Regulation of Selenium Metabolism and Selenoprotein Synthesis - PROJECT SUMMARY Selenium is an essential micronutrient that functions through its incorporation into selenoproteins, a unique family of proteins containing the amino acid selenocysteine (Sec). These proteins maintain redox balance, detoxify reactive oxygen species, regulate metabolism, and protect cells under stress. Key selenoproteins such as glutathione peroxidases and thioredoxin reductases provide potent antioxidant defense, and disruption of selenium metabolism has been linked to cancer, cardiovascular disease, diabetes, thyroid disorders, neurodegeneration, and autoimmune conditions. Despite decades of study, fundamental questions about selenium metabolism remain unanswered. Clinical trials of selenium supplementation have yielded inconclusive results, suggesting that outcomes may depend on the chemical form of selenium provided, its delivery to specific tissues, or the presence or absence of transporters and regulatory factors. How distinct dietary forms of selenium are absorbed and distributed remains unclear. Likewise, it is not understood how tissues prioritize selenium use under limiting conditions, or how the hierarchy of selenoprotein synthesis is dynamically regulated during oxidative stress. Addressing these questions requires integrated in vivo and functional genomic approaches that extend beyond traditional cell culture systems. Our preliminary studies reveal that oxidative stress activates the KEAP1/NRF2 pathway to enhance selenium uptake and that tissues and microenvironments exhibit distinct selenium profiles not reflected in standard culture conditions. Building on these insights, we will use animal models combined with advanced elemental analysis (ICP-MS and laser ablation ICP-MS) to generate a blueprint of selenium absorption, systemic distribution, and tissue-level preservation. Interstitial fluid profiling will complement these approaches by defining how dietary selenium influences the microenvironmental selenium pool. In parallel, to identify the key selenoprotein synthesis regulators, we will conduct genome-wide CRISPR knockout screens using a well- established GFP-based reporter. This reporter allows GFP fluorescence only upon successful selenocysteine insertion, providing a sensitive readout of selenoprotein synthesis. Screens will be performed under both steady state and oxidative stress conditions across multiple cell types, enabling identification of both core regulators and stress-specific pathways. Candidate genes will be validated using CRISPR knockout and a combination of assays, including selenoprotein expression, selenium uptake, and Sec tRNA modification profiling. Together, by integrating animal models, advanced trace-element mapping, and unbiased genetic screening, this work will resolve long-standing questions about selenium utilization and redox regulation. Ultimately, these insights will inform strategies to optimize dietary selenium interventions and identify new therapeutic opportunities for diseases driven by oxidative stress.
