complex (Figure 6B, step 2). How 90 min, coimmunoprecipitation experiments revealed that endogenous Hrd1 binds CTA less efficiently at both time points when ERdj5 is knocked down using ERdj5 #1 siRNA (Figure 6A, top panel, compare lane 2 with lane 1 and lane 4 with lane 3). "
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    ABSTRACT: Cholera toxin (CT) traffics from the host cell surface to the endoplasmic reticulum (ER) where the toxin's catalytic CTA1 subunit retro-translocates to the cytosol to induce toxicity. In the ER, CT is captured by the E3 ubiquitin ligase Hrd1 via an undefined mechanism to prepare for retro-translocation. Using loss- and gain-of function approaches, we demonstrate that the ER-resident factor ERdj5 promotes CTA1 retro-translocation, in part, via its J domain. This Hsp70 cochaperone regulates binding between CTA and the ER Hsp70 BiP, a chaperone previously implicated in toxin retro-translocation. Importantly, ERdj5 interacts with the Hrd1 adapter Sel1L directly through Sel1L's N-terminal lumenal domain, thereby linking ERdj5 to the Hrd1 complex. Sel1L itself also binds CTA and facilitates toxin retro-translocation. By contrast, EDEM1 and OS-9, two established Sel1L binding partners, do not play significant roles in CTA1 retro-translocation. Our results thus identify two ER factors that promote ER-to-cytosol transport of CTA1. They also indicate ERdj5, by binding to Sel1L, triggers BiP-toxin interaction proximal to the Hrd1 complex. We postulate this scenario enables the Hrd1-associated retro-translocation machinery to capture the toxin efficiently once the toxin is released from BiP.
    Molecular biology of the cell 01/2013; 24(6). DOI:10.1091/mbc.E12-07-0522 · 5.98 Impact Factor
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    • "S-nitrosation Inhibition of chaperone/isomerase activities Catalysis of transnitrosation [116–118,140] Neurotoxicity in Alzheimer disease and amyotrophic lateral sclerosis Glutathionylation Decreased reductase activity UPR signaling in cancer cells [164] Phosphorylation No change in isomerase activity Transient autophosphorylation, unknown function [165] Arginylation N-end rule signal for PDI ubiquitination/proteasomal degradation [166] [167] Cleavage Caspase-3 and -7 cleave PDI in vitro [168] [169] Possible marker of hepatocellular carcinoma 4-Hydroxynonenal addition Decreased reductase activity Possible PDI impairment during oxidative stress [170] O-linked N-acetylglucosamine addition Potential involvement in transcription, proliferation, apoptosis, and proteasomal degradation [171] Metal binding Ca 2+ : PDI is a major calcium binding protein of the ER via acidic residues; role unknown [172] [173] [174] [175] Cu + : PDI can bind 10 Cu + /mol and form a tetramer Zn 2+ : induces PDI dimers/oligomers with decreased isomerase activity; unknown function 1961 F.R.M. Laurindo et al. / Free Radical Biology & Medicine 52 (2012) 1954–1969 has been described with mutant huntingtin [93] in association with ER stress. Known effects of PDI in assisting cytosolic retrotranslocation of un/misfolded proteins for proteasome-mediated degradation may participate in those processes [143]. Ero1-mediated oxidation can, for example with cholera toxin, release retrotranslocating substrates from translocon-associated PDI [33]. "
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    ABSTRACT: Thiol proteins may potentially act as redox signaling adaptor proteins, adjusting reactive oxygen species intermediates to specific signals and redox signals to cell homeostasis. In this review, we discuss redox effects of protein disulfide isomerase (PDI), a thioredoxin superfamily oxidoreductase from the endoplasmic reticulum (ER). Abundantly expressed PDI displays ubiquity, interactions with redox and nonredox proteins, versatile effects, and several posttranslational modifications. The PDI family contains >20 members with at least some apparent complementary actions. PDI has oxidoreductase, isomerase, and chaperone effects, the last not directly dependent on its thiols. PDI is a converging hub for pathways of disulfide bond introduction into ER-processed proteins, via hydrogen peroxide-generating mechanisms involving the oxidase Ero1α, as well as hydrogen peroxide-consuming reactions involving peroxiredoxin IV and the novel peroxidases Gpx7/8. PDI is a candidate pathway for coupling ER stress to oxidant generation. Emerging information suggests a convergence between PDI and Nox family NADPH oxidases. PDI silencing prevents Nox responses to angiotensin II and inhibits Akt phosphorylation in vascular cells and parasite phagocytosis in macrophages. PDI overexpression spontaneously enhances Nox activation and expression. In neutrophils, PDI redox-dependently associates with p47phox and supports the respiratory burst. At the cell surface, PDI exerts transnitrosation, thiol reductase, and apparent isomerase activities toward targets including adhesion and matrix proteins and proteases. Such effects mediate redox-dependent adhesion, coagulation/thrombosis, immune functions, and virus internalization. The route of PDI externalization remains elusive. Such multiple redox effects of PDI may contribute to its conspicuous expression and functional role in disease, rendering PDI family members putative redox cell signaling adaptors.
    Free Radical Biology and Medicine 03/2012; 52(9):1954-69. DOI:10.1016/j.freeradbiomed.2012.02.037 · 5.71 Impact Factor
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    • "Similar studies in mammalian cells have been hampered by the plethora of homologues and by the difficulties in maintaining cell viability when PDI levels are constitutively depleted (Park et al., 2006). However, transient PDI knockdown experiments have implicated this enzyme in facilitating cholera toxin retrotranslocation from ER to cytosol whereas the related family members ERp57 and ERp72 either had no effect or an opposing effect, respectively (Forster et al., 2006). Furthermore, PDI, but not ERp57, has been shown to affect the production of infectious rotavirus particles (Maruri-Avidal et al., 2008) and the folding of human MHC class I heavy chains (Kang et al., 2009). "
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    ABSTRACT: To examine the relationship between protein disulfide isomerase family members within the mammalian endoplasmic reticulum, PDI, ERp57, ERp72, and P5 were depleted with high efficiency in human hepatoma cells, either singly or in combination. The impact was assessed on the oxidative folding of several well-characterized secretory proteins. We show that PDI plays a predominant role in oxidative folding because its depletion delayed disulfide formation in all secretory proteins tested. However, the phenotype was surprisingly modest suggesting that other family members are able to compensate for PDI depletion, albeit with reduced efficacy. ERp57 also exhibited broad specificity, overlapping with that of PDI, but with preference for glycosylated substrates. Depletion of both PDI and ERp57 revealed that some substrates require both enzymes for optimal folding and, furthermore, led to generalized protein misfolding, impaired export from the ER, and degradation. In contrast, depletion of ERp72 or P5, either alone or in combination with PDI or ERp57 had minimal impact, revealing a narrow substrate specificity for ERp72 and no detectable role for P5 in oxidative protein folding.
    Molecular biology of the cell 09/2010; 21(18):3093-105. DOI:10.1091/mbc.E10-04-0356 · 5.98 Impact Factor
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