Interaction of the TMEM127 tumor suppressor with the mTORC1 lysosomal activating complex

TMEM127 肿瘤抑制因子与 mTORC1 溶酶体激活复合物的相互作用

• 批准号: 9311059
• 负责人: PATRICIA Leal DAHIA
• 金额: $ 29.72万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-06-01 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9311059
• 关键词: Address; Amino Acids; Arginine; Binding; CRISPR/Cas technology; Cancer Control; Cell model; Cell physiology; Cells; Cellular Stress; Complement; Complex; Data; Docking; FRAP1 gene; Functional disorder; Genetic Models; Glucose; Glutamine; Growth; Growth Factor; Guanosine Triphosphate Phosphohydrolases; Homeostasis; Human; In Vitro; Individual; Inherited; Knock-out; Knowledge; Leucine; Light; Lysosomes; Malignant Neoplasms; Mediating; Metabolism; Modeling; Mouse Strains; Multiprotein Complexes; Mutate; Mutation; Neuroendocrine Tumors; Nutrient; Osmolalities; Oxidative Stress; Pathogenesis; Pathway interactions; Patients; Pheochromocytoma; Primary Neoplasm; Process; Property; Proteins; Publishing; Recruitment Activity; Regulation; Regulatory Pathway; Renal carcinoma; Role; Sampling; Signal Transduction; Small Interfering RNA; Specificity; Starvation; Stress; Surface; System; TSC2 gene; Testing; Tissues; Transmembrane Domain; Tumor Suppressor Genes; Tumor Suppressor Proteins; Ursidae Family; Variant; Vertebrates; Vitronectin; base; cancer cell; clinically relevant; cohort; design; detection of nutrient; experimental study; gene discovery; genome editing; in vivo; insight; knock-down; lysosomal proteins; malignant endocrine gland neoplasm; mutant; novel; novel therapeutics; protein complex; response; sensor; tool; tumor; vacuolar H+-ATPase

The mTORC1 pathway regulates multiple cellular processes to promote a switch from catabolic to anabolic metabolism and is thus under tight regulatory control by growth factor signaling and nutrient sensing pathways. Dysregulation of this complex machinery is implicated in many cancers, so defining the key mechanisms by which mTORC1 senses changes in cellular homeostasis to activate growth signals is of great relevance. Spatial regulation of mTORC1 signaling has been recognized as a major mechanism that influences the cellular response to nutrients and the lysosome is central to this process. We found that the tumor suppressor TMEM127, a poorly characterized lysosomal protein, is a component of the lysosome-anchored multiprotein complex involved in the mTORC1 response to amino acids. In this proposal we seek to define the mechanisms through which the interaction between TMEM127 and the mTORC1 regulatory machinery is regulated and how this can impact on mTORC1 inhibition. We previously identified TMEM127 as a tumor suppressor gene mutated in hereditary neuroendocrine tumors, pheochromocytomas, and in renal cancers, and found that mutant tumors display increased mTORC1 signaling. Our earlier studies revealed that TMEM127 loss leads to lysosomal expansion with redistribution of mTOR toward the lysosome. The lysosome functions as a docking platform for mTORC1 signaling in response to amino acids through the assembly of a multi‐protein complex involving Rag GTPases, Ragulator (LAMTOR1‐5) and vacuolar ATPase (v‐ATPase). Using multiple in vivo and in vitro approaches developed in our lab, we found that TMEM127 physically associates with ragulator, vATPase and Rags, and in its absence the interaction between ragulator and Rags is enhanced and mTOR recruitment by Rags is increased. Furthermore, our preliminary data support of an effect of TMEM127 in nutrient sensing. Based on these observations, our general hypothesis is that TMEM127 disrupts mTOR recruitment to the lysosomal‐centered protein complex through inhibition of the ragulator-Rag interaction in response to amino acids. To test this hypothesis, we propose to define the signals that regulate the TMEM127-ragulator-Rag-mTORC1 interaction, including its response to individual amino acids, to other nutrients (e.g. glucose), to cellular stresses including starvation, osmolality, oxidative stress, and growth factor signaling. Furthermore, we propose to systematically define the components of the lysosomal multiprotein assembly that are required for TMEM127 binding, and conversely, which domains of TMEM127 are necessary for this interaction. Finally, we will explore the emerging notion that the nutrient sensing and the growth-factor signaling branches of mTORC1 activation are integrated at the lysosome by defining TMEM127's contribution to the mTOR inhibitory actions by TSC2 at the lysosomal surface.To carry out these experiments we will take advantage of tools and models that were developed in our lab, i.e. an in vivo mouse strain of targeted Tmem127 deletion, cell models of human TMEM127 knockout (by CRISPR-Cas9-based genome editing), knockdown (by siRNA), as well as a cohort of primary tumor samples with TMEM127 mutations, along with mutant constructs and genome edited cells that mimic naturally-occurring TMEM127 mutations identified in patients with cancer. In addition, genetic models of activation or loss of ragulator, Rags and mTOR components will complement our analysis. Given its tumor suppressor role in humans, understanding the contribution of TMEM127, a novel lysosomal protein that participates in the mTORC1 lysosomal assembly should provide insights into how nutrient and growth signals are integrated and can be coopted by cancer cells to promote uncontrolled proliferation.

MTORC1途径调节多个细胞过程，以促进从分解代谢到 合成代谢的代谢，因此受到生长因子信号和养分的严格调节控制 感知途径。这种复杂机械的失调在许多癌症中实现，因此定义 MTORC1感应细胞稳态变化以激活生长的关键机制 信号具有很大的意义。 MTORC1信号的空间调节已被认为是主要的 影响细胞对养分和溶酶体反应的机制至关重要 过程。我们发现肿瘤抑制剂TMEM127是一种溶酶体蛋白质不佳的肿瘤TMEM127，是一个 MTORC1响应涉及的溶酶体锚定的多蛋白复合物的成分 氨基酸。在此提案中，我们试图定义与之相互作用之间相互作用的机制 TMEM127和MTORC1调节机制受到调节，以及这会如何影响MTORC1 抑制。 我们先前鉴定出TMEM127是在遗传神经内分泌中突变的肿瘤抑制基因 肿瘤，嗜铬细胞瘤和肾癌中，发现突变肿瘤显示出增加 MTORC1信号传导。我们较早的研究表明，TMEM127损失导致溶酶体扩张 将mTOR重新分布向溶酶体。溶酶体作为一个对接平台 MTORC1信号传导通过组装多蛋白质复合物响应氨基酸 涉及RAG GTPases，Ragulator（Lamtor1-5）和真空ATPase（V-ATPase）。使用多个英寸 体内和体外方法在我们的实验室中开发出来，我们发现TMEM127与 窗帘，vatpase和rags，在缺乏间rag和rags之间的相互作用是 破布增加和MTOR募集。此外，我们的初步数据支持 TMEM127在营养敏感性中的影响。基于这些观察，我们的一般假设是 TMEM127通过 响应氨基酸抑制间窗帘的相互作用。为了检验这一假设，我们 定义调节TMEM127间rag-rag-mtorc1相互作用的信号的建议，包括 它对单个氨基酸的反应，对其他营养素（例如葡萄糖）的反应 饥饿，渗透压，氧化应激和生长因子信号传导。此外，我们建议 系统地定义溶酶体多蛋白组件的成分 TMEM127结合，相反，TMEM127的哪个域对于这种相互作用是必需的。 最后，我们将探讨营养感应和生长因子信号传导的新兴概念 通过定义TMEM127对TMEM127的贡献，MTORC1激活的分支在溶酶体上集成 TSC2在溶酶体表面进行的MTOR抑制作用。要进行这些实验，我们将 利用我们实验室开发的工具和模型，即 靶向TMEM127缺失，人类TMEM127敲除的细胞模型（通过CRISPR-CAS9 基因组编辑），敲低（由siRNA）以及与TMEM127的原发性肿瘤样品的队列 突变以及突变构建体和基因组编辑的细胞，这些细胞模仿天然存在 TMEM127突变在癌症患者中鉴定出来。另外，激活或丧失的遗传模型 摊贩，抹布和mTOR组件的分析将补充我们的分析。 鉴于其在人类中的肿瘤抑制作用，了解TMEM127的贡献，这是一种新颖 参与MTORC1溶酶体组装的溶酶体蛋白应提供见解 养分和生长信号如何整合，可以由癌细胞促进以促进 不受控制的增殖。