Characterization of the Group II Chaperonin TriC derived from Human Cervical Adenocarcinoma (HeLa) Cells

Abstract

Molecular chaperones are key elements in protein folding pathways, and there are numerous proteins which are unable to fold in the absence of one or more molecular chaperones. Eukaryotic chaperones can be broadly separated into two families: chaperones, which sequester and protect unfolded proteins, but do not facilitate folding, and chaperonins, which require ATP, and actively fold nacent or misfolded proteins. The mammalian chaperonin TRiC, in addition to being required for actin and tubulin folding, binds and refold several disease causing proteins in vitro, including those associated with Alzheimer's disease, Von Hippel Landau tumor, and Huntington's disease. This refolding activity has revealed TRiC as a possible therapeutic agent for the treatment and prevention of aggregation diseases. We have purified TriC from cervical adenocarcinoma cells. To assess the properties of Human TriC, we are characterizing its interactions of with human γ Crystallins. Crystallins are a family of structural proteins found in the lens of the human eye, and aggregation of these proteins is thought to be the cause of cataract. Methanococcus marapaludis chaperonin Mm-Cpn, a homolog of human TriC, has been shown to both suppress aggregation of Human γ Crystallins, and refold the Crystallins to a native-like conformation. Suppression of γ Crystallin aggregation is being investigated using UV/Vis spectroscopy, and the ability of TriC to restore unfolded γ Crystallin to a native fold investigated using size exclusion chromatography. In collaboration with fellow members of the Center for Protein Folding Machinery, we are investigating the possibility of visualizing the crystallin substrate in the chaperonin/substrate complex by Cryo-EM.Supported by an NIH Roadmap Award to the Center for Protein Folding Machinery (http://ncmi.bcm.tmc.edu/nanomedicine).