Decoding DUB regulation in Golgi reassembly and protein degradation

Abstract

The ubiquitin proteasome system (UPS) plays a critical role in maintaining protein homeostasis in cells – regulating both protein synthesis and protein degradation. Embedded in the UPS are proteins that (1) add ubiquitin enabling different signals, (2) remove ubiquitin signals allowing for substrate rescue and (3) recognize poly-ubiquitin signals for subsequent proteasomal degradation. Targeting different components of the UPS is of high interest for drug discovery in hopes to uncover novel therapeutic targets for regulating protein homeostasis for a wide range of diseases. My thesis work investigates interactions of deubiquitylating enzymes (DUBs) to various players in the UPS to explore the possibility of targeting specific DUBs, with in-house DUB inhibitors, to regulate substrate fate. In Chapter 2, I utilized structural biology, biochemistry and cellular biology to elucidate the interaction between VCPIP1, a DUB of interest, with VCP, a key player in the UPS, and p47, a VCP adaptor protein, in the context of Golgi reassembly. In Chapter 3, I employed biochemistry to understand the function of VCPIP1 at the C-terminal end of VCP in the context of the UPS. In the presence of a poly-ubiquitylated substrate, we used fluorescence-based assays and SDS-PAGE gels to assess the effect of VCPIP1 domain truncations as well as VCPIP1 and p97 inhibitors on VCP and VCPIP1 function. We provide initial structural analysis of a substrate-engaged VCP-VCPIP1C219A-UN complex. In Chapter 4, I applied mass spectrometry proteomics and structural biology to characterize the structural features of USP family of DUBs, specifically USP7, USP25 and USP48, and explored their interactome to discover unknown binding partners for the DUBs. The multi-faceted discoveries from my thesis work provide new structural and functional understanding of a few DUBs of interest using cryo-EM, biochemistry, cellular biology and proteomics. Taken together, this unveiled new binding regions for inhibitor development demonstrated by the newly characterized bivalent interaction of VCPIP1 to VCP and/or revealed novel interactions with binding partners between DUBs and E3 ligases observed in the proteomics data that can be leveraged for targeted protein degradation.