TSC2 Localization and Function at the Peroxisome

描述（由申请人提供）：恶性硬化复合物2（TSC 2）肿瘤抑制因子在PI 3 K信号通路中作为mTOR活性的看门人发挥作用。mTOR激酶调节几种细胞功能，包括蛋白质合成、细胞生长和自噬，TSC 2作为变阻器通过整合促有丝分裂信号与能量可用性来调节mTOR。有趣的是，TSC 2缺陷细胞具有异常的氧化还原稳态，我们的初步数据表明TSC 2是活性氧（ROS）抑制mTOR所必需的，这使我们假设TSC 2抑制mTOR以响应氧化应激。此外，我们发现细胞损伤传感器ATM被ROS激活，并且在没有ATM或TSC 2的情况下，细胞不能适当地抑制mTOR以响应ROS。已知TSC 2定位于细胞内膜，并且膜定位调节TSC 2功能。然而，这种肿瘤抑制因子定位的特定内膜尚未确定。重要的是，我们发现ATM和TSC 2都定位于过氧化物酶体，这是细胞中脂肪酸2-氧化和ROS产生的主要位点。这些数据使我们提出了一个假设，即ATM和TSC 2参与了过氧化物酶体的应激反应途径，该途径响应ROS以抑制mTOR信号传导。由于mTOR是自噬的负调节因子，我们假设TSC 2介导的mTOR抑制对ROS的响应的生理作用是调节自噬和过氧化物酶体周转。 该提议的首要假设是，TSC 2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，以抑制mTOR信号传导并调节自噬。为了检验这一假设，我们提出了以下具体目标：具体目标1：检验TSC 2和该mTOR调节节点的组分（TSC 2激活配偶体TSC 1、其GAP靶标Rheb和mTOR）定位于过氧化物酶体的假设。具体目标2：确定在过氧化物酶体调节TSC 2功能的信号通路。具体目标3：检验过氧化物酶体TSC 2抑制mTOR以通过自噬调节自噬和/或过氧化物酶体周转的假设。 本申请中提出的实验将使我们能够测试我们的假设，即TSC 2在过氧化物酶体处参与ATM下游的新型应激反应途径以抑制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的途径具有生理学意义，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，他们将为未来的研究奠定基础，以了解过氧化物酶体的细胞信号传导如何参与致癌作用，并为开发针对过氧化物酶体的策略以调节肿瘤细胞中的细胞信号传导和自噬提供可能性。 公共卫生相关性：该提议的首要假设是TSC 2参与过氧化物酶体的新型应激反应途径，以抑制mTOR信号传导并调节细胞自噬。了解调节自噬过程或影响细胞启动该过程的决定的机制对致癌作用和癌症治疗具有重要意义。更好地了解控制这一过程的机制有可能有助于设计针对过氧化物酶体和定位于该细胞器的信号通路的预防策略。此外，在未来，它们可能有助于设计治疗方法，限制肿瘤细胞利用自噬作为生存途径和/或促进自噬诱导的程序性细胞死亡的能力。

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. PUBLIC HEALTH RELEVANCE: The overarching hypothesis of this proposal is that TSC2 participates in a novel stress response pathway at the peroxisome to repress mTOR signaling and regulate cellular autophagy. Understanding the mechanisms that regulate the process of autophagy or influence the cell's decision to initiate this process has important implications for carcinogenesis and cancer therapy. A better understanding of the mechanisms that control this process has the potential to aid in the design of prevention strategies targeting the peroxisome and signaling pathways that localize to this organelle. In addition, in the future they may aid in the design of therapeutics that limit the ability of tumor cells to utilize autophagy as a survival pathway and/or promote autophagy-induced programmed cell death in response to therapy.