Program Director/Principal Investigator (Lasl, Fitst, MItldle): Morimoto, Richard I., PL Project 4-Kelly, Jeffery W. PROJECT SUMMARY (See inslrucUons): The maintenance of protein homeostasis, or proteostasis, involves balancing protein biosynthesis, folding, vesicular trafficking, degradation, etc., which we hypothesize is critical for healthy aging. Since the demands on subcellular compartments to maintain proteostasis change with aging and due to environmental stresses, stress-responsive signaling pathways have evolved to quickly adjust subcellular proteostasis network capacity to meet demand. Herein, we focus on the development and utilization of cell-based reporter screens to discover small molecules that can activate stress-responsive signaling pathways selectively. A dual luminescence reporter cell line is proposed to discover arm-selective unfolded protein response activators affecting proteostasis in the endoplasmic reticulum. A prior cell-based heat shock response (influencing cytosolic proteostasis) activator reporter screen has generated numerous small molecule leads and the same is expected from the arm-selective UPR activator screen. A plan is outlined to discern the stress responsive signaling pathway activation selectivity of these leads, to establish their therapeutic index, to identify the transcriptional and proteomic changes of select activators, and to identify and validate the target(s) of the highly ranked activators. We also propose to utilize small molecule-regulated destabilized domain-FOXO transcription factor fusions to deveiop a sensitive and selective cell-based reporter assay for eventual screening of small molecule activators of FOXO signaling. FOXO influences metabolic and proteostatic control. Selective stress-responsive signaling pathway activators will make it possible for the other investigators in this program project to learn which subcellular compartments are most important to maintain proteostasis in for healthy aging. Organelle-selective proteostasis enhancement in cell and animal models, coupled with the systems biology characterization of the proteostasis network as a function of aging and activator treatment should provide a clear answer regarding the influence of organelle-specific proteostasis on healthspan, the period of life where individuals are disease free-owing to the enhanced fitness of the proteome. RELEVANCE (See inslrucUons): The molecules produced in Project 4 and their utilization in testing the hypothesis that maintenance of the proteome over the lifespan of an organism is critical for healthspan represents a paradigm shift in aging research, which heretofore has largely focused on genome instability.