WHAT IS THE STRUCTURAL CORRELATE OF INTRACELLULAR POLYGLUTAMINE TOXICITY?

这个子项目是众多研究子项目之一

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Expanded polyglutamine (polyQ) stretches greater than 36-40 glutamines in length are the only common feature in twelve unrelated proteins that cause neurodegenerative disease. Evidence indicates that abnormally long polyQ stretches confer new toxic functions that may be associated with the presence of soluble monomeric or oligomeric forms of the polyQ-containing protein. Although it is known that these forms eventually aggregate, it is not which of the soluble species are toxic. Identification of these toxic species will be a major breakthough in understanding the biological basis of these neurodegenerative diseases. We plan to use SAXS to determine the molecular envelope and monomeric or oligomeric state of polyQ-containing proteins in complexes with Fabs that bind polyQ. To identify toxic species, the information from the SAXS experiments will be correlated with the ability of the antibody staining to predict degeneration of neurons in cell culture experiments. Our preliminary experiments with complexes of the 3B5H10 Fab, an Fab specific for disease-associated stretches of polyQ, and an N-terminal fragment of the huntingtin protein with a 39 glutamine repeat indicated that the purified complex was well-behaved in solution at concentrations of 1.5- 5 mg/ml with no signs of aggregation. Data from this study was used to obtain of the molecular envelope of the monomeric conformation of the huntingtin fragment in the complex. We plan to extend these experiments by using longer polyQ repeats in the huntingtin fragment as well as constructs of ataxin-3 with variable polyglutamine repeats. In addition, Fabs such as the MW1 Fab which may promote polyglutamine toxicity in cell culture will be studied. A polyQbinding Fab that is ineffective at predicting polyglutamine toxicity will be studied as a control.