Molecular and Biochemical Basis of mTORC1-mediated Feedback Loops

 描述(申请人提供)：雷帕霉素复合体1(MTORC1)的作用靶点是一种进化上保守的丝氨酸/苏氨酸激酶复合体。它是细胞合成代谢过程的中心调节器，通过整合各种上游输入，包括生长因子、营养物质、能量和氧气状态。人们越来越认识到，mTORC1的下游目标可以通过各种反馈回路与其上游监管机构进行沟通。这些反馈机制在稳定整个网络方面起着至关重要的作用。它们在人类的多种疾病中也具有重要意义。例如，mTORC1的异常激活(例如，慢性过量喂养的结果)可能会导致胰岛素抵抗，这是2型糖尿病的一个特征。反馈环在癌症生物学中也特别相关。MTORC1在人类癌症的一个主要派别中被过度激活。不幸的是，使用雷帕霉素类似物(雷帕洛格)作为单一药物抑制mTORC1具有细胞抑制作用，但不具有细胞毒性。这主要是由于上游促进生存的激酶(如Akt)在mTORC1抑制时被激活。了解如何避免缓解反馈回路对于克服雷帕霉素耐药治疗人类癌症至关重要。为此，我们最近使用了一种定量的磷酸蛋白质组学方法来系统地表征mTOR调节的磷酸蛋白质组。对一种新的mTORC1底物Grb10的详细生化特征表明，mTORC1介导的磷酸化稳定了该蛋白，导致其积聚，随后抑制了胰岛素和IGF1信号转导。我们未发表的结果进一步表明，mTORC1超活性的细胞还分泌一种能够阻断IGF1信号转导的蛋白质(S)。在这个方案中，我们将研究mTORC1如何通过重塑分泌体来抑制IGF1的功能。在目标1中，我们将使用定量质谱学方法来全面表征mTORC1调节的分泌组。在目标2中，我们将阐明mTORC1调节这一分泌蛋白表达的分子机制，并确定其在mTORC1介导的反馈抑制IGF1信号转导中的作用。在Aim3中，我们将研究这一非细胞自主功能 分泌蛋白及其在体内与Grb10的相互作用。我们将通过多学科方法实现我们的目标，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mTORC1介导的反馈环的理解。我们在这项提案中获得的知识也将是必要的，以进一步探索我们如何更好地针对这些反馈机制来治疗各种人类疾病。

 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.