Project Summary The Blackwell lab studies mechanisms that maintain metabolic, protein, and lipid homeostasis, primarily by leveraging the advantages of the model organism C. elegans. Much of our work involves SKN-1, the C. elegans ortholog of the NRF (NF-E2-related factor) transcription factors, which respond to oxidative, xenobiotic, proteasomal, and certain metabolic stresses. NIGMS has supported our efforts almost continuously since 1994. Having pioneered work on SKN-1 as an NRF protein model, we have shown that these proteins play important roles in promoting longevity and elucidated a number of intriguing and conserved aspects of their functions. This MIRA covers the majority of our work on SKN-1, aside from a separate project investigating specific functions of the SKN-1A isoform (NRF1 ortholog) in lipid homeostasis. During the next five years this MIRA research will address a set of exciting and interrelated aspects of SKN-1 functions, building upon opportunities provided by our recent findings. It has become clear that fully delineating the functions of the two major SKN-1 isoforms (SKN-1A and SKN-1C) is critical and will be very informative because they correspond directly to NRF proteins with distinct functions and regulation (NRF1 and NRF2, respectively). To this end, we will generate necessary mutations and address the unmet need of identifying SKN-1C functions in a variety of contexts where skn-1 is critical. We have determined that when the endoplasmic reticulum (ER) is subject to stress from impairment of disulfide crosslinking the response is distinct from the canonical ER unfolded protein response, apparently because reactive oxygen species generation is elevated. Elucidating this response, which we have named the ER disulfide relay response (DRRER), and which involves SKN-1 isoforms directly, will yield new insights into an unanticipated aspect of ER homeostasis. No analyses of stress responses have investigated their effects on the redoxome, the universe of oxidatively modified Cys residues and redox-regulated processes. Having recently mapped the C. elegans redoxome at unprecedented coverage, we will thereby elucidate the redox effects of the DRRER and SKN-1 isoforms. This effort will reveal a new dimension of stress response effects, identify mechanisms subject to redox regulation, and uncover processes through which reactive oxygen species can unexpectedly increase lifespan. These approaches will be informative for understanding how stress resistance or lifespan can be increased by any intervention. We will thereby also investigate effects of inhibiting the master growth regulator mTORC1 (mechanistic target of rapamycin). C. elegans provides an unparalleled system for unbiased genetic investigation of stress defense mechanisms, an approach we will continue during the next five years by focusing on how proteasomal stress regulates SKN-1A. These efforts will yield new paradigms of biological regulation and significantly deep...