A number of severe human neurodegenerative diseases, including Huntington's Disease and several Spinocerebellar ataxia?s (SCA's), result from mutation (expansion) of a polyglutamine (polyQ) tract in a protein diagnostic for each disorder. How polyQ expansion leads to neuronal cell death remains an enigma, and there are no effective treatments for these ailments. This application will test the hypothesis that expanded polyQ tracts are poor substrates for the cell?s primary proteolytic enzyme- the proteasome, and that the net accumulation of polyQ fragments is cytotoxic. To test this hypothesis, normal lengthed and expanded polyQ versions of the ataxin-7 protein (SCA7) will be targeted to the proteasome, and degradation rates of both protein versions will be assayed in vitroand in vivo. For in vitroassays, 35S-labelled protein products will be transcribed/translated in reticulocyte lysate, and subjected to degradation in extracts enriched for 26S proteasomes. In vivo half-lives of SCA7 proteins will be measured in cell culture using modified pulse chase assays that utilize a post-translationally cleavable ubiquitin moiety as an internal standard. Additionally, attempts will be made to facilitate hydrolysis of polyQ proteins in vivo using proteasomal activators (REGs). Preliminary data in our laboratory suggest that distinct REG molecules differentially affect the proteasome?s ability to degrade polyQ peptides in vitro. Thus, as part of this application, degradation and cell viability assays will be carried out in cell lines expressing various REGs to explore if these observations hold true in vivo. If so, REGs will become an attractive therapeutic target in polyQ diseases.
| Gorbea, Carlos; Goellner, Geoffrey M; Teter, Ken et al. (2004) Characterization of mammalian Ecm29, a 26 S proteasome-associated protein that localizes to the nucleus and membrane vesicles. J Biol Chem 279:54849-61 |
| Goellner, Geoffrey M; Rechsteiner, Martin (2003) Are Huntington's and polyglutamine-based ataxias proteasome storage diseases? Int J Biochem Cell Biol 35:562-71 |
