Characterization of a ubiquitin-independent pathway for proteasomal degradation

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PROJECT SUMMARY/ABSTRACT The proteasome is the main intracellular protease and is responsible for degrading thousands of different proteins. The main signal for degradation by the proteasome is thought to be the post-translational attachment of ubiquitin molecules onto a substrate protein. While some proteasome substrates have been reported to be targeted to the proteasome without the use of ubiquitin, the extent and mechanism of ubiquitin-independent proteasomal degradation remain unclear. We have recently identified hundreds of peptides that, when fused to the C-terminus of enhanced green fluorescent protein (EGFP), are degraded by the proteasome in a ubiquitin- independent manner. Here, we propose to investigate the mechanism of this ubiquitin-independent degradation and determine the endogenous substrates of this pathway. First, we will determine if the EGFP fusion substrates are recognized by the proteasome directly, or if they are shuttled to the proteasome by an intervening factor (Aim 1). Next, we will characterize the interaction between the receptor and substrates using a combination of genetic and biochemical methods (Aim 2). Finally, we will identify endogenous substrates of the ubiquitin-independent pathway (Aim 3). Overall, these experiments will characterize in detail this newly-uncovered ubiquitin-independent proteasome pathway, which may have profound implications for our understanding of the proteasome and of protein homeostasis more generally. I will perform this research as a postdoctoral fellow under the mentorship of Dr. Stephen Elledge at Brigham and Women’s Hospital/Harvard Medical School (HMS) and into my future independent research career at a top U.S. institution. To accomplish my proposed aims, I will need to learn essential skills in the K99 period from Dr. Elledge pertaining to advanced genetic screening techniques. I have assembled a team of collaborators and advisors from around HMS (Dr. Alfred Goldberg, Dr. Wade Harper, Dr. Eric Fisher, Dr. Phil Cole, and Dr. Eugene Oh) who complement my skill set and will help me effectively accomplish my proposed projects and prepare for my future independent career. The local training environment in the Elledge lab and at HMS will support me with all the materials, equipment, and professional development opportunities necessary to become a world-class researcher. Receiving the K99/R00 Pathway to Independence Award would provide me with critical resources to realize my project and career goals.