Keywords

Cholera toxin, Cytosol, Endoplasmic reticulum, Heat shock proteins

Abstract

AB-type protein toxins such as cholera toxin (CT) consist of a catalytic A subunit and a cell-binding B subunit. CT proceeds through the secretory pathway in reverse, termed retrograde trafficking, and is delivered to the endoplasmic reticulum (ER). In order for the catalytic A1 subunit to become active it must separate from the rest of the holotoxin, and this dissociation event occurs in the ER lumen. CTA1 assumes an unfolded conformation upon dissociation from the holotoxin and is recognized by ERassociated degradation (ERAD), a quality control system that recognizes and exports misfolded proteins to the cytosol for degradation by the 26S proteasome. CTA1 is not degraded by the 26S proteasome because it has few sites for poly-ubitiquination, which is recognized by the cap of the 26S proteasome for degradation. Thus, CTA1 escapes the degradation of ERAD while at the same time using it as a transport mechanism into the cytosol. It was originally proposed that CTA1 is thermally stable and that ER chaperones actively unfolded CTA1 for translocation to the cytosol. In contrast, we hypothesized that the dissociated CTA1 subunit would unfold spontaneously at 37°C. This study focused on the three conditions linked to CTA1 instability and translocation: (i) CTA1 dissociation from the holotoxin, (ii) the translocation-competent conformation of CTA1, and the extraction of CTA1 from the ER into the cytosol. Disruption of any of these events will confer resistance to the toxin. The original model suggested that PDI actively unfolds CTA1 to allow for translocation. However, Fourier transform infrared iv spectroscopy (FTIR) and surface plasmon resonance (SPR) data we have gathered demonstrated that PDI dislodges CTA1 from the rest of the holotoxin without unfolding CTA1. Once released by the holotoxin, CTA1 spontaneously unfolds. PDI is thus required for the toxicity of CT, but not as an unfoldase as originally proposed. CTA1 must maintain an unfolded conformation to keep its translocation-competent state. Based on our model, if CTA1 is stabilized then it will not be able to activate the ERAD translocation system. Our SPR and toxicity results demonstrated that treatment with 4- phenylbutyrate (PBA), a chemical chaperone, stabilizes the structure of CTA1. This stabilization resulted in a decrease in translocation from the ER to the cytosol and a block of intoxication, which makes it a viable candidate for a therapeutic. Because CTA1 exits the ER in an unfolded state, there must be a driving force for this translocation. We hypothesized that Hsp90, a cytosolic chaperone, is responsible for the translocation of CTA1 across the membrane. Previous research had shown Hsp90 to be present on the cytosolic face of the ER and had also shown that Hsp90 will refold exogenously added proteins that enter the cytosol. Using drug treatments and RNAi, we found that Hsp90 is required for the translocation of CTA1 from the ER lumen to the cytosol, a brand new function for this chaperone. We have provided evidence to support a new, substantially different model of CTA1 translocation. CTA1 does not masquerade as a misfolded protein in order to utilize ERAD for entry into the cytosol; it actually becomes misfolded and is treated as any other ERAD substrate. The spontaneous unfolding of CTA1 is the key to its v recognition by ERAD and ultimately its translocation into the cytosol. Host factors play very important roles in intoxication by AB toxins and are targets for blocking intoxication.

Notes

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Graduation Date

2011

Semester

Spring

Advisor

Teter, Kenneth

Degree

Doctor of Philosophy (Ph.D.)

College

College of Graduate Studies

Department

Burnett School of Biomedical Sciences

Format

application/pdf

Identifier

CFE0003733

URL

http://purl.fcla.edu/fcla/etd/CFE0003733

Language

English

Length of Campus-only Access

None

Access Status

Doctoral Dissertation (Open Access)

Subjects

Dissertations, Academic -- Graduate Studies, Graduate Studies -- Dissertations, Academic

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