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负责转化的分子过程 一个细胞从正常状态转变为病态的过程。与DNA和RNA的快速发展相比， 测序技术，表征的最后，可以说是最可行的元素的中心 教条，蛋白质，已经落后了。细胞基因组与蛋白质组之间的动态关系 目前还知之甚少，反映了转录/转录后调控的多个层面。特别是 人类蛋白质组的复杂性因约400种不同类型的蛋白质翻译后 修改（PTM）。各种PTM事件，单独或组合（即，“cross-talk”），表示 调节蛋白质功能的强大机制（例如，活性、稳定性和定位）， 这些信息的集合在信令网络内传达信息，这些信息是各种通信系统中复杂特性的基础。 病理生理条件。然而，由于许多固有的技术困难， 蛋白质PTM的分析（例如，化学多样性，不稳定和低丰度），一个完整的描述 任何给定细胞的后修饰蛋白质组仍然是一项艰巨的任务。首要的使命是 我们的计划是：（1）发展尖端的定量蛋白质组学方法，系统地识别和 表征新的PTM，（2）全面询问由磷酸化调节的信号传导事件 (mTOR途径）和ADP-核糖基化（PARP途径），和（3）联合收割机这些系统生物学 通过经典的生物化学、细胞生物学和动物实验， 细胞生长和应激反应的基础是由这两个重要的途径控制的。到 为了实现这些目标，我们将利用我们的初步结果（包括大量独特的命中，试剂 方法），围绕以下六个方面开展工作。一是大力发展创新型大众 光谱技术，具有显著改进的性能，可用于全球、定量和特定地点的 新型PTM的分析。其次，我们将研究IGFBP 5（最近鉴定的细胞外靶点）的作用。 的mTORC 1）作为mTORC 1的“非细胞自主”功能的介体。第三，我们将确定角色 EGR 1（从我们以前的MS筛选中鉴定出的一种新的命中）作为mTORC 1依赖性的主要调节因子。 反馈回路第四，我们将生成mTORC 1磷酸化底物的组织特异性图谱， 这样做，询问该重要途径对不同组织的生理学的非均匀影响。第五， 我们将开发一种大规模的MS方法，对D/E-单ADP-核糖基化的 最后，我们将开发一种大规模的方法来测量绝对蛋白质PAR化 化学计量总之，这些研究为MS鉴定提供了全面的框架， 与细胞生长控制和应激反应相关的PTM事件的功能表征。

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.