Physiological role of the TNKS-PI31 dependent proteasome regulation Protein degradation by the ubiquitin-proteasome system is central to cell homeostasis and survival. Defects in this process are associated with many human diseases, including cancer and neurodegenerative disorders. The 26S proteasome is a large protease complex that degrades ubiquitinated proteins. We recently showed that ADP-ribosylation promotes 26S proteasome activity in both Drosophila and human cells. We identified the ADP-ribosyltransferase Tankyrase (TNKS) and the 19S assembly chaperones as direct binding partners of the proteasome regulator PI31 (Proteasome Inhibitor of 31kDA). TNKS-mediated ADP-ribosylation of PI31 promotes 26S assembly by modifying its affinity for proteasome proteins. Inhibition of TNKS by a small-molecule inhibitor, XAV939, blocks this process - revealing a novel mechanism of proteasome regulation that can be targeted with existing small-molecule inhibitors - as well as attenuating the growth of multiple myeloma cells that are particularly sensitive to fluctuations in proteasome activity. These results suggest a potential new link between NAD+, formation of toxic protein aggregates, cancer growth and proteasome regulation that likely play a central role in protein homeostasis. Here, we propose aims to further dissect this regulatory mechanism. Specifically, during the K99/R00 phase of the award, we intend to characterize the newly identified Archipelago (Ago; Drosophila ortholog of Fbw7)-PI31 interaction and develop tools to understand the physiological role of the TNKS- dependent proteasome activation. Furthermore, by exploiting the TNKS-PI31 pathway, we will be able to rigorously explore the translational research potential of proteasome in various human disorders that arise secondary to dysfunctional protein homeostasis. For these purposes, we will use a multi-disciplinary approach that integrates genetics, cell biology, biochemistry and structural biology in the context of human disease models in Drosophila and mice, as well as in human multiple myeloma and colon adenocarcinoma cell lines. We expect that this project will fundamentally advance our understanding of how protein degradation is regulated and provide new insights into how we can manipulate this process to treat human diseases.