Understanding Compartmentalized Leucine Metabolism Downstream of mTORC1 Signaling

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Project Summary/Abstract: The mechanistic target of rapamycin complex 1 (mTORC1) kinase coordinates cell growth and metabolism, integrating signals from growth factors, nutrients, and stresses. Many diseases, including cancers, diabetes, and epilepsy, have been associated with aberrations in mTORC1 signaling. Understanding the inputs for and outputs of mTORC1 signaling is of critical importance in driving innovations in new therapeutic interventions for these diseases. Recently, it has been appreciated that mTORC1 signaling is important for compartmentalized amino acid metabolism; this is particularly true with respect to the essential amino acid leucine. While leucine activates mTORC1 in the cytosol, mTORC1 also regulates leucine metabolism in the cytosol, mitochondria, and lysosomes. Recent work from our lab has demonstrated a key role for mTORC1 signaling in regulating the efflux of leucine and other essential amino acids from lysosomes as part of the cellular response to starvation, but how mTORC1 signaling affects leucine levels in the cytosol and mitochondria, the two other important sites of leucine metabolism in cells, remains unknown. Answering this question could help to illuminate how mTORC1 rewires amino acid metabolism in response to starvation. We will determine how mTORC1 signaling affects compartmentalized leucine metabolism through the following specific aims: 1) optimize methods to measure compartmentalized leucine levels, 2) determine how mTORC1 activity changes leucine levels in the cytosol and mitochondria, and 3) characterize how mTORC1 activity affects the rate of leucine catabolism. We will use a multidisciplinary approach, drawing on tools including genetically encoded fluorescent biosensors, organellar metabolomics, and isotope tracing, to systematically address how mTORC1 signaling affects leucine levels in the cytosol and mitochondria. Our results will be important for understanding the role of mTORC1 in regulating adaptive responses to starvation, and may identify previously unappreciated metabolic vulnerabilities in diseases with dysregulated mTORC1 signaling. The proposed work, which will take place at the MIT Biology Department/Whitehead Institute— outstanding intellectual environments that are dedicated to scientific training, includes a strong and comprehensive training plan. This plan emphasizes the development of experimental skills in cutting-edge biochemical approaches including protein engineering, fluorescence imaging, and metabolomics; critical thinking abilities; oral and written scientific communication skills; and science education experience.