One of the most fundamental issues in cell biology is how cells integrate growth-stimulating and inhibitory signals to ultimately regulate a diversity of key cellular functions, including gene expression, autophagy, organelle biogenesis, and cell growth. mTOR is a serine/threonine kinase that regulates proliferation, cell cycle, and autophagy in response to energy levels, growth factors, and nutrients. mTOR responds to numerous stresses and its dysregulation leads to cancer, metabolic disease, and diabetes. In cells, mTOR exists as two structurally and functionally distinct complexes termed mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). mTORC1 couples energy and nutrient abundance to cell growth and proliferation by balancing anabolic (protein synthesis and nutrient storage) and catabolic processes (autophagy and the utilization of energy stores). Active mTORC1 localizes to late endosomes/lysosomes and this distribution is thought to be critical for the ability of mTORC1 to sense and respond to variations in the levels of amino acids. mTORC1 is considered a transcription-independent regulator of autophagy. Under rich-nutrient conditions, mTORC1 is active and directly phosphorylates and inhibits Atg proteins involved in autophagy induction such as Atg13 and Atg1 (ULK1/2). Under starvation conditions when mTORC1 is inactivated, mTORC1 dissociates from the ULK complex, thus leading to autophagy induction. Recently, a new transcription-dependent mechanism regulating autophagy has been identified. The transcription factor EB (TFEB) is a member of the basic helix-loop-helix leucine-zipper family of transcription factors that controls lysosomal biogenesis and autophagy by positively regulating genes belonging to the Coordinated Lysosomal Expression and Regulation (CLEAR) network. Importantly, we have found that mTORC1 controls the activity and cellular localization of TFEB. Under nutrient-rich conditions, mTORC1 phophorylates TFEB in S211, thus promoting binding of TFEB to the cytosolic chaperone 14-3-3 and retention of TFEB in the cytosol. Upon amino acids deprivation, dissociation of the TFEB/14-3-3 complex results in delivery of TFEB to the nucleus and up-regulation of genes that leads to induction of autophagy, biogenesis of lysosomes, and increased lysosomal degradation. We also found that TFEB is recruited to lysosomes through direct interaction with active Rag GTPases. This Rag-mediated redistribution of TFEB to the lysosomal surface facilitates the phosphorylation of TFEB by mTORC1 and constitutes an efficient way to link nutrient availability to TFEB inactivation. Inhibition of the interaction between TFEB and Rags results in accumulation of TFEB in the nucleus and constitutive activation of autophagy under nutrient rich conditions, thus indicating that recruitment of TFEB to lysosomes is critical for the proper control of this transcription factor. Overall, our work provides new insight for understanding the novel and exciting role of lysosomes as signaling centers that synchronize environmental cues with gene expression, energy production, and cellular homeostasis. In collaboration with the group of Dr. Andrea Ballabio, we previously reported that over-expression of TFEB induces lysosomal exocytosis and leads to cellular clearance in several Lysosomal Storage Disorders. We have now extended these observations and found that TFEB is a promising novel therapeutic target for the treatment of Pompe disease. In collaboration with the laboratory of Nina Raben we observed that over-expression of TFEB is sufficient to dramatically reduce lysosomal size and intra-lysosomal glycogen accumulation in Pompe disease myotubes. This work emphasizes how the elucidation of novel basic cellular processes may potentially lead to the development of new approaches for treatment of human disease.

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Project End
Budget Start
Budget End
Support Year
2
Fiscal Year
2013
Total Cost
$483,312
Indirect Cost
Name
National Heart, Lung, and Blood Institute
Department
Type
DUNS #
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Puertollano, Rosa (2014) mTOR and lysosome regulation. F1000Prime Rep 6:52
Martina, Jose A; Diab, Heba I; Lishu, Li et al. (2014) The nutrient-responsive transcription factor TFE3 promotes autophagy, lysosomal biogenesis, and clearance of cellular debris. Sci Signal 7:ra9
Feeney, Erin J; Spampanato, Carmine; Puertollano, Rosa et al. (2013) What else is in store for autophagy? Exocytosis of autolysosomes as a mechanism of TFEB-mediated cellular clearance in Pompe disease. Autophagy 9:1117-8
Martina, Jose A; Puertollano, Rosa (2013) Rag GTPases mediate amino acid-dependent recruitment of TFEB and MITF to lysosomes. J Cell Biol 200:475-91
Martina, Jose A; Puertollano, Rosa (2013) RRAG GTPases link nutrient availability to gene expression, autophagy and lysosomal biogenesis. Autophagy 9:928-30