Project Grant F31AG104562

MECHANISMS OF K29-LINKED UBIQUITIN SIGNALING IN PROTEOSTASIS AND STRESS RESILIENCE - PROJECT SUMMARY CELLS RELY ON THE PROTEIN HOMEOSTASIS (PROTEOSTASIS) NETWORK (PN), A COORDINATED SYSTEM OF CHAPERONES, DEGRADATION PATHWAYS, AND STRESS-RESPONSIVE TRANSCRIPTION FACTORS, TO MAINTAIN THE INTEGRITY OF THE PROTEOME. THE CAPACITY OF THE PN DECLINES MARKEDLY WITH AGE, LEADING TO THE ACCUMULATION OF MISFOLDED AND AGGREGATED PROTEINS THAT DRIVE NEURODEGENERATION, CARDIOMYOPATHY, AND OTHER AGE-ASSOCIATED DISEASES. UBIQUITIN SIGNALING IS CENTRAL TO PROTEOSTASIS, YET MOST STUDIES HAVE FOCUSED ON CANONICAL UBIQUITIN CHAIN LINKAGES SUCH AS K48 AND K63. IN CONTRAST, THE FUNCTIONS OF NONCANONICAL LINKAGES, INCLUDING THE EVOLUTIONARILY CONSERVED AND STRESS-INDUCIBLE K29 UBIQUITIN CHAIN, REMAIN ESSENTIALLY UNKNOWN. UNDERSTANDING HOW THESE ALTERNATIVE LINKAGES CONTRIBUTE TO STRESS RESILIENCE WILL REVEAL FUNDAMENTAL MECHANISMS THAT MAINTAIN CELLULAR HEALTH ACROSS THE LIFESPAN. THE OVERALL GOAL OF THIS PROJECT IS TO DETERMINE HOW K29-LINKED UBIQUITIN IS SPATIALLY ORGANIZED IN CELLS AND HOW IT FUNCTIONS IN PLASMA MEMBRANE PROTEIN QUALITY CONTROL (PMQC) DURING STRESS. PRELIMINARY PROTEOMICS DATA FROM SACCHAROMYCES CEREVISIAE REVEAL STRONG HEAT SHOCK-DEPENDENT INTERACTIONS BETWEEN K29 CHAINS AND THE ART-RSP5 PMQC NETWORK, SUGGESTING THAT K29-LINKED UBIQUITIN MAY MARK DAMAGED MEMBRANE PROTEINS FOR ADAPTIVE CLEARANCE. ADDITIONALLY, NEW TOOLS INCLUDING HIGH-AFFINITY K29-SPECIFIC SYNTHETIC ANTIBODY FRAGMENTS (K29- SABS), LIVE-CELL FLUORESCENT UBIQUITIN REPORTERS, AND A NOVEL N-DEGRON-BASED TARGETED PROTEIN DEGRADATION PLATFORM PROVIDE AN UNPRECEDENTED OPPORTUNITY TO DEFINE K29-LINKED UBIQUITIN SIGNALING PATHWAYS IN CELLS. AIM 1 WILL ESTABLISH THE SPATIOTEMPORAL DYNAMICS OF K29-LINKED UBIQUITIN DURING HEAT SHOCK AND RECOVERY USING COMPLEMENTARY FIXED-CELL AND LIVE-CELL IMAGING APPROACHES. THESE STUDIES WILL DETERMINE THE SUBCELLULAR LOCALIZATION OF K29 CHAINS THAT ACCUMULATED DURING STRESS, HOW THEIR LOCALIZATION CHANGES OVER TIME, AND HOW THEY INTERFACE WITH CHAPERONES, ORGANELLES, AND THE PROTEASOME. AIM 2 WILL DEFINE THE FUNCTIONAL ROLE OF K29- LINKED UBIQUITIN IN THE PMQC PATHWAY BY PERTURBING KEY LIGASES AND DEUBIQUITINASES, EXAMINE K29 CHAIN LOCALIZATION AND PMQC-SUBSTRATE TURNOVER IN MUTANT BACKGROUNDS, AND EVALUATE THE PHENOTYPIC CONSEQUENCES OF ABOLISHING K29 LINKAGE FORMATION. TOGETHER, THESE STUDIES WILL UNCOVER HOW K29-LINKED UBIQUITIN CONTRIBUTES TO PROTEOSTASIS MAINTENANCE AND STRESS RESILIENCE. BECAUSE PROTEOSTASIS FAILURE IS A HALLMARK OF AGING, ELUCIDATING THIS OVERLOOKED ASPECT OF UBIQUITIN SIGNALING HAS THE POTENTIAL TO REVEAL NEW MOLECULAR TARGETS FOR PRESERVING CELLULAR FUNCTION AND MITIGATING AGE-RELATED DECLINE. THE PROPOSED TRAINING PLAN PROVIDES ME WITH AMPLE RESEARCH, EDUCATIONAL, AND CAREER OPPORTUNITIES THROUGH THE SPONSORSHIP OF DR. DAVID PINCUS AND DR. MINGLEI ZHAO. THESE OPPORTUNITIES WILL FUEL MY TRAINING GOALS, PROVIDING ME WITH THE NECESSARY SKILLS AND EXPERTISE TO SUCCEED IN ACHIEVING MY LONG-TERM GOAL OF BECOMING A PRINCIPAL INVESTIGATOR AT AN R1 RESEARCH INSTITUTION.