The over-arching goal of this project is to understand the molecular and cellular mechanisms that drive glutamine-repeat (polyQ) protein aggregation. Such polyQ sequences are found in at least twelve human genetic diseases including Huntington's disease and Machado-Joseph disease. It is well established from in vitro experiments that stable intermediates, or oligomers, may be present during the assembly of polyQ proteins into fibrils. Such oligomers have been shown to have cytotoxic and pathogenic properties in cells. We propose that native sequences flanking the polyQ stretches in mutant proteins will greatly influence the distribution of aggregation species in the context of the cell, and that the activities of molecular machinery that mitigate protein aggregation may be influenced by such flanking sequences. A recent study, using crude extracts from a mouse neuroblastoma cell line (2), has identified the presence of oligomers using the newly developed analytical ultracentrifuge with fluorescence detection capability. This technique will be used, combined with FRAP (fluorescence recovery after photobleaching, carried out using a confocal microscope), to probe the aggregation states of polyQ-containing proteins both in vivo and in crude extracts in the worm, C. elegans. The effect of protein flanking sequences will be explored on the relative distribution of oligomers, and its correlation to cytotoxicity. To further assess biological significance for the role of various aggregation states in disease, D. melanogaster will be used to discover how two well-studied chaperones (Hsp104 and Hsp70) might mitigate the effects of intermediate aggregation states, using the same set of fluorescence techniques. Key reagents and fly lines are being developed while the principal investigator is on sabbatical leave in the laboratory of Dr. Nancy Bonini, an HHMI investigator at the University of Pennsylvania. The Bonini laboratory has expertise in the study of the role of several polyQ- containing proteins on fly development and aging (3-8). Preliminary work with C. elegans will be carried out in the laboratory of Dr. Christopher Link, at the University of Colorado, Boulder in the summer of 2012. He has expertise in the use of this animal model to study protein aggregative diseases (9-14). The products of this critical work will be brought back to Haverford, and will provide an important set of tools for undergraduate students to use in their senior research work. Two faculty in the Biology Department at Haverford College already use these animal models for their research, so there is adequate infrastructure support in place, which will allow for a sharing of resources, and provide new synergies in the existing research programs. By involving undergraduate researchers in all aspects of the project, this award will also contribute significantly to the education and training of future generations of biomedical scientists.
Twelve human diseases involve polyQ-driven aggregation, and the pathway to sequestration into inclusion bodies or nuclear inclusions has been shown to be complex, involving the accumulation of toxic oligomers. New biophysical and imaging techniques have been recently developed that allow scientists to probe aggregation both in vivo and in crude extracts, and the PI plans to use fluorescence techniques (using a confocal microscope, and characterization of molecular populations using an analytical ultracentrifuge) to probe polyQ aggregation using D. melanogaster and C. elegans model systems. The ability to mitigate potentially toxic intermediate populations will be explored using chaperones, proteins whose function is to prevent or reduce protein aggregation.
|Kartvelishvili, Ekaterine; Peretz, Moshe; Tworowski, Dmitry et al. (2016) Chimeric human mitochondrial PheRS exhibits editing activity to discriminate nonprotein amino acids. Protein Sci 25:618-26|
|Kokona, Bashkim; May, Carrie A; Cunningham, Nicole R et al. (2016) Studying polyglutamine aggregation in Caenorhabditis elegans using an analytical ultracentrifuge equipped with fluorescence detection. Protein Sci 25:605-17|