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Lysosome biogenesis and homeostasis

溶酶体生物发生和稳态

基本信息

批准号：
10929152
负责人：
rosa puertollano
金额：
$ 157.37万
依托单位：
NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10929152
关键词：
Alanine Animals Anisomycin Antioxidants Autophagocytosis Binding Binding Sites Biogenesis Biological Response Modifiers Caenorhabditis elegans Cell Nucleus Cell physiology Cells Chromatin Complex Cues Cytosol DNA Damage Data Ensure Exocytosis Family Family member Gene Expression Generations Genes Genetic Transcription Goals Growth Factor Guanosine Triphosphate Phosphohydrolases Heterodimerization Histones Homeostasis Immune Immune response Immunoprecipitation Impairment Infection Inflammasome Inflammatory Knock-in Laboratories Leucine Zippers Light Lysosomes MAP Kinase Gene Macrophage Mass Spectrum Analysis Mediating Mitochondria Modification Molecular Molecular Chaperones Mutate Nuclear Translocation Nucleosomes Organelles Organism Output Oxidation-Reduction Oxidative Stress Pathway interactions Phosphoric Monoester Hydrolases Phosphorylation Physical Exercise Play Post-Translational Protein Processing Protein Dephosphorylation Protein-Serine-Threonine Kinases Proteins Regulation Reporting Research Role Serine Signal Pathway Stimulus Stress Surface TFE3 gene Transcription Coactivator Transcription Elongation Transcriptional Regulation Up-Regulation Viral Work betacoronavirus biological adaptation to stress cell type chemokine chromatin remodeling cytokine epigenetic regulation member monocyte novel nutrient deprivation overexpression p38 Mitogen Activated Protein Kinase pathogen preservation prevent programs promoter recruit response stressor transcription factor transcriptome sequencing

项目摘要

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. 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 damage, physical exercise, and increased cytosolic Ca2+. These observations clearly suggest 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. This work reveals 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. Finally, we seek to identify novel regulators of TFEB and TFE3 transcriptional activity. For this, we performed Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins (RIME) and identified a novel interaction between TFEB/TFE3 and the Facilitating Chromatin Transcription (FACT) complex, a heterodimeric histone chaperone that mediates nucleosome disassembly to facilitate rapid transcriptional elongation of target genes. We found that several stimuli, including nutrient deprivation, Torin1-induced mTORC1 inactivation and oxidative stress, induced nuclear translocation of TFEB and TFE3, which then associated with the FACT complex to regulate stress-induced gene transcription. Depletion or inactivation of FACT did not affect TFEB/TFE3 activation, stability, or ability to bind to the promoter of target genes. In contrast, by using a combination or RNA-seq and q-PCR, we found that the TFEB-mediated induction of lysosomal and antioxidant genes was significantly impaired in FACT-depleted cells. Furthermore, the transcriptional elongation rates of numerous TFEB/TFE3 targets were decreased by FACT depletion or inactivation, thus suggesting that the FACT complex functions as a TFEB/TFE3 transcriptional activator. This work highlights the importance of chromatin remodeling for a sustained and efficient stress response and sheds new light on the epigenetic regulation of redox homeostasis and lysosomal biogenesis.

动物有能力适应许多内部和外部的扰动，从而确保生物体在其一生中保持稳态。近年来，MiT/TFE家族的基本螺旋环螺旋亮氨酸拉链转录因子已经成为细胞对应激反应的关键组成部分。MiT/TFE家族包括MITF、TFEB、TFE3和TFEC四个成员，它们存在于大多数后生动物中，并且可以相互异二聚。相比之下，在黑腹线虫和秀丽隐杆线虫中只存在一个家族成员，分别称为Mitf和HLH-30。