Molecular and Biochemical Basis of mTORC1-mediated Feedback Loops

 描述（由申请人提供）：雷帕霉素复合物1（mTORC 1）的机制靶标是一种进化上保守的丝氨酸/苏氨酸激酶复合物。它是细胞合成代谢过程的中央调节器，通过整合各种上游输入，包括生长因子，营养素，能量和氧气状态。越来越多的人认识到，mTORC 1的下游靶标可以通过各种反馈回路与其上游调节器进行通信。这些反馈机制在稳定整个网络方面发挥着关键作用。它们在多种人类疾病中也具有重要意义。例如，异常的mTORC 1激活（例如，由于慢性过度喂养）可导致胰岛素抵抗，这是2型糖尿病的标志。反馈回路在癌症生物学中也特别相关。mTORC 1在人类癌症的主要部分中被过度活化。不幸的是，使用雷帕霉素类似物（rapalogs）作为单一药剂抑制mTORC 1是细胞抑制性的，但不是细胞毒性的。这主要是由于mTORC 1抑制后上游促存活激酶（例如Akt）的激活。了解如何避免反馈回路的缓解对于克服雷帕霉素耐药性治疗人类癌症至关重要。为此，我们最近使用了定量磷酸化蛋白质组学方法系统地表征mTOR调节的磷酸化蛋白质组。一种新的mTORC 1底物Grb 10的详细生物化学表征表明，mTORC 1介导的磷酸化稳定了这种蛋白质，导致其积累，随后抑制胰岛素和IGF 1信号传导。我们未发表的结果进一步表明，具有过度活跃mTORC 1的细胞也分泌能够阻断IGF 1信号传导的蛋白质。在本研究中，我们将研究mTORC 1如何通过重塑分泌蛋白质组来抑制IGF 1的功能。在目标1中，我们将使用定量质谱方法来全面表征mTORC 1调节的分泌组。在目标2中，我们将阐明mTORC 1调节这种分泌蛋白表达的分子机制，并确定其在mTORC 1介导的IGF 1信号反馈抑制中的作用。在Aim 3中，我们将研究这种非细胞自主功能， 分泌的蛋白质，及其与Grb 10在体内的相互作用。我们将通过多学科的方法来实现我们的目标，利用包括蛋白质组学，生物化学，分子生物学和动物模型在内的工具。这些结果将极大地促进我们对mTORC 1介导的反馈回路的理解。我们在这项提案中获得的知识也将是进一步探索我们如何更好地针对这些反馈机制治疗各种人类疾病所必需的。

 DESCRIPTION (provided by applicant): The mechanistic target of rapamycin complex 1 (mTORC1) is an evolutionarily conserved serine/threonine kinase complex. It is a central regulator of cellular anabolic processes by integrating a variety of upstream inputs, including growth factors, nutrients, energy and oxygen status. It is being increasingly appreciated that downstream targets of mTORC1 can communicate with its upstream regulators, through various feedback loops. These feedback mechanisms play a critical role in stabilizing the entire network. They also have great significance in a variety of human diseases. For example, aberrant mTORC1 activation (e.g. as a result of chronic overfeeding) can cause insulin resistance, a hallmark of type 2 diabetes. The feedback loops are also particularly relevant in cancer biology. mTORC1 is hyperactivated in a major faction of human cancers. Unfortunately, inhibition of mTORC1 using rapamycin analogs (rapalogs) as single agents is cytostatic but not cytotoxic. This is mainly due to the activation of upstream, pro-survival kinases (e.g. Akt) upon mTORC1 inhibition. Understanding how to avoid the relief of feedback loops will be critical to overcome rapamycin resistance for treating human cancer. To this end, we recently used a quantitative phosphoproteomic approach to systematically characterizing the mTOR-regulated phosphoproteome. Detailed biochemical characterization of a novel mTORC1 substrate, Grb10, showed that mTORC1-mediated phosphorylation stabilized this protein, which led to its accumulation, and subsequent inhibition of insulin and IGF1 signaling. Our unpublished results further indicate that cells with hyperactive mTORC1 also secrete a protein(s) that is able to block IGF1 signaling. In this proposal, we will investigate how mTORC1 may inhibit the function of IGF1 through remodeling the secretome. In Aim 1, we will use a quantitative mass spectrometry approach to comprehensively characterizing the mTORC1-regulated secretome. In Aim 2, we will elucidate the molecular mechanism by which mTORC1 regulates the expression of this secreted protein, and determine its role in mTORC1-mediated feedback inhibition of IGF1 signaling. In Aim3, we will investigate the non-cell autonomous function of this secreted protein, and its interplay with Grb10 in vivo. We will accomplish our goals with a multi- disciplinary approach, utilizing tools including proteomics, biochemistry, molecular biology and animal models. The results will greatly facilitate our understanding of mTORC1-mediated feedback loops. Knowledge we garner in this proposal will also be necessary to further explore how we might better target these feedback mechanisms for treating various human diseases.