One neuropathological hallmark of numerous age-related disorders, including Alzheimer's disease and Parkinson's disease, is the aggregation of aberrantly folded proteins. The in vivo molecular mechanisms that facilitate this toxic protein aggregation are poorly understood. However, several cellular pathways are implicated in misfolded protein accumulation, two being the ER stress response and protein modification by the small ubiquitin-like protein SUMO. The ER stress response is a homeostatic control system that functions to maintain proper folding conditions within the organelle. Stresses such as accumulation of unfolded proteins and lipid imbalances within the ER membrane activate a signaling cascade that restores equilibrium within the ER. Although the triggers of the ER stress response are well-characterized, further understanding the regulation of this pathway will contribute to our understanding of age-dependent, atypical protein aggregation. Recent genetic data from our lab highlights a novel connection between the ER stress response and the SUMO pathway in the model eukaryote Saccharomyces cerevisiae. Yet, the molecular mechanisms connecting these two cellular processes are not known, and furthermore, how the interplay of these pathways relates to aging is still to be elucidated. Thus, I aim to further define the link between the SUMO pathway and the ER stress response and determine how these pathways contribute to aging in S. cerevisiae. To achieve this, I will first use a novel microfluidic devicedesigned to isolate and monitor individual yeast mother cells throughout their lifespan. Using this device, I will quantify the replicative lifespan (RLS) of wild-type yeast and SUMO pathway mutant strains, in the presence and absence of ER stress reagents. Additionally, I will monitor the efficiency of the ER stress response in individual aging wild-type cells and SUMO mutant cells using the microfluidic device and attached fluorescence microscope. Taken together, these experiments will elucidate the role of the SUMO pathway in regulating the ER stress response and will help identify links between regulation of the ER stress response and aging-associated occurrences, such as aberrant protein accumulation. For my second aim, I will identify proteins that lie at the interface of the ER stress response and SUMO pathways using two complementary approaches. To begin with, I will employ a high-copy suppressor screen of the tunicamycin sensitivity (an ER stress-inducing reagent) of the SUMO protease mutant ulp2-delta. Secondly, I will use immuno-purification and tandem mass spectrometry to isolate proteins that interact with the SUMO protease Ulp2 both under normal growth conditions and during ER stress. Using these two approaches, I anticipate discovering proteins that link the SUMO pathway and the ER stress response. Moreover, I will begin to define the SUMO-mediated mechanisms regulating the ER stress response and will understand the association of this regulation with cellular aging.
The proposed project seeks to elucidate the connection between SUMO-mediated regulation of the ER stress response and aberrant protein aggregation. Aggregation of misfolded proteins within the cell is a hallmark of many age-related neurodegenerative disorders; including Alzheimer's and Parkinson's Disease. Thus; better understanding the cellular mechanisms that regulate protein aggregation will provide insight into one consequence of aging and will lead to the discovery of potential therapeutic treatments for aging-associated disorders.