In vivo targeting of unfolded protein to a phase-separated Huntingtin inclusion

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All major neurodegenerative disorders are characterized by the accumulation of large aggregates of unfolded protein in the central nervous system. Among neurodegenerative disorders, the etiology of Huntington's Disease (HD) is uniquely simple: the sole cause of HD is a class of dominant mutations in the huntingtin gene, which lead to the expansion of a polyglutamine repeat region in the huntingtin protein (Htt). The length of the polyglutamine tract correlates with increased instability and unfolding of mutant Htt protein, causing aggregation. Although Htt protein is widely expressed in all tissues throughout life, only a subset of cells degenerate; most cells are able to tolerate mutant Htt without significant ill effect. How do cells cope with large quantities of unfolded protein? Many studies have found that the presence of large inclusions of aggregated protein correlates poorly with cell death; in contrast, the concentration of small oligomeric aggregates is more predictive of cell death. Similarly, in S. cerevisiae, cells that form a single ovoid mutant Htt inclusion grow normally, whereas numerous widely distributed Htt aggregates are toxic to the cell. These observations have led many researchers to conclude that inclusions are cytoprotective, a cellular mechanism for collecting and quarantining harmful small aggregative species. Our studies have shown that the mHtt inclusion is a mobile phase-separated compartment that grows through the coalescence of the inclusion with small aggregates that diffuse through the cytoplasm. Evidence suggests that the formation of an inclusion body (IB) is nucleated and occurs even when the ubiquitin-proteasome system has excess capacity. Our goal is to elucidate the mechanisms by which inclusions are initiated and material is incorporated into them. Typically, a yeast cell develops a single ovoid mutant Htt IB, but IBs fail to form in a number of strains carrying single-gene deletions. Two genes that are absolutely required to form single ovoid inclusions are chaperone proteins; our evidence suggests that chaperones cooperate with other proteins in vivo to target material to IBs. The proposed work will help us better understand the interactions and additional functions of chaperone systems in the complex cellular milieu. Importantly, this project will also facilitate the continued development of the PI, as well as maintaining a highly active research lab that has provided training for 17 York College undergraduates in the past three years.