Project Summary In this project we will study two important homeostasis/stress responses we have discovered in C. elegans. Each of these responses is mediated by the conserved transcription factor SKN-1A, the ortholog of human Nrf1 (NF-E2-related factor 1). SKN-1A/Nrf1 resides in the ER and canonically maintains proteasome levels. However, in a pathway we term the SKN-1A/Nrf1 lipid homeostasis response, SKN-1A is activated independently of proteasome activity by the monounsaturated fatty acid oleic acid (OA), through effects on ER membrane and metabolic mechanisms. In turn, SKN-1A reduces fat levels, enhances proteostasis, and extends lifespan. In the second response, SKN-1A is activated by ribosomal assembly stress which, unexpectedly, induces a metabolic crisis in which lipids and specific amino acids (AAs) are depleted. SKN-1A counteracts this stress by increasing AA levels, improving proteostasis, and supporting translation. Ribosomal stress can also be ameliorated by AA feeding, which partially reverses these metabolic deficits. Our findings add a new dimension to our understanding of protein synthesis homeostasis. They are also likely relevant to human ribosomopathies such as Diamond Blackfan Anemia (DBA), a genetic disease resulting from ribosomal subunit mutations, and suggest potential metabolic treatment strategies for such diseases. Each of these SKN-1A functions provides a window into mechanisms that are fundamentally important for metabolism and aging. In Aim 1 we will focus on the SKN-1A/Nrf1 lipid homeostasis response. We will investigate how SKN-1A acts through specific mechanisms and in certain tissues to promote health and lifespan in response to OA, and perform cell culture experiments to test our model that key features of this response are conserved in humans. In Aims 2 and 3 we will further develop our models for how ribosomal stress affects the organism and is counteracted by SKN-1A, and examine conservation of these mechanisms. In Aim 2 we will test our hypothesis that the metabolic demands of impaired ribosomal assembly induce the metabolic crisis we have observed. We will elucidate the cause(s) of these metabolic demands through collaborative stable isotope tracing metabolomic pathway analyses, and lipidomics. Our metabolomics will be guided in part by identification of specific AA combinations that can rescue effects of ribosomal stress and the lack of SKN-1A. We will also determine whether the translation deficits that result from ribosomal stress and lack of SKN-1A are mediated in part through decreased AA levels affecting mTORC1 signaling and/or ribosome stalling. In Aim 3 we will investigate the importance of SKN-1A and AA availability for lifespan extensions induced by ribosomal perturbation. We will also determine whether the SKN-1A/Nrf1 response to ribosomal stress is conserved in human cells, including investigating collaboratively whether human Nrf1 and AA supplementation are beneficial in an in vitro human ...