In the expanded CAG repeat diseases, such as Huntington's Disease, abnormally long polyglutamine sequences have been implicated as the principle causative factor of the disease. The mechanism by which these molecules promote neurodegeneration is not clear, but a large body of evidence suggests that the mechanism has something to do with the enhanced ability of lengthened polyglutamine sequences to self-associate into oligomers or aggregates. While there is an imperfect correlation of disease physiology with the appearance of large aggregates in cellular nuclei, there is also a lot of indirect evidence, such as the widely reported involvement of cellular chaperones, that a misfolding / aggregation process is involved in these diseases. One limitation in our ability to progress more deeply into the disease mechanism is our ignorance of the details of the process and products of polyglutamine aggregation. For example, improved correlation of disease development with polyglutamine aggregation might be obtained if it were possible to distinguish different sizes and types of aggregates. There is significant information on the behavior of polyglutamine sequences from cellular expression experiments, but, owing to the very poor solubility properties of these molecules, there is much less mechanistic and structural information on defined polyglutamine sequences at the in vitro chemical level. This application is based on recent advances in this laboratory in the ability to solubilize these molecules and control their aggregation in vitro. A series of chemically synthesized polyglutamine sequences of different lengths will be developed and used to pursue the following aims. The length dependence of the kinetics and thermodynamics of the aggregation process will be characterized. The length dependence of aggregate structure will also be characterized. Mutational analysis o f the polyglutamine sequence will be conducted in order to better define the unique role of glutamine in aggregation and to better understand aggregate structure at the atomic level. Finally, the abilities of a series of molecular chaperones to retard and reverse the aggregation process will be characterized in vitro. The work should lead to a better understanding of the role of polyglutamine self-association in the disease mechanism and the nature of the toxic entity in turn; this should stimulate new work on the therapeutic intervention.
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