Proline Isomerization as a Key Determinant for Hsp90-Toxin Interactions.

The A chains of ADP-ribosylating toxins exploit Hsp90 for translocation into the host cytosol. Here, we hypothesize that cis proline residues play a key role in toxin recognition by Hsp90. Our model is largely derived from studies on the unusual interplay between Hsp90 and the catalytic A1 subunit of cholera toxin (CTA1), including the recent identification of an RPPDEI-like binding motif for Hsp90 in CTA1 and several other bacterial toxins. Cis/trans proline isomerization is known to influence protein-protein interactions and protein structure/function, but it has not yet been proposed to affect Hsp90-toxin interactions. Our model thus provides a new framework to understand the molecular basis for Hsp90 chaperone function and Hsp90-driven toxin translocation.

A binding motif for Hsp90 in the A chains of ADP-ribosylating toxins that move from the endoplasmic reticulum to the cytosol.

Cholera toxin (Ctx) is an AB-type protein toxin that acts as an adenosine diphosphate (ADP)-ribosyltransferase to disrupt intracellular signalling in the target cell. It moves by vesicle carriers from the cell surface to the endoplasmic reticulum (ER) of an intoxicated cell. The catalytic CtxA1 subunit then dissociates from the rest of the toxin, unfolds, and activates the ER-associated degradation system for export to the cytosol. Translocation occurs through an unusual ratchet mechanism in which the cytosolic chaperone Hsp90 couples CtxA1 refolding with CtxA1 extraction from the ER. Here, we report that Hsp90 recognises two peptide sequences from CtxA1: an N-terminal RPPDEI sequence (residues 11-16) and an LDIAPA sequence in the C-terminal region (residues 153-158) of the 192 amino acid protein. Peptides containing either sequence effectively blocked Hsp90 binding to full-length CtxA1. Both sequences were necessary for the ER-to-cytosol export of CtxA1. Mutagenesis studies further demonstrated that the RPP residues in the RPPDEI motif are required for CtxA1 translocation to the cytosol. The LDIAPA sequence is unique to CtxA1, but we identified an RPPDEI-like motif at the N- or C-termini of the A chains from four other ER-translocating toxins that act as ADP-ribosyltransferases: pertussis toxin, Escherichia coli heat-labile toxin, Pseudomonas aeruginosa exotoxin A, and Salmonella enterica serovar Typhimurium ADP-ribosylating toxin. Hsp90 plays a functional role in the intoxication process for most, if not all, of these toxins. Our work has established a defined RPPDEI binding motif for Hsp90 that is required for the ER-to-cytosol export of CtxA1 and possibly other toxin A chains as well.

HSC70 and HSP90 chaperones perform complementary roles in translocation of the cholera toxin A1 subunit from the endoplasmic reticulum to the cytosol.

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.

Avirulins, a Novel Class of HIV-1 Reverse Transcriptase Inhibitors Effective in the Female Reproductive Tract Mucosa.

While extensive research efforts have decreased human immunodeficiency virus (HIV) transmissions and mortalities, new challenges have arisen in the fight to eradicate HIV. Drug resistance to antiretroviral therapy threatens infected individuals, while the prevalence of heterosexual transmission creates an urgent need for therapies effective in the female reproductive tract (FRT) mucosa. We screened a library of 2095 small molecule compounds comprising a unique chemical space, purchased from Asinex Corporation, for antiviral activity against human immunodeficiency virus type 1 (HIV-1) strain BaL and identified several molecular representatives of a unique class of HIV-1 inhibitors, which we termed "Avirulins." We determined that Avirulins were active against clinical isolates of HIV-1 from genetically variant subtypes, several of which have reduced sensitivity to other antivirals. Avirulins displayed specific dose-dependent inhibition of the HIV-1 drug target, reverse transcriptase (RT). Avirulins were effective against several nucleoside RT-inhibitor resistant strains of HIV-1, as well as one nonnucleoside RT-inhibitor resistant strain containing a 106A mutation, suggesting a noncompetitive mechanism of action. Drugs, which are damaging to the FRT, can increase the risk of HIV-1 transmission. We therefore explored the cytotoxicity of Avirulins against epithelial cells derived from the FRT and found no significant toxicity, even at the highest concentrations tested. Importantly, Avirulin antiviral activity was not diminished in human cervico-vaginal fluid, suggesting retained potency in the milieu of the FRT. Based on these promising results, Avirulins should be valuable chemical scaffolds for development into next-generation treatments and preventatives that target HIV-1.