Project Summary/Abstract My research program is focused on the intersection of pro-folding chaperones and the pro-degradation 26S proteasome. Protein Quality Control (PQC) is the balance of protein folding, unfolding and degradation, which is central to human health and disease. An outstanding question in the PQC field is “how do chaperone proteins triage substrate proteins between the pro-folding and pro-degradation pathways?” In my graduate and postdoctoral training, I have gained experience using in vitro techniques to study both chaperone proteins and the 26S proteasome. This positions me to explore how chaperone activity and substrate processing affect downstream degradation by the proteasome. This work will bridge the gap between two major cellular pathways that, to-date, have been characterized independently. Our goal over the next five years will be to use a multi-disciplinary approach to study how the chaperone complex, Hsp70/CHIP/BAG-1, affects proteasomal degradation. Hsp70 is a molecular chaperone and ATPase that binds and releases substrates throughout an ATP-hydrolysis cycle that is regulated by co- chaperone proteins. Two such co-chaperones are C-terminus of Hsc70-interacting protein (CHIP), an E3 ubiquitin ligase, and Bcl-2 associated athanogene-1 (BAG-1), a nucleotide exchange factor. The Hsp70/CHIP/BAG-1 complex has been shown to target human disease substrates, such as mutant huntingtin and immature BCR-ABL oncoproteins, to the proteasome for degradation. Therefore, this is an ideal chaperone complex for developing my research program. I propose to use in vitro characterization to dissect how chaperones influence each step of proteasomal degradation, including ubiquitination, substrate binding, and degradation. In addition, we will employ cryo- electron microscopy to determine first-of-its kind structures that directly observe the substrate handoff mechanism from chaperones to the 26S proteasome. These techniques will be paired with single molecule Fluorescence Resonance Energy Transfer (smFRET) experiments that report on the conformational state of Hsp70 during substrate processing. Tracking the changes in Hsp70 will demonstrate how interactions with co-chaperones and the proteasome affect the conformational landscape during substrate processing. Combining these in vitro techniques to rigorously study how chaperones mediate proteasomal degradation will provide unique insights into the molecular mechanisms that affect substrate handoff and degradation. Furthermore, I expect that our data will lead to novel strategies for targeting this process in human disease.