Faculty Opinions recommendation of Protein disulfide isomerase acts as a redox-dependent chaperone to unfold cholera toxin.

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

The Journal of Cell Biology ◽

10.1083/jcb.200207120 ◽

2002 ◽

Vol 159 (2) ◽

pp. 207-216 ◽

Cited By ~ 98

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.

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Faculty Opinions recommendation of Unfolded cholera toxin is transferred to the ER membrane and released from protein disulfide isomerase upon oxidation by Ero1.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1010341.151107 ◽

2002 ◽

Author(s):

Tom Stevens

Keyword(s):

Cholera Toxin ◽

Protein Disulfide Isomerase ◽

Disulfide Isomerase ◽

Protein Disulfide ◽

Er Membrane

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The nucleotide exchange factors Grp170 and Sil1 induce cholera toxin release from BiP to enable retrotranslocation

Molecular Biology of the Cell ◽

10.1091/mbc.e15-01-0014 ◽

2015 ◽

Vol 26 (12) ◽

pp. 2181-2189 ◽

Cited By ~ 14

Author(s):

Jeffrey M. Williams ◽

Takamasa Inoue ◽

Grace Chen ◽

Billy Tsai

Keyword(s):

Cholera Toxin ◽

Ubiquitin Ligase ◽

Protein Disulfide Isomerase ◽

Nucleotide Exchange ◽

Binding Studies ◽

Disulfide Isomerase ◽

Binding Partner ◽

Nucleotide Exchange Factors ◽

Protein Disulfide ◽

J Protein

Cholera toxin (CT) intoxicates cells by trafficking from the cell surface to the endoplasmic reticulum (ER), where the catalytic CTA1 subunit hijacks components of the ER-associated degradation (ERAD) machinery to retrotranslocate to the cytosol and induce toxicity. In the ER, CT targets to the ERAD machinery composed of the E3 ubiquitin ligase Hrd1-Sel1L complex, in part via the activity of the Sel1L-binding partner ERdj5. This J protein stimulates BiP's ATPase activity, allowing BiP to capture the toxin. Presumably, toxin release from BiP must occur before retrotranslocation. Here, using loss-and gain-of-function approaches coupled with binding studies, we demonstrate that the ER-resident nucleotide exchange factors (NEFs) Grp170 and Sil1 induce CT release from BiP in order to promote toxin retrotranslocation. In addition, we find that after NEF-dependent release from BiP, the toxin is transferred to protein disulfide isomerase; this ER redox chaperone is known to unfold CTA1, which allows the toxin to cross the Hrd1-Sel1L complex. Our data thus identify two NEFs that trigger toxin release from BiP to enable successful retrotranslocation and clarify the fate of the toxin after it disengages from BiP.

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Substrate-Induced Unfolding of Protein Disulfide Isomerase Displaces the Cholera Toxin A1 Subunit from its Holotoxin

Biophysical Journal ◽

10.1016/j.bpj.2013.11.2668 ◽

2014 ◽

Vol 106 (2) ◽

pp. 472a ◽

Cited By ~ 1

Author(s):

Michael Taylor ◽

Tuhina Banerjee ◽

Supriyo Ray ◽

Helen Burress ◽

Suren A. Tatulian ◽

...

Keyword(s):

Cholera Toxin ◽

Protein Disulfide Isomerase ◽

Disulfide Isomerase ◽

Protein Disulfide

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

Bioscience Reports ◽

10.1042/bsr20181320 ◽

2018 ◽

Vol 38 (5) ◽

Cited By ~ 4

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.

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Holotoxin Disassembly by Protein Disulfide Isomerase Is Less Efficient for Escherichia Coli Heat-labile Enterotoxin Than Cholera Toxin

10.21203/rs.3.rs-918594/v1 ◽

2021 ◽

Author(s):

Albert Serrano ◽

Jessica L. Guyette ◽

Joel B. Heim ◽

Michael Taylor ◽

Patrick Cherubin ◽

...

Keyword(s):

Escherichia Coli ◽

Cholera Toxin ◽

Protein Disulfide Isomerase ◽

Protein Docking ◽

Disulfide Isomerase ◽

Mechanisms Of Toxicity ◽

Stable Domain ◽

Heat Labile ◽

Structural Differences ◽

Protein Disulfide

Abstract Cholera toxin (CT) and Escherichia coli heat-labile enterotoxin (LT) are structurally similar AB5-type protein toxins. They move from the cell surface to the endoplasmic reticulum where the A1 catalytic subunit is separated from its holotoxin by protein disulfide isomerase (PDI), thus allowing the dissociated A1 subunit to enter the cytosol for a toxic effect. Despite similar mechanisms of toxicity, CT is more potent than LT. The difference has been attributed to a more stable domain assembly for CT as compared to LT, but this explanation has not been directly tested and is arguable as toxin disassembly is an indispensable step in the cellular action of these toxins. We show here that PDI disassembles CT more efficiently than LT, which provides a possible explanation for the greater potency of the former toxin. Furthermore, direct examination of CT and LT domain assemblies found no difference in toxin stability. Using novel analytic geometry approaches, we provide a detailed characterization of the positioning of the A subunit with respect to the B5 pentamer and demonstrate significant differences in the interdomain architecture of CT and LT. Protein docking analysis further shows that these global structural differences result in distinct modes of PDI-toxin interactions. Our results highlight previously overlooked structural differences between CT and LT that provide a new molecular explanation for the PDI-assisted disassembly and differential potency of these toxins.

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