Lysosome biogenesis and homeostasis

细胞生物学中最基本的问题之一是细胞如何整合生长刺激信号和抑制信号，最终调控多种关键细胞功能，包括基因表达、自噬、细胞器生物发生和细胞生长。MTOR是一种丝氨酸/苏氨酸激酶，调节细胞增殖、细胞周期和自噬，以响应能量水平、生长因子和营养物质。MTOR对大量压力做出反应，其失调会导致癌症、代谢性疾病和糖尿病。在细胞中，mTOR以两种结构和功能不同的复合体存在，称为mTOR复合体1(MTORC1)和mTOR复合体2(MTORC2)。MTORC1通过平衡合成代谢(蛋白质合成和营养储存)和分解代谢(自噬和能量储存)，将丰富的能量和营养与细胞的生长和增殖联系在一起。活性的mTORC1定位于晚期的内切体/溶酶体，这种分布被认为是mTORC1感知和响应氨基酸水平变化的能力的关键。MTORC1被认为是自噬的非转录调节因子。在营养丰富的条件下，mTORC1是活性的，直接磷酸化并抑制参与自噬诱导的ATG蛋白，如ATg13和Atg1(ULK1/2)。在饥饿条件下，当mTORC1失活时，mTORC1从ULK复合体中解离，从而导致自噬诱导。 转录因子EB(TFEB)是基本的螺旋-环-螺旋-亮氨酸拉链转录因子家族的成员，通过正向调节溶酶体表达与调节(CLEAR)网络中的基因来控制溶酶体的生物发生和自噬。重要的是，我们发现mTORC1控制着TFEB的活性和细胞定位。在营养丰富的条件下，mTORC1磷酸化S211中的TFEB，从而促进TFEB与胞液伴侣14-3-3的结合和TFEB在胞浆中的保留。当氨基酸缺乏时，TFEB/14-3-3复合体的解离导致TFEB被运送到细胞核，基因上调导致自噬、溶酶体的生物发生和溶酶体降解的增加。我们还发现，TFEB通过与活性的Rag GTP酶直接相互作用而被招募到溶酶体中。这种RAG介导的TFEB在溶酶体表面的重新分布促进了mTORC1对TFEB的磷酸化，并构成了一种将营养可利用性与TFEB失活联系起来的有效途径。抑制TFEB和RAGS之间的相互作用会导致TFEB在细胞核内积聚，并在营养丰富的条件下激活自噬，从而表明TFEB在溶酶体内的募集对该转录因子的适当控制至关重要。 最近，我们发现转录因子E3(TFE3)是溶酶体形成和功能的新调节因子。与TFEB类似，TFE3在溶酶体内的募集是由活性的Rag GTP酶介导的，这一步骤对于mTORC1介导的TFE3的磷酸化和在胞浆中的滞留是至关重要的。TFE3的过度表达导致自噬增加和溶酶体生物发生增强，溶酶体数量和溶酶体活性的增加证明了这一点。相反，内源TFE3的耗尽完全取消了细胞对饥饿的反应，从而证实了TFE3在营养感知和能量代谢中的关键作用。 我们还描述了TFE3是一个新的和非常有前途的治疗溶酶体储存障碍的靶点，在庞贝病的细胞模型中，过表达的TFE3增加了溶酶体钙通道MCOLN1的丰度，触发了溶酶体的胞吐，并促进了有效的细胞清除。鉴于内源性TFE3在脑和肌肉等关键组织中的高水平表达，TFE3诱导细胞清除的能力具有潜在的临床意义。 最后，我们的工作揭示了Rag GTP酶作为对接位置，根据不同的激活状态将不同的效应物招募到溶酶体表面。通过蛋白质组学的方法，我们成功地鉴定了毛囊蛋白，它是mTORC1途径的一个新的调节因子。我们目前正在验证通过激活或非激活的RAG GTP酶选择性地招募到溶酶体的其他新的候选基因。

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. 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. More recently we identified the transcription factor E3 (TFE3) as novel regulator of lysosomal formation and function. Similar to TFEB, the recruitment of TFE3 to lysosomes is mediated by active Rag GTPases and this step is critical for mTORC1-mediated phosphorylation of TFE3 and retention in the cytosol. Over-expression of TFE3 results in increased autophagy and enhanced lysosomal biogenesis, as evidenced by an increase in the number of lysosomes and lysosomal activity. In contrast, depletion of endogenous TFE3 entirely abolishes the cellular response to starvation, thus confirming the crucial role of TFE3 in nutrient sensing and energy metabolism. We also described that TFE3 is a novel and very promising therapeutic target for the treatment of Lysosomal Storage Disorders by showing that overexpressed TFE3 increases the abundance of the lysosomal calcium channel MCOLN1, triggers lysosomal exocytosis, and promotes efficient cellular clearance in cellular model of Pompe disease. Given the high level of expression of endogenous TFE3 in critical tissues, such as brain and muscle, the ability of TFE3 to induce cellular clearance is of potential clinical relevance. Finally, our work revealed that Rag GTPases function as docking sites for the recruitment of different sets of effectors to the lysosomal surface depending on their activation state. By using proteomic approaches we have successfully identified folliculin, as a novel regulator of the mTORC1 pathway. We are currently validating additional novel candidates that are selectively recruited to lysosomes by either active or inactive Rag GTPases.