Discovery and characterization of chaperone-independent substrates of the CHIP ubiquitin ligase

Protein turnover through endolysosomal degradation and the ubiquitin proteasome system are critical for maintaining protein homeostasis. These pathways protect the cell from accumulation of misfolded proteins, coordinate critical signaling processes, and facilitate recycling pathways that are central to cellular health. An integral member of these systems is the E3 ubiquitin ligase CHIP, which facilitates the turnover of damaged or terminally misfolded proteins. Canonically, substrate recognition by CHIP is dependent on Hsp70 or Hsp90 chaperones, which serve as intermediaries between a misfolded client and CHIP. However, emerging evidence suggests that CHIP also has the capacity to recognize substrates independent of a chaperone binding partner. In this dissertation, we explore the biological relevance of such chaperone-independent substrates by exploring interactions predicted by a biophysical scoring function we previously developed, termed CHIPscore.In the first chapter, I and others describe one such interaction between CHIP and the relatively uncharacterized, membrane-anchored protein CHIC2. We find that CHIC2 binding strongly attenuates CHIP activity, and that CHIC2 knockout phenocopies CHIP knockout in certain cell types, implying that chaperone-independent interactions can sometimes predominate CHIP’s biological functions. Furthermore, loss of the CHIP-CHIC2 interaction induces neurodegeneration and shortens lifespan in C. elegans, demonstrating that formation of this chaperone-independent complex is important in animals. We propose that CHIC2 attenuates CHIP activity at the membrane, offering a novel mechanism by which this ubiquitin ligase can be regulated.In the second chapter, I explore additional chaperone-independent interactors beyond CHIC2 that are predicted by CHIPscore. In preliminary results, I demonstrate interactions between CHIP and three additional proteins. These proteins are completely uncharacterized and reside at different subcellular localizations, suggesting that CHIP may be regulated at various locations within the cell through mechanisms that are not yet understood. This work opens substantial new avenues upon which future studies should be based