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 very little is known about he regulation of ubiquitin homeostasis and its steady state concentration in the cell. Importantly, ubiquitin homeostasis is often disrupted in human diseases - particularly diseases associated with protein misfolding and neurodegeneration. Thus, there is a critical need to understand the basic biochemical mechanisms responsible for regulating ubiquitin metabolism and to define cellular mechanisms that maintain proper ubiquitin homeostasis. Recently, we identified a signaling mechanism in yeast which regulates ubiquitin metabolism in the cell by controlling the phosphorylation of ubiquitin itself. Our preliminary data indicates that phosphorylation of ubiquitin increases its rate of degradation and the rate of endocytic trafficking in the cell by preventing recognition by deubiquitylating enzymes (DUBs) along the endocytic route. We hypothesize that ubiquitin homeostasis is significantly impacted by the rate of ubiquitin flux through the endocytic pathway and by signaling pathways that determine whether ubiquitin is recycled or degraded in the vacuole. The experiments outlined here have strong potential to define mechanisms that regulate ubiquitin metabolism in the cell, which will likely improve our broader understanding of degradation pathways and global protein stability.
Aim 1 : Dissect the mechanism of Ppz-mediated regulation of ubiquitin phosphorylation. We hypothesize that Ppz phosphatases specifically recognize and de-phosphorylate Ser57 phospho-ubiquitin that is conjugated to proteins at the plasma membrane. Here, we will use in vitro biochemistry, genetics and live cell imaging experiments to dissect the mechanism of Ppz-mediated regulation of ubiquitin phosphorylation. In addition to uncovering new pathways that regulate ubiquitin phosphorylation, this aim also has strong potential to reveal biochemical mechanisms that regulate ubiquitin metabolism and homeostasis.
Aim 2 : Determine how Ser57 phosphorylation of ubiquitin regulates deubiquitylase activities. We hypothesize that Ser57 phosphorylation of ubiquitin confers resistance to deubiquitiylation by DUBs along the endocytic pathway, thereby facilitating bypass of DUB checkpoints and preventing the recycling of ubiquitin. Here, we will use a combination of biophysical methodologies, in vitro biochemistry, and live cell imaging to dissect the mechanism of DUB bypass by Ser57 phosphorylated ubiquitin. These experiments will drive new understanding of how phosphorylation of ubiquitin can alter its function in the context of ubiquitin recycling and endosomal sorting. These experiments will also elucidate in biochemical detail a regulatory decision that contributes to the fine tuning of ubiquitin levels in the cell.

Public Health Relevance

Cellular protein degradation capacity is often aberrant in human diseases - particularly in diseases of protein misfolding like neurodegeneration where the protein degradation capacity of disease cells is diminished. In this project, we investigate mechanisms for controlling the abundance of a critical protein degradation determinant called ubiquitin, with the long term goal of developing strategies for adjusting ubiquitin homeostasis in order to 'tune-up' protein degradation pathways in disease cells.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Membrane Biology and Protein Processing Study Section (MBPP)
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Barski, Oleg
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Vanderbilt University Medical Center
Anatomy/Cell Biology
Schools of Medicine
United States
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Xu, Peng; Hankins, Hannah M; MacDonald, Chris et al. (2017) COPI mediates recycling of an exocytic SNARE by recognition of a ubiquitin sorting signal. Elife 6:
Lee, Sora; Tumolo, Jessica M; Ehlinger, Aaron C et al. (2017) Ubiquitin turnover and endocytic trafficking in yeast are regulated by Ser57 phosphorylation of ubiquitin. Elife 6: