Wednesday, February 5, 2025
2/5/2025
PROJECT SUMMARY The mechanistic Target of Rapamycin Complex 1 (mTORC1) is master regulator of cell growth and metabolism. Dysregulation of mTORC1 is observed in sporadic and familial cancers. mTORC1 inhibition is an established treatment for renal cell carcinoma (RCC). Despite great efforts to target mTORC1 in cancer, adverse effects limit the use of mTORC1 inhibitors in the clinic. Recent work from our lab and collaborators has revealed the existence and structural basis of substrate-specific regulatory pathways that may be targeted with greater precision than heretofore. mTORC1 is activated on the surface of lysosomes in response to nutrient signals by conversion of the nucleotide state of the Rag GTPases from inactive (RagA/BGDP-RagC/DGTP) to active (RagA/BGTP- RagC/DGDP). The Rags are targeted to the lysosome by the Ragulator complex. Rag states are interconverted by the RagC/D GAP FLCN-FNIP and the RagA/B GAP GATOR1. Subunits of these complexes, and the Rags, are mutated in cancer. RagA/BGTP and GATOR1 inactivation is required for phosphorylation of all mTORC1 substrates. RagC/DGDP and FLCN-FNIP activity is only required for phosphorylation of non-canonical substrates, which include TFEB, the key transcriptional regulator of lysosome biogenesis and autophagy. Cryo-EM studies of the Rag, Ragulator, and FLCN-FNIP pathway from our laboratory provided a start-to-finish structural explanation for the repression and reactivation of FLCN GAP activity in starvation and refeeding. These studies contributed to the discovery that RagC/DGDP uniquely regulates TFEB and MiT-TFE transcription factors, which in turn explained the tumor suppressor activity of FLCN in Birt-Hogg-Dubé (BHD) syndrome. This pathway has now been linked to RHEB activity and Tuberous Sclerosis Complex (TSC). We then demonstrated the existence and determining the structure and function of the mTORC1-TFEB-Rag-Ragulator “megacomplex”, containing a full mTORC1 dimer, two copies of TFEB, and four copies of the heptameric complex of active Rags and Ragulator, showing how RagC/DGDP specifically recruits TFEB. In aims 1 and 2, we will explore the new avenues opened up the analysis of the megacomplex. We will determine how the megacomplex is turned over following TFEB phosphorylation, and whether the principles of substrate specific activation seen for TFEB and RagC also apply to canonical substrates. Findings will be followed up in BHD and TSC cell lines and a BHD mouse xenograft model. While we now have a start-to-finish structural mapping of FLCN/RagC/D pathway, the still mysterious regulatory mechanisms operating in the GATOR1/RagA/B pathway will be elucidated in aim 3, and the cancer implications explored in knock-out and Glioblastoma cell lines.
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