Regulation of the mTOR Pathway by Hypoxia and the REDD1 Protein

描述（由申请人提供）：哺乳动物雷帕霉素靶点复合物1 （mTORC1）是细胞生长和代谢的关键调节剂，将细胞内和细胞外的各种信号与蛋白质翻译机制结合在一起。来自生长因子和能量储存的信号通过结节硬化复合体蛋白1 （TSC1）和2 （TSC2）传递给mTORC1，形成具有肿瘤抑制功能的复合体（TSC1/TSC2）。我们已经发现通过氧水平调节mTORC1也需要TSC1/TSC2复合体。在TSC1/ tsc2缺陷细胞中，抑制mTORC1以应对缺氧的失败会导致细胞增殖异常，并可能促进肿瘤生长。最近，我们发现调控发育和DNA损伤1 （REDD1）的基因是缺氧抑制mTORC1所必需的，这是一个迄今为止功能未知的基因。在缺氧条件下，REDD1是转录诱导的，并且REDD1过表达足以抑制mTORC1。REDD1编码一个保守的25 kDa蛋白，没有可识别的结构域或功能域，与其他已知功能的蛋白没有同源性。本文的数据显示，REDD1形成了一个包含单个REDD1单体的复合物，并提出了实验来评估该复合物在REDD1信号传导中的作用。结构-功能分析揭示了REDD1中功能所需的两个结构域的存在，并提出了实验来测试这些结构域如何起作用。此外，本文还概述了实验来评估REDD1复合体是直接调控mTORC1还是通过TSC1/TSC2调控mTORC1。本文还提供了表征REDD1亚细胞定位的初步数据，并提出了实验来评估REDD1亚细胞分布的调控机制及其功能意义。最后，建立了一种新的小鼠品系，并概述了实验来表征red1在小鼠缺氧信号传导中的调节和作用机制。mTORC1在许多病理条件下不受调节，了解mTORC1如何受缺氧和REDD1调节可能对人类健康有影响。

DESCRIPTION (provided by applicant): Mammalian target of rapamycin complex 1 (mTORC1) is a critical regulator of cell growth and metabolism that integrates a variety of signals, both intracellular and extracellular, with the protein translation machinery. Signals from growth factors and energy stores are relayed to mTORC1 through the tuberous sclerosis complex proteins 1 (TSC1) and 2 (TSC2), which form a complex (TSC1/TSC2) with tumor suppressor function. We have discovered that mTORC1 regulation by oxygen levels also requires the TSC1/TSC2 complex. Failure to inhibit mTORC1 in TSC1/TSC2-deficient cells in response to hypoxia results in abnormal cell proliferation and might contribute to tumor growth. Recently, we established that the gene regulated in development and DNA damage 1 (REDD1), a gene of hitherto unknown function, was necessary for mTORC1 inhibition by hypoxia. REDD1 is transcriptionally induced in response to hypoxia and REDD1 overexpression is sufficient to inhibit mTORC1. REDD1 encodes a conserved 25 kDa protein with no recognizable structural or functional domains and no homology to other proteins of known function. Herein, data is presented showing that REDD1 forms a complex that contains a single REDD1 monomer and experiments are proposed to evaluate the role of the complex in REDD1 signaling. Structure-function analyses have revealed the existence of two domains in REDD1 that are required for function, and experiments are presented to test how these domains act. In addition, experiments are outlined to assess whether the REDD1 complex regulates mTORC1 directly, or through TSC1/TSC2. Preliminary data is also presented characterizing the subcellular localization of REDD1 and experiments are proposed to evaluate the mechanism that governs REDD1 subcellular distribution and its functional significance. Finally, a novel mouse strain has been generated and experiments are outlined to characterize the regulation and mechanism of REDD1 action in hypoxia signaling in the mouse. mTORC1 is deregulated in many pathological conditions and understanding how mTORC1 is regulated by hypoxia and REDD1 might have implications for human health. PUBLIC HEALTH RELEVANCE: This project seeks to understand the mechanism whereby cells adapt to changes in their environment. In particular we are interested in understanding how cells adapt to low oxygen levels. This process involves the inhibition of a cellular protein complex called mammalian target of rapamycin complex 1 (mTORC1) and this complex is implicated in multiple pathological processes. mTORC1 inhibitors have, in fact, been approved by the FDA for (1) the treatment of cancer, (2) to prevent transplant rejection, and (3) to prevent coronary artery stent occlusions. Thus, understanding how mTORC1 is regulated has profound implications for human health.