TSC2 Localization and Function at the Peroxisome

描述（由申请人提供）：结节性硬化症复合体2 （TSC2）肿瘤抑制因子在PI3K信号通路中作为mTOR活性的守门人。mTOR激酶调节多种细胞功能，包括蛋白质合成、细胞生长和自噬，TSC2作为变阻器通过整合有丝分裂信号和能量可用性来调节mTOR。有趣的是，缺乏TSC2的细胞具有异常的氧化还原稳态，我们的初步数据表明，TSC2是活性氧（ROS）抑制mTOR所必需的，这使我们假设TSC2在氧化应激下抑制mTOR。此外，我们发现细胞损伤传感器ATM被ROS激活，并且在ATM或TSC2缺失的情况下，细胞无法适当抑制mTOR以响应ROS。已知TSC2定位于细胞内膜，膜定位调节TSC2的功能。然而，这种肿瘤抑制因子定位的特异性内膜尚未确定。重要的是，我们发现ATM和TSC2都定位于过氧化物酶体，过氧化物酶体是细胞中脂肪酸2-氧化和生成ROS的主要位点。这些数据使我们提出了ATM和TSC2参与过氧化物酶体的应激反应途径的假设，该过氧化物酶体响应ROS抑制mTOR信号传导。由于mTOR是自噬的负调节因子，我们假设tsc2介导的mTOR抑制对ROS的生理作用是调节自噬和过氧化物酶体的转换。该提议的总体假设是，TSC2肿瘤抑制因子参与ATM下游过氧化物酶体的新型应激反应途径，抑制mTOR信号传导并调节自噬。为了验证这一假设，我们提出了以下具体目的：具体目的1：验证TSC2和mTOR调控节点的成分（TSC2激活伙伴TSC1，其GAP靶点Rheb和mTOR）定位于过氧化物酶体的假设。特异性目标2：确定调节过氧化物酶体上TSC2功能的信号通路。具体目的3：验证过氧化物酶体TSC2通过抑制mTOR调节自噬和/或过氧化物酶体通过噬噬进行转换的假设。本申请中提出的实验将允许我们验证我们的假设，即TSC2参与ATM下游过氧化物酶体的新型应激反应途径，抑制mTOR并调节细胞自噬。如果这一假设是正确的，并且我们成功地证明了这种过氧化物酶体定位途径具有生理意义，这些研究将首次将注意力集中在过氧化物酶体作为信号细胞器上。此外，它们将为未来研究了解过氧化物酶体的细胞信号传导如何参与癌变奠定基础，并为开发靶向过氧化物酶体调节肿瘤细胞信号传导和自噬的策略提供可能性。

DESCRIPTION (provided by applicant): The Tuberous Sclerosis Complex 2 (TSC2) tumor suppressor functions as a gatekeeper for mTOR activity in the PI3K signaling pathway. The mTOR kinase regulates several cellular functions, including protein synthesis, cell growth and autophagy, with TSC2 functioning as a rheostat to regulate mTOR by integrating mitogenic signals with energy availability. Interestingly, TSC2-deficent cells have aberrant redox homeostasis, and our preliminary data indicate TSC2 is required for mTOR repression by reactive oxygen species (ROS), leading us to hypothesize that TSC2 represses mTOR in response to oxidative stress. In addition, we found that the cellular damage sensor ATM is activated by ROS, and that in the absence of either ATM or TSC2, cells are unable to appropriately repress mTOR in response to ROS. It is known that TSC2 localizes to cellular endomembranes, and that membrane localization regulates TSC2 function. However, the specific endomembrane(s) to which this tumor suppressor localizes has not been determined. Importantly, we found that both ATM and TSC2 localize to the peroxisome, a major site for 2-oxidation of fatty acids and generation of ROS in the cell. These data have led us to propose the hypothesis that ATM and TSC2 participate in a stress response pathway at the peroxisome that responds to ROS to repress mTOR signaling. As mTOR is a negative regulator of autophagy, we hypothesize that the physiological role for TSC2-mediated mTOR repression in response to ROS is to regulate autophagy and peroxisome turnover. The overarching hypothesis of this proposal is that the TSC2 tumor suppressor participates downstream of ATM in a novel stress response pathway at the peroxisome to repress mTOR signaling and regulate autophagy. To test this hypothesis, we propose the following Specific Aims: Specific Aim 1: Test the hypothesis that TSC2 and components of this mTOR regulatory node (the TSC2 activation partner TSC1, its GAP target Rheb and mTOR) localize to the peroxisome. Specific Aim 2: Identify the signaling pathway(s) that regulate TSC2 function at the peroxisome. Specific Aim 3: Test the hypothesis that peroxisomal TSC2 represses mTOR to regulate autophagy and/or peroxisomal turnover by pexophagy. The experiments proposed in this application will allow us to test our hypothesis that TSC2 participates downstream of ATM in a novel stress response pathway at the peroxisome to repress mTOR and regulate cellular autophagy. If this hypothesis is correct, and we are successful at demonstrating that this peroxisome- localized pathway has physiological significance, these studies will for the first time focus attention on the peroxisome as a signaling organelle. In addition, they will lay the groundwork for future studies to understand how cell signaling at the peroxisome participates in carcinogensis and open the possibility for the development of strategies targeting the peroxisome to regulate cell signaling and autophagy in tumor cells.