scholarly journals Unfolded cholera toxin is transferred to the ER membrane and released from protein disulfide isomerase upon oxidation by Ero1

2002 ◽  
Vol 159 (2) ◽  
pp. 207-216 ◽  
Author(s):  
Billy Tsai ◽  
Tom A. Rapoport
Keyword(s):  
Toxic Effect ◽  
Cholera Toxin ◽  
Disulfide Bond ◽  
Protein Disulfide Isomerase ◽  
Reduced Form ◽  
Target Cells ◽  
Disulfide Isomerase ◽  
Toxin Complex ◽  
Protein Disulfide ◽  
Er Membrane

The toxic effect of cholera toxin (CT) on target cells is caused by its A1 chain. This polypeptide is released from the holotoxin and unfolded in the lumen of the ER by the action of protein disulfide isomerase (PDI), before being retrotranslocated into the cytosol. The polypeptide is initially unfolded by binding to the reduced form of PDI. We show that upon oxidation of the COOH-terminal disulfide bond in PDI by the enzyme Ero1, the A1 chain is released. Both yeast Ero1 and the mammalian Ero1α isoform are active in this reaction. Ero1 has a preference for the PDI–toxin complex. We further show that the complex is transferred to a protein at the lumenal side of the ER membrane, where the unfolded toxin is released from PDI by the action of Ero1. Taken together, our results identify Ero1 as the enzyme mediating the release of unfolded CT from PDI and characterize an additional step in retrotranslocation of the toxin.

scholarly journals Protein disulfide isomerase does not act as an unfoldase in the disassembly of cholera toxin

2018 ◽  
Vol 38 (5) ◽  
Author(s):  
Patrick Cherubin ◽  
Jessica Guyette ◽  
Michael Taylor ◽  
Morgan O’Donnell ◽  
Laura Herndon ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Cholera Toxin ◽  
Disulfide Bond ◽  
Protein Disulfide Isomerase ◽  
Functional Role ◽  
Cellular Activity ◽  
Cell Binding ◽  
Disulfide Isomerase ◽  
Protein Disulfide ◽  
Multiple Conditions

Cholera toxin (CT) is composed of a disulfide-linked A1/A2 heterodimer and a ring-like, cell-binding B homopentamer. The catalytic A1 subunit must dissociate from CTA2/CTB5 to manifest its cellular activity. Reduction of the A1/A2 disulfide bond is required for holotoxin disassembly, but reduced CTA1 does not spontaneously separate from CTA2/CTB5: protein disulfide isomerase (PDI) is responsible for displacing CTA1 from its non-covalent assembly in the CT holotoxin. Contact with PDI shifts CTA1 from a protease-resistant conformation to a protease-sensitive conformation, which is thought to represent the PDI-mediated unfolding of CTA1. Based solely on this finding, PDI is widely viewed as an ‘unfoldase’ that triggers toxin disassembly by unfolding the holotoxin-associated A1 subunit. In contrast with this unfoldase model of PDI function, we report the ability of PDI to render CTA1 protease-sensitive is unrelated to its role in toxin disassembly. Multiple conditions that promoted PDI-induced protease sensitivity in CTA1 did not support PDI-mediated disassembly of the CT holotoxin. Moreover, preventing the PDI-induced shift in CTA1 protease sensitivity did not affect PDI-mediated disassembly of the CT holotoxin. Denatured PDI could still convert CTA1 into a protease-sensitive state, and equal or excess molar fractions of PDI were required for both efficient conversion of CTA1 into a protease-sensitive state and efficient disassembly of the CT holotoxin. These observations indicate the ‘unfoldase’ property of PDI does not play a functional role in CT disassembly and does not represent an enzymatic activity.

scholarly journals Structure-Function Analysis of the Endoplasmic Reticulum Oxidoreductase TMX3 Reveals Interdomain Stabilization of the N-terminal Redox-active Domain

2007 ◽  
Vol 282 (46) ◽  
pp. 33859-33867 ◽  
Author(s):  
Johannes Haugstetter ◽  
Michael Andreas Maurer ◽  
Thomas Blicher ◽  
Martin Pagac ◽  
Gerhard Wider ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Disulfide Isomerase ◽  
Function Analysis ◽  
Transmembrane Protein ◽  
Reduced Form ◽  
Disulfide Isomerase ◽  
Binding Domains ◽  
Redox Active ◽  
Protein Disulfide ◽  
Protein Disulfide Isomerase Family

Disulfide bond formation in the endoplasmic reticulum is catalyzed by enzymes of the protein disulfide-isomerase family that harbor one or more thioredoxin-like domains. We recently discovered the transmembrane protein TMX3, a thiol-disulfide oxidoreductase of the protein disulfide-isomerase family. Here, we show that the endoplasmic reticulum-luminal region of TMX3 contains three thioredoxin-like domains, an N-terminal redox-active domain (named a) followed by two enzymatically inactive domains (b and b′). Using the recombinantly expressed TMX3 domain constructs a, ab, and abb′, we compared structural stability and enzymatic properties. By structural and biophysical methods, we demonstrate that the reduced a domain has features typical of a globular folded domain that is, however, greatly destabilized upon oxidization. Importantly, interdomain stabilization by the b domain renders the a domain more resistant toward chemical denaturation and proteolysis in both the oxidized and reduced form. In combination with molecular modeling studies of TMX3 abb′, the experimental results provide a new understanding of the relationship between the multidomain structure of TMX3 and its function as a redox enzyme. Overall, the data indicate that in addition to their role as substrate and co-factor binding domains, redox-inactive thioredoxin-like domains also function in stabilizing neighboring redox-active domains.

scholarly journals Protein Disulfide Isomerase Acts as a Redox-Dependent Chaperone to Unfold Cholera Toxin

Cell ◽  
2001 ◽  
Vol 104 (6) ◽  
pp. 937-948 ◽  
Author(s):  
Billy Tsai ◽  
Chiara Rodighiero ◽  
Wayne I. Lencer ◽  
Tom A. Rapoport
Keyword(s):  
Cholera Toxin ◽  
Protein Disulfide Isomerase ◽  
Disulfide Isomerase ◽  
Protein Disulfide

scholarly journals Quercetin-3-Rutinoside Blocks the Disassembly of Cholera Toxin by Protein Disulfide Isomerase

Toxins ◽  
2019 ◽  
Vol 11 (8) ◽  
pp. 458 ◽  
Author(s):  
Jessica Guyette ◽  
Patrick Cherubin ◽  
Albert Serrano ◽  
Michael Taylor ◽  
Faisal Abedin ◽  
...  
Keyword(s):  
Cholera Toxin ◽  
Protein Disulfide Isomerase ◽  
Cytopathic Effect ◽  
Thrombus Formation ◽  
Inhibitory Effect ◽  
Dependent Manner ◽  
Oxidoreductase Activity ◽  
Disulfide Isomerase ◽  
Ricin Toxin ◽  
Protein Disulfide

Protein disulfide isomerase (PDI) is mainly located in the endoplasmic reticulum (ER) but is also secreted into the bloodstream where its oxidoreductase activity is involved with thrombus formation. Quercetin-3-rutinoside (Q3R) blocks this activity, but its inhibitory mechanism against PDI is not fully understood. Here, we examined the potential inhibitory effect of Q3R on another process that requires PDI: disassembly of the multimeric cholera toxin (CT). In the ER, PDI physically displaces the reduced CTA1 subunit from its non-covalent assembly in the CT holotoxin. This is followed by CTA1 dislocation from the ER to the cytosol where the toxin interacts with its G protein target for a cytopathic effect. Q3R blocked the conformational change in PDI that accompanies its binding to CTA1, which, in turn, prevented PDI from displacing CTA1 from its holotoxin and generated a toxin-resistant phenotype. Other steps of the CT intoxication process were not affected by Q3R, including PDI binding to CTA1 and CT reduction by PDI. Additional experiments with the B chain of ricin toxin found that Q3R could also disrupt PDI function through the loss of substrate binding. Q3R can thus inhibit PDI function through distinct mechanisms in a substrate-dependent manner.

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