One of the cellular hallmarks of aging and neurodegeneration is a general decline in the protein degradation capacity of the cell, resulting in the accumulation of damaged and misfolded proteins which can threaten cellular integrity and viability. A key player in most cellular degradation pathways is ubiquitin ? a 76 amino acid peptide that is covalently conjugated to proteins in order to target their degradation. Despite its central role in global protein stability we know very little about the regulation of ubiquitin itself. Thus, there is a critical need to understand the basic biochemical mechanisms responsible for regulating ubiquitin metabolism and to explore the relationship between ubiquitin homeostasis and cellular aging. Recently, we identified a novel signaling mechanism in yeast which regulates ubiquitin metabolism in the cell by controlling the phosphorylation of ubiquitin itself. Importantly, we have found that yeast cells expressing phosphomimetic ubiquitin exhibit a significantly extended post-mitotic lifespan. Given our preliminary results, we hypothesize that phosphorylation of ubiquitin not only alters its metabolism but generally accelerates global protein degradation and in doing so extends the post-mitotic life span of the cell. The experiments outlined in this proposal will explore this hypothesis in the following specific aims:
Aim 1. Determine how ubiquitin phosphorylation extends yeast life span. We hypothesize that the life span extension associated with ubiquitin phosphorylation is due to a global increase in protein degradation. To test this, we will perform genetic analysis to define the degradation pathway that confers life span extension in the presence of phospho-ubiquitin. We will also leverage newly-developed reagents to define and quantify how ubiquitin phosphorylation affects the ubiquitin-modified proteome and how such changes may finely tune the activity of the ubiquitin-proteasome system during an aging time course. These experiments will elucidate the biochemical mechanism of life span extension associated with ubiquitin phosphorylation.
Aim 2. Identify ubiquitin kinases and test their ability to modulate yeast aging. We hypothesize that the kinase activity responsible for Ser57 phosphorylation of ubiquitin will also play an important role in yeast chronological aging. To identify yeast ubiquitin kinase(s), we will adopt both genetic and biochemical screening approaches. Candidate kinases will be tested for membrane trafficking and cellular aging phenotypes. Identification of the yeast ubiquitin kinase(s) will significantly advance our understanding of ubiquitin metabolism and its relationship to the regulation of membrane traffic and post-mitotic aging. Together, the experiments outlined in this proposal have a strong potential to define the relationship between ubiquitin homeostasis, global protein stability, and cellular aging. Ultimately, this will contribute to our understanding of longevity in post-mitotic cells and may lead to the identification of new pathways that generally alter global protein stability.
Given the mounting evidence that protein degradation capacity declines with aging, there is a critical need to understand how cells regulate protein degradation and how these pathways can be restored in aging cells. The research plan outlined here will explore a novel mechanism for extending cellular life span that involves ?re-tuning? protein degradation mechanisms, with the long-term goal of identifying therapeutic strategies to delay post-mitotic cellular aging.