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

mTORC1通路调节多种细胞过程，以促进从分解代谢到代谢的转换。 合成代谢，并因此受到生长因子信号传导和营养物质的严格调控。 感知路径这种复杂机制的失调与许多癌症有关，因此定义 mTORC 1感知细胞内稳态变化以激活生长的关键机制 信号是非常重要的。mTORC1信号传导的空间调节已被认为是一个主要的 一种影响细胞对营养物质反应的机制，而溶酶体是其中的核心 过程我们发现肿瘤抑制因子TMEM127是一种特征不明显的溶酶体蛋白， 溶酶体锚定的多蛋白复合物的组成部分，参与mTORC 1对 个氨基酸在这一建议中，我们试图界定各种机制， TMEM127和mTORC1调节机制受到调节，以及这如何影响mTORC1 抑制作用 我们先前鉴定TMEM127是一个在遗传性神经内分泌肿瘤中突变的肿瘤抑制基因， 肿瘤，嗜铬细胞瘤和肾癌，并发现突变型肿瘤显示增加 mTORC1信号传导。我们早期的研究表明，TMEM127缺失导致溶酶体扩增， mTOR向溶酶体的重新分布。溶酶体的功能是作为一个对接平台， mTORC1信号通过多蛋白复合物的组装响应氨基酸 涉及Rag GTP酶、Ragulator（LAMTOR 1 - 5）和液泡ATP酶（v-ATP酶）。使用多个 在我们实验室开发的体内和体外方法中，我们发现TMEM127与 ragulator、vATPase和Rags之间的相互作用，并且在其不存在的情况下，ragulator和Rags之间的相互作用是 加强和mTOR征聘的Rags增加。此外，我们的初步数据支持， TMEM127在营养感测中的作用。根据这些观察，我们的一般假设是， TMEM127通过以下途径破坏mTOR向以溶酶体为中心的蛋白质复合物的募集 响应于氨基酸的调节子-Rag相互作用的抑制。为了验证这个假设，我们 建议定义调节TMEM 127-调节子-Rag-mTORC 1相互作用的信号，包括 它对单个氨基酸、其他营养素（如葡萄糖）、细胞应激（包括 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装所需的组分， TMEM127结合，以及相反，TMEM127的哪些结构域是这种相互作用所必需的。 最后，我们将探讨新出现的概念，即营养传感和生长因子信号 mTORC 1激活的分支通过定义TMEM 127对溶酶体的贡献而整合在溶酶体中。 TSC2在溶酶体表面的mTOR抑制作用。为了进行这些实验， 利用我们实验室开发的工具和模型，即体内小鼠品系， 靶向Tmem127缺失，人TMEM127敲除的细胞模型（通过基于CRISPR-Cas9的 基因组编辑）、敲低（通过siRNA）以及具有TMEM127的原发性肿瘤样品的队列 突变，沿着突变构建体和基因组编辑的细胞， 在癌症患者中发现的TMEM127突变。此外，激活或丢失的遗传模型 ragulator、Rags和mTOR成分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，理解TMEM127的贡献， 参与mTORC1溶酶体组装的溶酶体蛋白应该提供以下见解： 营养和生长信号是如何整合的，以及如何被癌细胞吸收， 不受控制的扩散。

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.