Summary The long-term 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 either fold or degrade them. How does Hsp70 make this choice? Why does it sometimes make the “wrong” decision? 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 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 cycles, we made substantial progress towards this goal. Specifically, we found that prolonged binding of Hsp70 to polyQ-AR or MAPT/tau creates a “signal” that recruits the E3 ubiquitin ligase, CHIP, and triggers a degradation cascade. These new insight were revealed using our chemical probes that selectively control protein-protein interactions (PPIs). Namely, we have developed molecules that inhibit PPIs between Hsp70 and its major co-chaperones, the J-domain proteins (JDPs) and nucleotide exchange factors (NEFs). In the course of this R01 (2008-present), we have shipped these reagents to 100+ laboratories worldwide, enabling important discoveries about the role of Hsp70 sub-networks in many biological 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 identified which peptide sequences in polyQ-AR and MAPT/tau bind to Hsp70 and the JDP, DnaJA2, a potent suppressor of aggregation Taking advantages of these insights and leveraging new technical innovations, we propose to create “molecular glues” that promote selective and potent binding of polyQ- AR and MAPT/tau to (SA1) Hsp70 and (SA2) DnaJA2. We also propose to (SA3) create “molecular glues” that promote binding of Hsp70s and JDPs to each other. As envisioned, these drug-like compounds will stabilize PPI contacts in the Hsp70 sub-network, promoting selective recognition of polyQ-AR and MAPT/tau and enhancing their degradation. Thus, we anticipate that these new chemical probes could be the starting point for exciting, new therapeutics. There is considerable enthusiasm for “molecular glues” in oncology and we are enthusiastic about taking the lessons (and technologies) from that emerging field and applying them in new ways to probe Hsp70 sub-networks in models of neurodegeneration. More fundamentally, we expect that these studies will reveal the molecular logic by which Hsp70 systems conduct PQC and maintain protein homeostasis.