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。 最近，人们发现了一种新的调控自噬的转录依赖机制。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。总体而言，我们的工作为理解溶酶体作为信号中心的新的和令人兴奋的角色提供了新的见解，它将环境信号与基因表达、能量产生和细胞动态平衡同步。 我们之前与Andrea Ballabio博士合作，报道了TFEB的过度表达诱导溶酶体胞吐，并导致几种溶酶体储存障碍的细胞清除。我们现在已经扩展了这些观察，发现TFEB是治疗庞贝病的一个有前途的新的治疗靶点。在与Nina Raben实验室的合作中，我们观察到TFEB的过度表达足以显着减少Pompe病肌管中溶酶体的大小和溶酶体内糖原的积聚。这项工作强调了对新的基本细胞过程的阐明可能如何潜在地导致治疗人类疾病的新方法的发展。

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. Recently, a new transcription-dependent mechanism regulating autophagy has been identified. 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. Overall, our work provides new insight for understanding the novel and exciting role of lysosomes as signaling centers that synchronize environmental cues with gene expression, energy production, and cellular homeostasis. In collaboration with the group of Dr. Andrea Ballabio, we previously reported that over-expression of TFEB induces lysosomal exocytosis and leads to cellular clearance in several Lysosomal Storage Disorders. We have now extended these observations and found that TFEB is a promising novel therapeutic target for the treatment of Pompe disease. In collaboration with the laboratory of Nina Raben we observed that over-expression of TFEB is sufficient to dramatically reduce lysosomal size and intra-lysosomal glycogen accumulation in Pompe disease myotubes. This work emphasizes how the elucidation of novel basic cellular processes may potentially lead to the development of new approaches for treatment of human disease.