Lysosome biogenesis and homeostasis

溶酶体生物发生和稳态

• 批准号: 8939900
• 负责人: Rosa Puertollano-Moro
• 金额: $ 85.73万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 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/8939900
• 关键词: Amino Acids; Autophagocytosis; Binding; Biogenesis; Brain; Calcium Channel; Catabolic Process; Cell Cycle; Cell Nucleus; Cell Proliferation; Cell model; Cell physiology; Cells; Cellular biology; Complex; Couples; Cytosol; Diabetes Mellitus; Disease; Dissociation; Docking; Energy Metabolism; Equilibrium; Exocytosis; Family; Folliculin; Gene Expression; Genes; Genetic Transcription; Glycogen storage disease type II; Goals; Growth; Growth Factor; Guanosine Triphosphate Phosphohydrolases; Helix-Turn-Helix Motifs; Homeostasis; Leucine Zippers; Link; Lysosomes; Malignant Neoplasms; Mediating; Metabolic Diseases; Molecular; Molecular Chaperones; Muscle; Nutrient; Organelles; Pathway interactions; Phosphorylation; Protein Biosynthesis; Protein-Serine-Threonine Kinases; Proteins; Proteomics; Recruitment Activity; Regulation; Research; Role; Signal Transduction; Site; Starvation; Stress; Surface; Tissues; Up-Regulation; Variant; Work; cell growth; clinically relevant; deprivation; detection of nutrient; human FRAP1 protein; human TFE3 protein; late endosome; member; novel; overexpression; response; therapeutic target; transcription factor

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.

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和抑制信号，以最终调节多种关键细胞功能，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，其响应于能量水平、生长因子和营养物调节增殖、细胞周期和自噬。mTOR对许多压力做出反应，其失调导致癌症、代谢疾病和糖尿病。在细胞中，mTOR作为两种结构和功能不同的复合物存在，称为mTOR复合物1（mTORC 1）和mTOR复合物2（mTORC 2）。mTORC 1通过平衡合成代谢（蛋白质合成和营养储存）和分解代谢过程（自噬和能量储存的利用）将能量和营养丰度与细胞生长和增殖相结合。活性mTORC 1定位于晚期内体/溶酶体，这种分布被认为对于mTORC 1感知和响应氨基酸水平变化的能力至关重要。mTORC 1被认为是自噬的转录非依赖性调节因子。在营养丰富的条件下，mTORC 1是活跃的，并直接磷酸化和抑制参与自噬诱导的Atg蛋白，如Atg 13和Atg 1（ULK 1/2）。在饥饿条件下，当mTORC 1失活时，mTORC 1从ULK复合物中解离，从而导致自噬诱导。 转录因子EB（TFEB）是碱性螺旋-环-螺旋亮氨酸拉链转录因子家族的成员，其通过正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。重要的是，我们发现mTORC 1控制TFEB的活性和细胞定位。在营养丰富的条件下，mTORC 1磷酸化S211中的TFEB，从而促进TFEB与胞质伴侣14-3-3的结合和TFEB在胞质中的保留。在氨基酸剥夺后，TFEB/14-3-3复合物的解离导致TFEB递送至细胞核和基因的上调，这导致诱导自噬、溶酶体的生物发生和增加的溶酶体降解。我们还发现TFEB通过与活性Rag GTP酶直接相互作用被募集到溶酶体。这种Rag介导的TFEB向溶酶体表面的再分布促进了mTORC 1对TFEB的磷酸化，并构成了将营养可用性与TFEB失活联系起来的有效方式。TFEB和Rags之间相互作用的抑制导致TFEB在细胞核中的积累和在营养丰富的条件下自噬的组成性激活，从而表明TFEB向溶酶体的募集对于该转录因子的适当控制是至关重要的。 最近，我们确定了转录因子E3（TFE 3）作为新的调节溶酶体的形成和功能。与TFEB类似，TFE 3向溶酶体的募集由活性Rag GTP酶介导，该步骤对于mTORC 1介导的TFE 3磷酸化和保留在胞质溶胶中至关重要。TFE 3的过表达导致增加的自噬和增强的溶酶体生物发生，如通过溶酶体数量和溶酶体活性的增加所证明的。相反，内源性TFE 3的耗尽完全消除了细胞对饥饿的反应，从而证实了TFE 3在营养感测和能量代谢中的关键作用。 我们还描述了TFE 3是一种新的和非常有前途的治疗靶点，用于治疗溶酶体贮积症，通过显示过表达的TFE 3增加溶酶体钙通道MCOLN 1的丰度，触发溶酶体胞吐，并促进有效的细胞清除庞贝氏症的细胞模型。鉴于内源性TFE 3在关键组织如脑和肌肉中的高水平表达，TFE 3诱导细胞清除的能力具有潜在的临床相关性。 最后，我们的工作表明，Rag GTP酶的功能作为对接站点的招聘不同套的效应器的溶酶体表面取决于他们的激活状态。通过使用蛋白质组学方法，我们成功鉴定了毛囊素，作为mTORC 1途径的新型调节因子。我们目前正在验证其他新的候选人，选择性地招募到溶酶体的活性或非活性的拉格GTP酶。