PROJECT SUMMARY Errors associated with DNA replication, transcription, mRNA processing, and protein biogenesis result in the continuous production of potentially toxic defective proteins. The error-prone nature of these essential processes requires robust quality control (QC) systems to effectively triage and destroy defective translation products. Protein quality control is an essential component within the larger protein homeostasis (proteostasis) system and proteostasis dysfunction has been implicated in human aging-related pathologies. On one hand, elevated protein QC function is needed to enable neoplastic cell proliferation in cells with high mutational burdens or chromosomal abnormalities. Conversely, impaired proteostasis and defects in protein QC function result in the enhanced production of misfolded and toxic aggregation prone proteins that typify many neurodegenerative disorders. These observations suggest that developing molecular strategies to predictably alter QC function to either enhance, or limit QC capacity as needed can improve aging-associated disorders and extend human healthspan. However, there is a surprising and substantial gap in our understanding of not only how QC systems selectively engage their substrates, but also how substrates evade detection during proteostasis dysfunction. To make substantive progress toward the goal of leveraging QC systems to combat aging-associated disorders, it is necessary to identify and characterize cellular and molecular mechanisms that enable detection and degradation of diverse QC substrates. Recent research progress from my lab has identified a spatially restricted QC pathway that acts on stalled and collided ribosomal complexes both before and after translation initiation to target defective translation products for degradation and recycle ribosomal complexes. Further, we have developed a systematic pipeline for biochemical, structural, and cellular interrogation of enigmatic but critical QC ubiquitin ligases that have been implicated in targeting diverse substrates for degradation by unknown mechanisms. We have focused our initial studies on the ubiquitin ligase HUWE1. Our recently described HUWE1 structure represents the first full-length structure of a HECT-domain ligase. We have generated a unique and powerful set of genome-edited cell lines and HUWE1 variants that have and will enable molecular dissection of HUWE1 function, HUWE1 substrate identification, and identification of cellular stress conditions that require HUWE1 for cellular survival and proliferation. Research outcomes achieved by the proposed studies will mechanistically determine how terminally stalled ribosomes are sensed and resolved and how ribosome-associated QC pathways can be manipulated to alter proteostasis function. Further, we will establish mechanisms by which QC ligases engage substrates under normal and stressed conditions. Successful completion of the proposed research will provide substantial progress...