title: Stabilization of the tertiary structure of the cholera toxin A1 subunit inhibits toxin dislocation and cellular intoxication.
abstract: Cholera toxin (CT) moves from the cell surface to the endoplasmic reticulum (ER) by retrograde vesicular transport. The catalytic subunit of CT (CTA1) then crosses the ER membrane and enters the cytosol in a process that involves the quality control mechanism of ER-associated degradation. The molecular details of this dislocation event have not been fully characterized. Here, we report that thermal instability in the CTA1 subunit-specifically, the loss of CTA1 tertiary structure at 37 degrees C-triggers toxin dislocation. Biophysical studies found that glycerol preferentially stabilized the tertiary structure of CTA1 without having any noticeable effect on the thermal stability of its secondary structure. The thermal disordering of CTA1 tertiary structure normally preceded the perturbation of its secondary structure, but in the presence of 10% glycerol the temperature-induced loss of CTA1 tertiary structure occurred at higher temperatures in tandem with the loss of CTA1 secondary structure. The glycerol-induced stabilization of CTA1 tertiary structure blocked CTA1 dislocation from the ER and instead promoted CTA1 secretion into the extracellular medium. This, in turn, inhibited CT intoxication. Glycerol treatment also inhibited the in vitro degradation of CTA1 by the core 20S proteasome. Collectively, these findings indicate that toxin thermal instability plays a key role in the intoxication process. They also suggest the stabilization of CTA1 tertiary structure is a potential goal for novel antitoxin therapeutic agents.
authors.affiliation: Burnett School of Biomedical Science, College of Medicine, University of Central Florida, Orlando, FL 32826, USA.
authors.forename: Shane
authors.initials: S
authors.lastname: Massey
authors.forename: Tuhina
authors.initials: T
authors.lastname: Banerjee
authors.forename: Abhay H
authors.initials: AH
authors.lastname: Pande
authors.forename: Michael
authors.initials: M
authors.lastname: Taylor
authors.forename: Suren A
authors.initials: SA
authors.lastname: Tatulian
authors.forename: Ken
authors.initials: K
authors.lastname: Teter
mesh_terms: D016923:Cell Death / Q000187:drug effects; D002772:Cholera Toxin / Q000737:chemistry* / Q000378:metabolism* / Q000633:toxicity; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D005990:Glycerol / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D010447:Peptide Hydrolases / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D055550:Protein Stability / Q000187:drug effects; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry* / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects; D013329:Structure-Activity Relationship; D013696:Temperature; D013997:Time Factors
chemical_list: D021122:Protein Subunits; D002772:Cholera Toxin; D010447:Peptide Hydrolases; D046988:Proteasome Endopeptidase Complex; D005990:Glycerol