Lysosome biogenesis and homeostasis

National Heart, Lung, And Blood Institute

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Abstract

Animals have the ability to adapt to numerous internal and external perturbations, thus ensuring organismal homeostasis throughout their lifetime. In recent years, the MiT/TFE family of basic helixloophelix leucine-zipper transcription factors has emerged as a critical component of the cellular response to stress. The MiT/TFE family includes four members, MITF, TFEB, TFE3, and TFEC, which are present in most metazoan organisms and can heterodimerize with each other. In contrast, only one member of the family is present in D. melanogaster and C. elegans, termed Mitf and HLH-30, respectively. The transcription factors TFEB and TFE3 control lysosomal biogenesis and autophagy by positively regulating genes belonging to the Coordinated Lysosomal Expression and Regulation (CLEAR) network. We previously described that the main regulatory mechanism for TFEB and TFE3 is the control of their translocation from the cytosol to the nucleus. Under basal (non-stressed) conditions, TFEB and TFE3 are recruited to the surface of lysosomes through interaction with active Rag GTPases. This brings TFEB and TFE3 in close proximity to the serine/threonine kinase mTORC1, which phosphorylates the transcription factors on multiple residues. mTORC1dependent phosphorylation of TFEB on serine 211 (S211) and TFE3 on serine 321 (S321) creates a binding site for 1433, resulting in sequestration of TFEB and TFE3 in the cytosol. Under stress conditions, dephosphorylation of TFEB and TFE3, either by inactivation of mTORC1 or activation of specific phosphatases, causes a rapid translocation of the transcription factors to the nucleus, where they activate multiple transcriptional networks with the goal of eliminating damaged organelles, preserving cellular functions and ultimately, restoring cellular homeostasis. We have identified a growing list of stressors that induce TFEB and TFE3 activation, including nutrient deprivation, pathogens, accumulation of unfolded proteins pathogens, and oxidative stress. Other groups have also reported TFEB and TFE3 activation in response to mitochondrial damage, physical exercise, and increased cytosolic Ca2+. These observations clearly suggest an essential role of these transcription factors in cellular response to stress. In agreement with this idea, we recently found that TFEB and TFE3 also regulate p53 dependent transcriptional programs in response to DNA damage. Treatment with a variety of compounds known to induce DNA damage, including etoposide, UV light or cisplatin, caused TFEB and TFE3 nuclear accumulation that was dependent on p53-mediated mTORC1 inactivation. The genotoxic stress response was severely altered in RAW 264.7 cells depleted of TFEB and TFE3, including reduced expression of p53 targets and decreased lysosomal permeability and apoptosis. Moreover, p53 half-live was significantly reduced in TFEB/TFE3 DKOs due to increased Mdm2 levels. Finally, expression of multiple genes implicated in cell cycle control was also altered in TFEB/TFE3-depleted cells, revealing a previously unrecognized role of these transcription factors in the regulation of cell cycle checkpoints in response to stress. While most studies have analyzed the role of TFEB and TFE3 in response to acute stress conditions, little is known about potential mechanisms to allow sustained TFEB and TFE3 activation under chronic stress. We recently characterized a novel mechanism of TFEB and TFE3 regulation. We identified a cysteine-based redox switch that controls the shift between TFEB and TFE3 oligomeric states. Detachment of 14-3-3 following stress exposes a single cysteine residue that undergoes ROS-dependent disulfide-bond formation, resulting in the assembly of TFEB and TFE3 oligomers. Oligomer formation is rapid and reversible and occurs in response to a variety of stresses both in vitro and in vivo. Oligomers are further stabilized under prolonged stress conditions and show increased resistance to inactivation. In C. elegans, inhibition of oligomers assembly also affects HLH-30 activity, resulting in deleterious phenotypes like developmental delay, altered dauer function, and increased pathogen susceptibility. These findings reveal a novel and evolutionary conserved mechanism important to maintain MiT/TFE transcription factors activation under prolonged stress conditions.