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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.

Cholera toxin (CT) travels by vesicle carriers from the cell surface to the endoplasmic reticulum (ER) where the catalytic A1 subunit of CT (CTA1) dissociates from the rest of the toxin, unfolds, and moves through a membrane-spanning translocon pore to reach the cytosol. Heat shock protein 90 (HSP90) binds to the N-terminal region of CTA1 and facilitates its ER-to-cytosol export by refolding the toxin as it emerges at the cytosolic face of the ER membrane. HSP90 also refolds some endogenous cytosolic proteins as part of a foldosome complex containing heat shock cognate 71-kDa protein (HSC70) and the HSC70/HSP90-organizing protein (HOP) linker that anchors HSP90 to HSC70. We accordingly predicted that HSC70 and HOP also function in CTA1 translocation. Inactivation of HSC70 by drug treatment disrupted CTA1 translocation to the cytosol and generated a toxin-resistant phenotype. In contrast, the depletion of HOP did not disrupt CT activity against cultured cells. HSC70 and HSP90 could bind independently to disordered CTA1, even in the absence of HOP. This indicated HSP90 and HSC70 recognize distinct regions of CTA1, which was confirmed by the identification of a YYIYVI-binding motif for HSC70 that spans residues 83–88 of the 192-amino acid CTA1 polypeptide. Refolding of disordered CTA1 occurred in the presence of HSC70 alone, indicating that HSC70 and HSP90 can each independently refold CTA1. Our work suggests a novel translocation mechanism in which sequential interactions with HSP90 and HSC70 drive the N- to C-terminal extraction of CTA1 from the ER.

The catalytic A1 subunit of cholera toxin (CTA1) has a disordered structure at 37°C. An interaction with host factors must therefore place CTA1 in a folded conformation for the modification of its Gsα target which resides in a lipid raft environment. Host ADP-ribosylation factors (ARFs) act as in vitro allosteric activators of CTA1, but the molecular events of this process are not fully characterized. Isotope-edited Fourier transform infrared spectroscopy monitored ARF6-induced structural changes to CTA1, which were correlated to changes in CTA1 activity. We found ARF6 prevents the thermal disordering of structured CTA1 and stimulates the activity of stabilized CTA1 over a range of temperatures. Yet ARF6 alone did not promote the refolding of disordered CTA1 to an active state. Instead, lipid rafts shifted disordered CTA1 to a folded conformation with a basal level of activity that could be further stimulated by ARF6. Thus, ARF alone is unable to activate disordered CTA1 at physiological temperature: additional host factors such as lipid rafts place CTA1 in the folded conformation required for its ARF-mediated activation. Interaction with ARF is required for in vivo toxin activity, as enzymatically active CTA1 mutants that cannot be further stimulated by ARF6 fail to intoxicate cultured cells.

Cholera toxin (CT) is an AB-type protein toxin that contains a catalytic A1 subunit, an A2 linker, and a cell-binding B homopentamer. The CT holotoxin is released into the extracellular environment, but CTA1 attacks a target within the cytosol of a host cell. We recently reported that grape extract confers substantial resistance to CT. Here, we used a cell culture system to identify twelve individual phenolic compounds from grape extract that inhibit CT. Additional studies determined the mechanism of inhibition for a subset of the compounds: two inhibited CT binding to the cell surface and even stripped CT from the plasma membrane of a target cell; two inhibited the enzymatic activity of CTA1; and four blocked cytosolic toxin activity without directly affecting the enzymatic function of CTA1. Individual polyphenolic compounds from grape extract could also generate cellular resistance to diphtheria toxin, exotoxin A, and ricin. We have thus identified individual toxin inhibitors from grape extract and some of their mechanisms of inhibition against CT.

The catalytic A1 subunit of cholera toxin (CTA1) has a disordered structure at 37°C. An interaction with host factors must therefore place CTA1 in a folded conformation for the modification of its Gsα target which resides in a lipid raft environment. Host ADP-ribosylation factors (ARFs) act as in vitro allosteric activators of CTA1, but the molecular events of this process are not fully characterized. Isotope-edited Fourier transform infrared spectroscopy monitored ARF6-induced structural changes to CTA1, which were correlated to changes in CTA1 activity. We found ARF6 prevents the thermal disordering of structured CTA1 and stimulates the activity of stabilized CTA1 over a range of temperatures. Yet ARF6 alone did not promote the refolding of disordered CTA1 to an active state. Instead, lipid rafts shifted disordered CTA1 to a folded conformation with a basal level of activity that could be further stimulated by ARF6. Thus, ARF alone is unable to activate disordered CTA1 at physiological temperature: additional host factors such as lipid rafts place CTA1 in the folded conformation required for its ARF-mediated activation. Interaction with ARF is required for in vivo toxin activity, as enzymatically active CTA1 mutants that cannot be further stimulated by ARF6 fail to intoxicate cultured cells.

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 Gsα 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.

Cholera toxin (CT) is an AB(5) toxin that moves from the cell surface to the endoplasmic reticulum (ER) by retrograde vesicular transport. In the ER, the catalytic A1 subunit dissociates from the rest of the toxin and enters the cytosol by exploiting the quality control system of ER-associated degradation (ERAD). The driving force for CTA1 dislocation into the cytosol is unknown. Here, we demonstrate that the cytosolic chaperone Hsp90 is required for CTA1 passage into the cytosol. Hsp90 bound to CTA1 in an ATP-dependent manner that was blocked by geldanamycin (GA), an established Hsp90 inhibitor. CT activity against cultured cells and ileal loops was also blocked by GA, as was the ER-to-cytosol export of CTA1. Experiments using RNA interference or N-ethylcarboxamidoadenosine, a drug that inhibits ER-localized GRP94 but not cytosolic Hsp90, confirmed that the inhibitory effects of GA resulted specifically from the loss of Hsp90 activity. This work establishes a functional role for Hsp90 in the ERAD-mediated dislocation of CTA1.

Cholera toxin (CT) moves from the cell surface to the endoplasmic reticulum (ER) where the catalytic CTA1 subunit separates from the holotoxin and unfolds due to its intrinsic instability. Unfolded CTA1 then moves through an ER translocon pore to reach its cytosolic target. Due to the instability of CTA1, it must be actively refolded in the cytosol to achieve the proper conformation for modification of its G protein target. The cytosolic heat shock protein Hsp90 is involved with the ER-to-cytosol translocation of CTA1, yet the mechanistic role of Hsp90 in CTA1 translocation remains unknown. Potential post-translocation roles for Hsp90 in modulating the activity of cytosolic CTA1 are also unknown. Here, we show by isotope-edited Fourier transform infrared spectroscopy that Hsp90 induces a gain-of-function in disordered CTA1 at physiological temperature. Only the ATP-bound form of Hsp90 interacts with disordered CTA1, and its refolding of CTA1 is dependent upon ATP hydrolysis. Surface plasmon resonance experiments found that Hsp90 does not release CTA1, even after ATP hydrolysis and the return of CTA1 to a folded conformation. The interaction with Hsp90 allowed disordered CTA1 to attain an active state and did not prevent further stimulation of toxin activity by ADP-ribosylation factor 6, a host cofactor for CTA1. Our studies suggest CTA1 translocation involves a ratchet mechanism which couples the Hsp90-mediated refolding of CTA1 with CTA1 extraction from the ER. Continued association of Hsp90 with refolded CTA1 allows the cytosolic toxin to attain an active conformation but does not protect it from proteasomal degradation.