Lysosome biogenesis and homeostasis

动物有能力适应许多内部和外部干扰，从而确保其一生中的有机体稳态。近年来，MiT/TFE家族的碱性螺旋螺旋亮氨酸拉链转录因子已成为细胞应激反应的关键组成部分。MiT/TFE家族包括四个成员，MITF，TFEB，TFE 3和TFEC，它们存在于大多数后生动物生物体中并且可以彼此异二聚化。与此相反，D. melanogaster和C. elegans，分别命名为Mitf和HLH-30。 转录因子TFEB和TFE 3通过正向调节属于协调溶酶体表达和调节（CLEAR）网络的基因来控制溶酶体生物发生和自噬。我们先前描述了TFEB和TFE 3的主要调节机制是控制它们从细胞质到细胞核的易位。在基础（非应激）条件下，TFEB和TFE 3通过与活性Rag GTP酶相互作用被募集到溶酶体表面。这使得TFEB和TFE 3非常接近丝氨酸/苏氨酸激酶mTORC 1，其在多个残基上磷酸化转录因子。TFEB在丝氨酸211（S211）上和TFE 3在丝氨酸321（S321）上的mTORC 1依赖性磷酸化产生1433的结合位点，导致TFEB和TFE 3在胞质溶胶中的螯合。在应激条件下，通过mTORC 1的失活或特定磷酸酶的激活，TFEB和TFE 3的去磷酸化导致转录因子快速易位到细胞核，在那里它们激活多个转录网络，目的是消除受损的细胞器，保护细胞功能并最终恢复细胞内稳态。 我们的实验室已经确定了越来越多的诱导TFEB和TFE 3激活的应激源，包括营养剥夺，病原体，未折叠蛋白质病原体的积累，DNA损伤和氧化应激。其他研究小组也报道了TFEB和TFE 3激活对线粒体损伤、体育锻炼和细胞溶质Ca 2+增加的响应。这些观察清楚地表明，这些转录因子在细胞应激反应中起着重要作用。 我们还研究了在慢性应激下维持持续TFEB和TFE 3激活所需的潜在机制。我们最近描述了TFEB和TFE 3调节的新机制。我们确定了一个基于半胱氨酸的氧化还原开关，控制TFEB和TFE 3寡聚状态之间的转换。应力后14-3-3的分离暴露了单个半胱氨酸残基，其经历ROS依赖性二硫键形成，导致TFEB和TFE 3寡聚体的组装。寡聚体的形成是快速和可逆的，并且在体外和体内响应于各种应力而发生。低聚物在长期强制降解条件下进一步稳定，并显示出对失活的抗性增加。In C.在线虫中，寡聚体组装的抑制也影响HLH-30活性，导致有害的表型，如发育延迟、改变的dauer功能和增加的病原体易感性。这些发现揭示了一种新的和进化上保守的机制，重要的是维持MiT/TFE转录因子在长期的应激条件下激活。 虽然TFEB/TFE 3核转位的几种机制已被很好地表征，但对调节其转录活性的核因子知之甚少。为了解决这个问题，我们最近进行了内源性蛋白质的快速免疫沉淀质谱（RIME），并确定了TFEB/TFE 3和促进染色质转录（FACT）复合物之间的一种新的相互作用，FACT复合物是一种异二聚体组蛋白伴侣，可介导核小体解体，以促进靶基因的快速转录延伸。我们发现，几种刺激，包括营养剥夺，Torin 1诱导的mTORC 1失活和氧化应激，诱导TFEB和TFE 3的核转位，然后与FACT复合物调节应激诱导的基因转录。FACT的耗尽或失活不影响TFEB/TFE 3的活化、稳定性或结合靶基因启动子的能力。相比之下，通过使用RNA-seq和q-PCR的组合，我们发现TFEB介导的溶酶体和抗氧化剂基因的诱导在FACT耗尽的细胞中显著受损。此外，许多TFEB/TFE 3靶标的转录延伸率通过FACT耗尽或失活而降低，从而表明FACT复合物作为TFEB/TFE 3转录激活剂起作用。这项工作突出了染色质重塑对持续有效的应激反应的重要性，并为氧化还原稳态和溶酶体生物发生的表观遗传调节提供了新的线索。

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. We have also investigated the potential mechanisms required to maintain 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. While several of the mechanisms of TFEB/TFE3 nuclear translocation are well characterized, little is known about the nuclear factors that modulate their transcriptional activity. To address this question, we recently 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.