| Protein aggregates are a common hallmark of a number of neurodegenerative diseases. Many of these diseases, such as Parkinson's and Huntington's, are accompanied by the accumulation of intracellular protein aggregates in cytoplasmic and nuclear inclusion bodies. Thus, it is important to study cellular responses to protein aggregation and explore means by which protein aggregates may be cytotoxic. The work in this thesis provides experimental evidence that protein aggregates impair the function of a major cellular proteolytic system, the ubiquitin proteasome system (UPS). By fusing a UPS specific degron to the carboxyl terminus of green fluorescent protein, we have created an in vivo UPS reporter called GFPu. Upon inhibition of the UPS, cells accumulate GFPu and exhibit an increase in green fluorescence. We have shown that multiple species of protein aggregates impair the function of the UPS, as evidenced by increased GFPu in cells containing protein aggregates. The exact mechanism of UPS impairment is unclear, but work in this thesis describes two additional projects aimed at gaining greater insight into this phenomenon. The first project describes the creation of an in vitro system to test if aggregates can impair the proteasome directly. The second project describes the creation of nuclear and cytoplasmic GFPu based reporters to determine if UPS impairment by cytoplasmic aggregates affects the nuclear UPS pool and vice versa. Because of the central role of the UPS in regulating fundamental cellular events such as cell division and apoptosis, our data suggest a potential mechanism linking protein aggregation to cellular disregulation and cell death. Thus, impairment of the UPS by protein aggregates may have direct relevance to a number of diseases in which protein aggregates are a pathological hallmark. |
