Characterization of De Novo Aggregation of Prions and Protein Misfolding Disease Proteins In Yeast

Many proteins spontaneously misfold, and escape from protein quality mechanisms in the cell. Accumulation of such proteins may become toxic to the host cell. The toxic conformers of some proteins often interact with other native forms of the same protein and convert them into the toxic state. These proteins can repeatedly cycle this self-sustaining loop, and exacerbate the cellular proteostatis network, resulting in death. Among such proteins, prions and ALS-linked proteins, FUS and TDP-43, are of great interest to understand the mechanism of de novo appearance of protein misfolding diseases. In this study, I have first studied the de novo appearance of a well-established prion in yeast, [PSI+], and showed that [PSI+] aggregates initially appear at a perivacuolar protein deposit site, where they are converted to amyloid by being cross-seeded via other heterologous protein aggregates. Such an interaction causes continued growth of the de novo [PSI+] fibrils, which requires the molecular chaperones. I also showed that the ALS-linked TDP-43, but not FUS protein can propagate as a self-seeding prion in yeast. Like yeast prions, TDP-43 can form self-seeding aggregates, which are cytoplasmically transmissible, and form detergent resistant oligomers. Unlike yeast prions, the TDP-43 prion causes growth inhibition, and does not require the Hsp104 chaperone for maintenance. In contrast to overexpressed TDP-43 or in vitro TDP-43 fibers, stress does not induce the formation of TDP-43 prions, but can partially cure cells of the TDP-43 prion. The study of the aggregation and toxicity of TDP-43 and FUS in yeast revealed that the yeast prion [PIN+] exacerbates FUS toxicity, and TDP-43 and FUS aggregation. I also showed that the level of the Hsp40 chaperone, Sis1 is vitally important to shear/dissolve and partially detoxify FUS and TDP-43 aggregates in yeast as such aggregates titrate the essential Sis1 protein away from the nucleus. This inhibits Sis1’s function in UPS-mediated delivery of cytosolic misfolded proteins to the nuclear proteasomes, leading to the accumulation of such proteins in the cytosol, and ultimately death. Taken together, insights gained here help us understand the de novo appearance of prions and prion-like disease protein aggregates, and how they cause toxicity in the cell.