UCSF

C-terminus of Heat-shock protein-70 interacting protein (CHIP) is an E3 ubiquitin ligase canonically known to mark misfolded Heat shock protein (Hsp) clients for degredation. A recent publication from the Craik lab demonstrated CHIP can directly interact with numerous proteins outside of Hsps. While the authors demonstrate the occurrence of Hsp-independent CHIP interactions in cells and that CHIP can ubiquitinate substrates without chaperones in biochemical assays, they were unable to show Hsp-independent substrate ubiquitination in cells. Successful demonstration of CHIP’s Hsp-independent enzymatic activity in cellular contexts could elucidate molecular underpinnings of multiple disease states, including neurodegeneration, cancer, and viral infections, as well as provide insights into the feasibility using Hsp-independent CHIP interactions for targeted degradation of proteins. Chapter 1 describes recombinant antibodies that function as CHIP inhibitors. These were shown to have distinct epitopes and were able to inhibit both CHIP substrate binding and ubiquitination. When reformatted into scFvs for intracellular expression, some antibodies can still bind to CHIP. The recombinant antibodies can be used to examine molecular mechanisms of CHIP interactions in disease states and enable structural studies. In chapter 2, a predicted, Hsp-independent, CHIP interaction with the viral protein ORF28 was examined. Data indicates this interaction occurs in cells, and preliminary results show this interaction may be a mechanism of host protein regulation. Further studies will allow for non-biased identification of interactors and determination of the impacts of these interactions on viral replication. Chapter 3 is a distinct effort from the rest of this thesis, driven by the continued need for the novel of COVID-19 antivirals. Stable, BSL2 models of SARS-CoV-2 replication (replicons) are needed to facilitate the screening and development of small molecule inhibitors, as well as probe the biology of SARS-CoV-2 replication in a non-BSL-3 lab. While multiple designs were attempted and gave some success in generating lines, none had enough assay window to successfully screen for compounds.