Functional Proteomics of mTOR-Regulated Nuclear Proteins

描述（由申请人提供）：雷帕霉素的哺乳动物靶标（mTOR）是协调细胞生长与营养可用性的信号传导途径的核心组分，是治疗糖尿病和肥胖症的有希望的靶标。越来越多的证据支持mTOR信号传导在代谢性疾病的发展中起着至关重要的作用，但目前还不可能对mTOR介导的疾病发病机制有清楚的了解。本申请的目的是鉴定将mTOR信号向下游传递到参与营养代谢和基因表达的关键核过程的蛋白质。这项研究的中心假设是，mTOR触发了一系列细胞过程，导致蛋白质的核定位，这些蛋白质激活或抑制重要代谢或调控基因的表达。这项研究的基本原理是，一旦发现在mTOR控制下在细胞核和细胞质之间穿梭的核因子，将对mTOR调节的营养代谢和代谢疾病的发病机制有重要的了解。具体目的是：（1）系统鉴定脂肪细胞中mTOR调控的核蛋白。本研究将利用一种新的定量蛋白质组学方法，重点鉴定脂肪细胞中由mTOR信号调节其核定位的核蛋白，脂肪细胞来源于脂肪组织，其控制全身能量稳态;（2）系统地鉴定脂肪细胞中mTOR调节的DNA结合蛋白。本研究的主要目的是通过定量蛋白质组学方法结合全染色质分离技术鉴定脂肪细胞中mTOR调控的DNA结合蛋白;（3）确定mTOR调控的核蛋白的功能。将从目标#1和#2中鉴定的蛋白质中选择约20种蛋白质，并将通过生物化学和遗传方法分析其亚核定位和功能，包括使用YFP蛋白和RNAi以及葡萄糖和脂肪代谢的生物化学和报告基因测定。本申请中提出的研究具有重要意义，因为它将使我们能够获得有关负责将mTOR活性与涉及代谢基因表达调控的核事件联系起来的机制的新知识，因此它将提供我们可以靶向治疗代谢疾病的关键调控蛋白。

DESCRIPTION (provided by applicant): Mammalian target of rapamycin (mTOR), a central component of a signaling pathway that coordinates cell growth with nutrient availability, is a promising target for the treatment of diabetes and obesity. Mounting evidence supports that mTOR signaling plays a crucial role in the development of the metabolic diseases, but it has not yet been possible to obtain a clear understanding of the mechanism underlying mTOR-mediated pathogenesis of the diseases. The objective of this application is to identify proteins that transmit mTOR signals downstream to the key nuclear processes involved in nutrient metabolism and gene expression. The central hypothesis for the proposed research is that mTOR triggers a cascade of cellular processes that lead to nuclear localization of proteins that activate or repress expression of important metabolic or regulatory genes. The rationale for the proposed research is that, once the nuclear factors that shuttle between nucleus and cytoplasm under the control of mTOR are discovered, a significant insight will be obtained into the mechanism responsible for mTOR-regulated nutrient metabolism and pathogenesis of metabolic diseases. The specific aims are: (1) To systematically identify mTOR-regulated nuclear proteins in adipocytes. The research will focus on the identification of nuclear proteins whose nuclear localization is regulated by mTOR signaling in adipocytes, the cells derived from the adipose tissue that governs a whole body energy homeostasis using a novel quantitative proteomics approach; (2) To systematically identify mTOR-regulated DNA-binding proteins in adipocytes. The aim of this study will focus on the identification of mTOR-regulated DNA-binding proteins in adipocytes, which will be achieved through quantitative proteomics approach coupled with whole chromatin isolation; (3) To determine functions of identified mTOR-regulated nuclear proteins. ~20 proteins will be selected among proteins identified in the aims #1 and #2, and their sub-nuclear localization and functions will be analyzed through biochemical and genetic approaches, which include the use of the YFP protein and RNAi as well as biochemical and reporter gene assay for glucose and fat metabolism. The research proposed in this application is significant, because it will allow us to obtain the new knowledge on the mechanism that is responsible for connecting mTOR activity to the nuclear events involving the regulation of metabolic gene expression, and therefore it will provide crucial regulatory proteins that we can target for the treatment of metabolic diseases.