Posttranslational Regulation of Cell Growth and Stress Responses

项目摘要 疾病生物学的一个中心目标是描述导致转化的分子过程。 将细胞从正常状态转变为病理状态。与DNA和RNA的快速发展相比 测序技术，最终也是最具可操作性的中央元素的特征 蛋白质这一信条已经落后了。细胞基因组与其蛋白质组之间的动态关系 人们对此知之甚少，反映了转录/转录后调控的多个层次。尤其是， 人类蛋白质组的复杂性被大约400种不同类型的蛋白质翻译后极大地扩大了 修改(PTM)。各种PTM事件单独或组合(即，串扰)表示 调节蛋白质功能的强大机制(例如，活性、稳定性和定位)， 它们的集合在信令网络内传递信息，该信令网络是各种 病理生理条件。然而，由于与 蛋白质PTM的分析(例如，化学多样性、不稳定和低丰度)，完整描述了 任何给定细胞的翻译后修饰蛋白质组仍然是一项艰巨的任务。的首要任务是 我们的计划是：(1)开发尖端的定量蛋白质组学方法，以系统地识别和 鉴定新的PTMS，(2)全面询问由磷酸化调控的信号事件 (mTOR通路)和ADP-核糖化(PARP通路)，以及(3)结合这些系统生物学 用经典的生化、细胞生物学和动物实验来破译分子 由这两条重要途径控制的细胞生长和应激反应的基础。至 为了实现这些目标，我们将利用我们的初步成果(包括大量独特的命中率、试剂 和方法)，并将努力集中在以下六个目标上。一是发展创新型群众 光谱技术显著提高了全球、定量和特定地点的性能 对新型PTMS的分析。第二，我们将研究Igfbp5(最近发现的细胞外靶标)的作用 MTORc1)作为mTORc1的“非细胞自主”功能的介体。第三，我们将确定角色 Egr1(从我们之前的MS屏幕中发现的一种新的热门)作为mTORC1依赖的主调节因子 反馈循环。第四，我们将生成mTORC1磷酸化底物的组织特异性图谱，并在 这样做，询问这一重要途径对不同组织的生理学的不一致影响。第五, 我们将开发一种大规模的MS方法来表征D/E-单-ADP-核糖化的位点特异性 蛋白质组，最后我们将开发一种大规模测量绝对蛋白质PAR化的方法 化学计量学。总而言之，这些研究为多发性硬化症的识别和 与细胞生长控制和应激反应相关的PTMS事件的功能特征。

Project Summary A central goal in disease biology is to describe the molecular processes responsible for transformation of a cell from a normal state into a pathological one. Compared to the rapid progresses in DNA and RNA sequencing technologies, characterization of the final and arguably most actionable element of the central dogma, protein, has lagged behind. The dynamic relationship between the genome of a cell and its proteome is poorly understood, reflecting multiple layers of transcriptional/post-transcriptional regulation. In particular, the complexity of the human proteome is greatly expanded by the ~400 different types of protein posttranslational modifications (PTMs). The various PTM events, either alone or in combination (i.e., “cross-talk”), represent powerful mechanisms to modulate the function of a protein (e.g., activity, stability and localization), the collection of which convey information within the signaling network that underlies the complex traits in various pathophysiological conditions. However, because of many inherent technical difficulties associated with the analysis of protein PTMs (e.g., chemically diverse, unstable and low abundance), a complete description of the posttranslationally modified proteome of any given cells remains a daunting task. The overarching mission of our program is to: (1) develop cutting-edge quantitative proteomic approaches to systematically identify and characterize novel PTMs, (2) comprehensively interrogate the signaling events regulated by phosphorylation (mTOR pathways) and ADP-ribosylation (PARP pathways), and (3) combine these systems biology approaches with classical biochemical, cell biology and animal experiments to decipher the molecular underpinnings of cell growth and stress responses that are controlled by these two important pathways. To accomplish these goals, we will leverage our preliminary results (including a large set of unique hits, reagents and methods), and center our efforts on the following six goals. First, we will develop innovative mass spectrometric technologies with dramatically improved performance for global, quantitative and site-specific analysis of novel PTMs. Second, we will investigate the role of IGFBP5 (a recently identified extracellular target of mTORC1) as a mediator of the “non-cell autonomous” function of mTORC1. Third, we will determine the role of EGR1 (a novel hit identified from our previous MS screen) as a master regulator of the mTORC1-dependent feedback loops. Fourth, we will generate a tissue-specific atlas of mTORC1 phosphorylation substrates, and in doing so, interrogate non-uniform effects of this important pathway on the physiology of different tissues. Fifth, we will develop a large-scale MS approach to site-specific characterization of the D/E-mono-ADP-ribosylated proteome, and finally we will develop a large-scale method to measure absolute protein PARylation stoichiometries. Together, these studies provide a comprehensive framework for the MS identification and functional characterization of PTMs events linked to cell growth control and stress responses.