Cholera toxin (CT) enters the cell via receptor-mediated endocytosis and travels in a retrograde fashion to the endoplasmic reticulum (ER). The catalytic A1 subunit (CTA1) is then displaced from the rest of the holotoxin, unfolds, and is exported to the cytosol where it regains an active conformation for the ADP-ribosylation of its G-protein target. We have shown that the cytosolic chaperones Hsp90 and Hsc70 are required for CTA1 translocation to the cytosol. We have also shown that both are able to independently bind and refold CTA1. Using libraries of CTA1-derived peptides, we have identified a single Hsc70 binding site, YYIYVI (CTA1 83-88), within the 192 amino acid protein, as well as two distinct Hsp90 binding sites: an N-terminal RPPDEI (CTA111-16) motif and a C-terminal LDIAPA (CTA1 153-158) motif. The LDIAPA motif is unique to CTA1, but an RPPDEI-like motif is present in four other ER-translocating ADP-ribosylating toxins: pertussis toxin, Pseudomonas aeruginosa exotoxin A, Escherichia coli heat-labile toxin, and Salmonella typhimurium ADP-ribosylating toxin. Using site-directed mutagenesis to further investigate the RPPDEI motif, we found that a modification of either proline residue blocks CTA1 translocation to the cytosol. Our work has identified, for the first time, specific amino acid sequences that are recognized by Hsp90/Hsc70 and are essential for toxin translocation from the ER to the cytosol. CT does not require prolyl isomerases for cellular activity, as is the case for Hsp90-dependent endosome-translocating toxins. We therefore hypothesize that the one or both of the prolines within the RPPDEI motif of CTA1 undergo an isomerization event as CTA1 unfolds in the ER. Furthermore, we predict that the trans- to cis- conformational change of proline(s) is the molecular determinate for the atypical Hsp90 interaction observed with CTA1 and related toxins. Additionally, we have identified Hsp90 and other host factors required for the translocation of pertussis toxin.
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Doctor of Philosophy (Ph.D.)
College of Medicine
Length of Campus-only Access
Doctoral Dissertation (Campus-only Access)
Kellner, Alisha, "It Takes Two to Tango: The Toxin-chaperone Relationship" (2019). Electronic Theses and Dissertations. 6513.