Abstract. The goal of this project is to understand the molecular mechanisms of chaperone-mediated, protein quality control (PQC). Heat shock protein 70 (Hsp70) is known to bind client proteins and make the “decision” to degrade them. How does Hsp70 make this choice? Why does it sometimes go wrong? Severe neurodegenerative disorders, including spinobulbar muscular atrophy (SBMA) and frontotemporal dementia (FTD), are associated with a failure of the Hsp70 system to adequately clear damaged clients, such as polyglutamine-expanded androgen receptor (polyQ-AR) and mutants of microtubule-associated protein tau (MAPT/tau). We envision that mechanistic knowledge of Hsp70- mediated PQC will uncover new drug targets that might be used to restore normal protein homeostasis and, ultimately, treat these diseases. Indeed, in the previous funding cycle, we made substantial progress in understanding this central mechanistic mystery. Specifically, our results suggest that prolonged binding of Hsp70 to polyQ-AR or MAPT/tau is a “signal” that recruits the E3 ubiquitin ligase, CHIP, and triggers a degradation cascade. This new model was only unlocked through our development of new chemical probes that selectively control protein-protein interactions (PPIs) in the Hsp70 system. In the course of this R01, we have shipped these reagents to 60+ laboratories worldwide, enabling important discoveries in many PQC models. Now, in this competitive renewal, we are excited to propose the next-generation of important mechanistic questions and PPI targets. In preliminary studies, we discovered that NEF family co-chaperones bind Hsp70 to antagonize turnover, while DnaJA2 seems to be involved in client selection. Thus, we will develop new chemical and genetic tools to reveal: (SA1) how the Hsp70-NEF complex controls affinity for polyQ-AR and MAPT/tau, (SA2) the mechanisms of client recruitment by Hsp70-DnaJA2 and (SA3) how broader chaperone networks collaborate to interact with these clients. In each aim, we will deploy innovative chemical and biochemical approaches, coupled with NMR-based structural methods. We anticipate that this work will have a fundamental impact on our understanding of how Hsp70 sub-networks maintain protein homeostasis.