The Ero1α-PDI Redox Cycle Regulates Retro-Translocation of Cholera Toxin

Moore, Paul C.; Bernardi, Kaleena M.; Tsai, Billy

doi:10.1091/mbc.e09-09-0826

Cited by 35 publications

(33 citation statements)

References 41 publications

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“…That model is based upon studies performed at temperatures below 37°C. While Ero1p clearly regulates the redox status of PDI (22,23,47,48), our results strongly suggest that Ero1p function is not required to release PDI from CTA1 at physiological temperature.…”

Section: Discussionmentioning

confidence: 57%

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Protein-disulfide Isomerase Displaces the Cholera Toxin A1 Subunit from the Holotoxin without Unfolding the A1 Subunit

Taylor

Banerjee

Ray

et al. 2011

Journal of Biological Chemistry

127

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Protein-disulfide isomerase (PDI) has been proposed to exhibit an "unfoldase" activity against the catalytic A1 subunit of cholera toxin (CT). Unfolding of the CTA1 subunit is thought to displace it from the CT holotoxin and to prepare it for translocation to the cytosol. To date, the unfoldase activity of PDI has not been demonstrated for any substrate other than CTA1. An alternative explanation for the putative unfoldase activity of PDI has been suggested by recent structural studies demonstrating that CTA1 will unfold spontaneously upon its separation from the holotoxin at physiological temperature. Thus, PDI may simply dislodge CTA1 from the CT holotoxin without unfolding the CTA1 subunit. To evaluate the role of PDI in CT disassembly and CTA1 unfolding, we utilized a real-time assay to monitor the PDI-mediated separation of CTA1 from the CT holotoxin and directly examined the impact of PDI binding on CTA1 structure by isotope-edited Fourier transform infrared spectroscopy. Our collective data demonstrate that PDI is required for disassembly of the CT holotoxin but does not unfold the CTA1 subunit, thus uncovering a new mechanism for CTA1 dissociation from its holotoxin. Cholera toxin (CT)2 is an AB 5 protein toxin that consists of a catalytic A moiety and a cell-binding B moiety (1, 2). The B subunit is pentameric ring-like structure that adheres to GM1 gangliosides on the plasma membrane of a target cell. The A subunit is initially synthesized as a 26 kDa protein that undergoes proteolytic nicking to generate a disulfide-linked A1/A2 heterodimer. The 21 kDa CTA1 polypeptide is an ADP-ribosyltransferase that modifies and activates Gs␣ in the host cell cytosol. CTA1 can be divided into three subdomains: the A1 1 subdomain contains the catalytic core of the toxin; the A1 2 subdomain is a short extended linker that connects the A1 1 and A1 3 subdomains; and the A1 3 subdomain is a globular structure with many hydrophobic residues as well as a cysteine residue involved with the single disulfide bridge between CTA1 and CTA2 (3). The 5 kDa CTA2 polypeptide maintains numerous non-covalent interactions with the central pore of the B pentamer and thereby anchors CTA1 to CTB 5 . A ribbon diagram of the CT holotoxin which highlights the subdomain structure of CTA1 is provided in supplemental Fig. S1.To reach its cytosolic Gs␣ target, CT moves from the cell surface to the endoplasmic reticulum (ER) by retrograde vesicular traffic (4). A C-terminal KDEL sequence in the CTA2 subunit is thought to target and/or retain CT in the ER (4, 5). Conditions in the ER lead to reductive cleavage of the CTA1/CTA2 disulfide bond and chaperone-assisted dissociation of CTA1 from CTA2/CTB 5 (6 -9). Unfolding of the free A1 subunit then activates the quality control system of ER-associated degradation (ERAD), thereby promoting CTA1 translocation to the cytosol (10, 11). Most exported ERAD substrates are degraded by the ubiquitin-proteasome system, but it was hypothesized that CTA1 and the A chains of other ER-translocating toxins avoid t...

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Section: Discussionmentioning

confidence: 57%

“…It was further posited that unfolding of the CTA1 polypeptide was directly coupled with toxin delivery to the Derlin-1 pore (21,22). Release of CTA1 from PDI in preparation for translocation through the Derlin-1 pore was thought to involve the oxidation of PDI by Ero1p (23).…”

Section: Cholera Toxin (Ct)mentioning

confidence: 99%

Protein-disulfide Isomerase Displaces the Cholera Toxin A1 Subunit from the Holotoxin without Unfolding the A1 Subunit

Taylor

Banerjee

Ray

et al. 2011

Journal of Biological Chemistry

127

View full text Add to dashboard Cite

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“…This, however, is a departure from a current model of CTA1 translocation in which PDI is required to unfold the supposedly stable CTA1 polypeptide (45). Moreover, PDI has been proposed to bypass the ERAD system and deliver unfolded CTA1 directly to the Derlin-1 pore for export to the cytosol (5,28). Our recent structural studies have challenged this model by demonstrating that PDI does not act as a CTA1 "unfoldase" (39) and that CTA1 will spontaneously unfold at physiological temperatures after its dissociation from the rest of the toxin (30).…”

Section: Discussionmentioning

confidence: 92%

Structural and Functional Interactions between the Cholera Toxin A1 Subunit and ERdj3/HEDJ, a Chaperone of the Endoplasmic Reticulum

et al. 2011

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Cholera toxin (CT) is endocytosed and transported by vesicle carriers to the endoplasmic reticulum (ER).The catalytic CTA1 subunit then crosses the ER membrane and enters the cytosol, where it interacts with its Gs␣ target. The CTA1 membrane transversal involves the ER chaperone BiP, but few other host proteins involved with CTA1 translocation are known. BiP function is regulated by ERdj3, an ER-localized Hsp40 chaperone also known as HEDJ. ERdj3 can also influence protein folding and translocation by direct substrate binding. In this work, structural and functional assays were used to examine the putative interaction between ERdj3 and CTA1. Cell-based assays demonstrated that expression of a dominant negative ERdj3 blocks CTA1 translocation into the cytosol and CT intoxication. Binding assays with surface plasmon resonance demonstrated that monomeric ERdj3 interacts directly with CTA1. This interaction involved the A1 2 subdomain of CTA1 and was further dependent upon the overall structure of CTA1: ERdj3 bound to unfolded but not folded conformations of the isolated CTA1 subunit. This was consistent with the chaperone function of ERdj3, as was the ability of ERdj3 to mask the solvent-exposed hydrophobic residues of CTA1. Our data identify ERdj3 as a host protein involved with the CT intoxication process and provide new molecular details regarding CTA1-chaperone interactions.Many toxins share a structural organization that consists of a catalytic A subunit and a cell-binding B subunit (32). These extracellular toxins attack targets within the eukaryotic cytosol and must therefore cross a membrane barrier in order to function. Some AB toxins, such as diphtheria toxin (DT), access the cytosol from acidified endosomes. Other AB toxins move from the plasma membrane to the endoplasmic reticulum (ER) before passage into the cytosol by a process involving the quality control system of ER-associated degradation (ERAD) (14, 21). For both endosome and ER translocation sites, holotoxin disassembly precedes or occurs concurrently with A chain entry into the cytosol.The process of ERAD-mediated toxin translocation is not completely defined, but some details have been elucidated for ERAD interactions with the catalytic A1 subunit of cholera toxin (CTA1). The single disulfide bond linking CTA1 to the rest of the cholera holotoxin (CT) is reduced at the resident redox state of the ER (22). Reduced CTA1 dissociates from the holotoxin with the aid of protein disulfide isomerase (PDI) (39, 45), and unfolding of the isolated CTA1 polypeptide then facilitates its passage into the cytosol through the Sec61 and/or Derlin-1 protein-conducting channels (5,11,33,34). Most ERAD substrates are efficiently degraded in the cytosol by the ubiquitin-proteasome system (47). CTA1 avoids this fate because it only has two lysine residues to serve as potential ubiquitin attachment sites (14,30,31). The translocated pool of CTA1 thus persists in the cytosol long enough to modify its Gs␣ target. A hydrophobic region in the C-terminal A1 3 subdomain o...

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“…It will also be instrumental to molecularly describe the mode(s) of interaction between Ero1a and ERp44 including the cysteine connectivities and subcellular localization of the complex (ER, ERGIC, or MAM) and compare it to the complex involving PDI (36). Since the catalytic Ero1a-PDI interaction plays a critical role during the PDI-assisted translocation of the cholera toxin A1 subunit from the ER to the cytosol (38), it also remains to be explored, if endogenous ER-associated degradation substrates require this interplay. Further, the mechanism of redox-driven activation of Ero1a/b remains to be elucidated, which-in conjunction with the aforementionedwill set the stage for more exciting Ero1 news in the future.…”

Section: Discussionmentioning

confidence: 99%

The Physiological Functions of Mammalian Endoplasmic Oxidoreductin 1: On Disulfides and More

Ramming

Appenzeller-Herzog²

2012

Antioxidants & Redox Signaling

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Significance: The oxidative process of disulfide-bond formation is essential for the folding of most secretory and membrane proteins in the endoplasmic reticulum (ER). It is driven by electron relay pathways that transfer two electrons derived from the fusion of two adjacent cysteinyl side chains onto various types of chemical oxidants. The conserved, ER-resident endoplasmic oxidoreductin 1 (Ero1) sulfhydryl oxidases that reduce molecular oxygen to generate an active-site disulfide represent one of these pathways. In mammals, two family members exist, Ero1a and Ero1b. Recent Advances: The two mammalian Ero1 enzymes differ in transcriptional and posttranslational regulation, tissue distribution, and catalytic turnover. A specific protein-protein interaction between either isoform and protein disulfide isomerase (PDI) facilitates the propagation of disulfides from Ero1 via PDI to nascent polypeptides, and inbuilt oxidative shutdown mechanisms in Ero1a and Ero1b prevent excessive oxidation of PDI. Critical Issues: Besides disulfide-bond generation, Ero1a also regulates calcium release from the ER and the secretion of disulfide-linked oligomers through its reversible association with the chaperone ERp44. This review explores the functional repertoire and possible redundancy of mammalian Ero1 enzymes. Future Directions: Systematic analyses of different knockout mouse models will be the most promising strategy to shed new light on unique and tissue-specific roles of Ero1a and Ero1b. Moreover, in-depth characterization of the known physical interactions of Ero1 with peroxidases and PDI family members will help broaden our functional and mechanistic understanding of Ero1 enzymes.

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The Ero1α-PDI Redox Cycle Regulates Retro-Translocation of Cholera Toxin

Abstract: Cholera toxin moves from the ER to the cytosol to intoxicate host cells. Here we demonstrate that the redox chaperones Ero1α and PDI play crucial roles in the ER-to-cytosol translocation of the toxin.

Cited by 35 publications

References 41 publications

Protein-disulfide Isomerase Displaces the Cholera Toxin A1 Subunit from the Holotoxin without Unfolding the A1 Subunit

Protein-disulfide Isomerase Displaces the Cholera Toxin A1 Subunit from the Holotoxin without Unfolding the A1 Subunit

Structural and Functional Interactions between the Cholera Toxin A1 Subunit and ERdj3/HEDJ, a Chaperone of the Endoplasmic Reticulum

The Physiological Functions of Mammalian Endoplasmic Oxidoreductin 1: On Disulfides and More

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