Lysosome biogenesis and homeostasis

National Heart, Lung, And Blood Institute

Investigators

Linked publications & trials

Abstract

Animals have the ability to adapt to numerous internal and external perturbations, thus ensuring organismal homeostasis throughout their lifetime. The MiT/TFE family of basic helix–loop–helix leucine-zipper transcription factors have 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. mTORC1‐dependent phosphorylation of TFEB on serine 211 (S211) and TFE3 on serine 321 (S321) creates a binding site for 14‐3‐3, 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. Our laboratory has identified a growing list of stressors that induce TFEB and TFE3 activation, including nutrient deprivation, pathogens, accumulation of unfolded proteins pathogens, DNA damage, and oxidative stress. Other groups have also reported TFEB and TFE3 activation in response to mitochondrial and lysosomal damage, physical exercise, and increased cytosolic Ca2+. These observations support an essential role of these transcription factors in cellular response to stress. However, how the output from complex microenvironmental cues is integrated by TFEB and TFE3 to regulate vastly different transcriptional programs in a wide variety of cell types is still poorly understood. We recently identified a novel mechanism of TFEB regulation through phosphorylation of TFEB-S401 by p38 MAPK. We found that multiple inputs that induced p38 MAPK activation, including oxidative stress, UVC light, growth factors, LPS, and anisomycin, dramatically increased S401 phosphorylation levels, while modification of this residue was prevented by p38 MAPK inhibition or depletion. Generation of THP1 knock-in clones in which endogenous TFEB-S401 was mutated to alanine demonstrated that the p38 MAPK/TFEB pathway plays a particularly relevant role during monocyte differentiation into macrophages. THP1 monocytes expressing TFEB-S401A failed to efficiently upregulate expression of multiple immune genes in response to PMA, including critical cytokines, chemokines, and growth factors. Polarization of M0 macrophages into M1 inflammatory macrophages was also aberrant in TFEB-S401A cells in terms of gene expression, cytokine and chemokine secretion, and inflammasome activation. These findings reveal a critical role of TFEB in the transcriptional control of monocyte differentiation and identifies phosphorylation of S401 as a novel post-translational modification that enables coordination of signaling pathways, gene expression, and lineage determination. The important function of TFEB and TFE3 in immune response was further corroborated by our data describing the contribution of these transcription factors to cellular response against beta-coronaviruses. We found that beta-coronavirus infection induced a robust and persistent activation of TFEB and TFE3. In the nucleus, TFEB and TFE3 bound to the promoter of multiple lysosomal and immune genes. Accordingly, MHV-induced upregulation of immune regulators was significantly decreased in TFEB/TFE3-depleted cells. Conversely, over-expression of either TFEB or TFE3 was sufficient to increase expression of several cytokines and chemokines. The reduced immune response observed in the absence of TFEB and TFE3 resulted in increased cellular survival of infected cells, but also in reduced lysosomal exocytosis and decreased viral infectivity. Therefore, modulation of TFEB/TFE3 activity might be a promising target for antiviral treatments. More recently, we showed that TFEB and TFE3 are also an important component of the cGAS-STING pathway. STING is an essential component of the innate immune defense against a wide variety of pathogens. Whereas induction of Type I interferon (IFN) responses is one of the best-defined functions of STING, our transcriptomic analysis revealed IFN-independent activities of STING in macrophages, including transcriptional upregulation of numerous lysosomal and autophagic genes. This upregulation was mediated by the STING-induced activation of the transcription factors TFEB and TFE3, and led to increased autophagy, lysosomal biogenesis, and lysosomal acidification. Unexpectedly, we observed that TFEB and TFE3 also modulated IFN-dependent STING signaling by controlling IRF3 activation. IFN production and cell death were increased in TFEB and TFE3 depleted iBMDMs. Conversely, TFEB over-expression led to reduced IRF3 activation and an almost complete inhibition of IFN synthesis and secretion, resulting in decreased caspase-3 activation and increased cell survival. Our work identifies and novel and key of TFEB and TFE3 as regulators of STING-mediated innate antiviral immunity. Given the important role played by TFEB and TFE3 in maintenance of cellular homeostasis, we investigated their potential contribution to embryonic development. We used CRISPR-CAS9 technology to deplete the one copy of tfeb and two copies of tfe3 present in zebrafish. Notably, we found that these transcription factors are essential for embryo survival during early development, with an almost complete lethality of the knockout larvae by 8-10 days post fertilization. By using a combination of scRNAseq, proteomics, confocal, and electron microscopy analysis, we characterized alterations in the pancreas, liver, and gut of KO animals. The most severe defects were found in exocrine pancreas, where the accumulation of abnormal zymogen granules led to acinar atrophy in embryos and pancreatitis-like phenotypes in adult fish. We also observed decreased proliferation and increased oxidative stress in KO hepatocytes. Finally, we showed that tfeb and tfe3 depletion increases embryos susceptibility to different stress conditions, including oxidative stress and heat shock treatment. These observations suggest an essential role of tfeb and tfe3 in maintaining tissue homeostasis during development.