Molecular mechanisms of SHP2 signaling dissected with designer binding proteins

 描述(由申请人提供)：本项目的目标是通过应用先进的蛋白质工程技术，促进对SH2结构域包含的磷酸酶2(SHP2)的细胞作用的高分辨率理解，并阐明其调控的分子机制。SHP2由PTPN11基因编码，在正常信号转导、肿瘤发生和发育性疾病中发挥重要作用。Shp2具有模块化的结构，至少有四个不同的SHP2区域可以分别作为信号节点：N-末端SH2结构域、C-末端SH2结构域、磷酸酶(PTP)结构域和非结构化C-末端区域中的Py基序。Shp2至少可以通过四种不同的机制发挥作用：(I)作为靶向PTP，从与SHP2SH2结构域或其C-末端Py基序相互作用的其他分子中去除Py；(Ii)作为一个接头，将含Py的蛋白质连接到Grb2/SOS；(Iii)作为Py基序与其他SH2结构域相互作用的竞争性抑制因子；以及(Iv)作为一个潜在的氧化还原传感器。这些机制的作用似乎依赖于信号/途径。由于SHP2中节点在功能上的重叠作用和整合的行为，定义这些机制对特定信号事件的贡献以及这些机制在疾病中是如何改变的一直是一个挑战。解决SHP2生物学和发病机制中的这些基本问题的一个主要障碍是缺乏选择性和有效的抑制剂。基因敲除不提供所需的节点级分辨率或快速时间分辨率。该项目将利用创新的蛋白质工程技术来产生高性能的SHP2信号节点结合蛋白，这些蛋白可以通过基因编码在细胞内使用。我们将利用我们在过去十年中开创和改进的设计师结合蛋白质平台，称为“单体”和“Py-Clip”。与传统的抗体及其片段不同，这些设计结合蛋白在细胞质还原的条件下很容易折叠成其功能形式。我们已经产生了识别SHP2信号节点的单体型和Py-钳，具有极高的特异性和高效性。我们将把这些初步的成功扩展到产生一套全面的遗传编码工具，用于生物化学控制细胞中的SHP2功能。利用这些工具，我们将(I)建立对SHP2调控的结构-功能关系的定量理解，(Ii)确定SHP2功能机制在不同信号背景和肿瘤发生中的细胞作用，以及(Iii)确定SHP2 PTP的直接底物及其在信号转导中的作用。能够探测单个磷酸酶的特定节点在特定信号通路中的作用的有效和选择性的分子工具将极大地帮助我们的 了解磷酸酶如何影响细胞生理和疾病发病机制，并为针对这类重要调控蛋白的药物发现工作提供信息。

 DESCRIPTION (provided by applicant): The goals of this project are to advance a high-resolution understanding of the cellular roles of the SH2 domain-containing phosphatase 2 (SHP2) and to elucidate molecular mechanisms underlying its regulation, by applying advanced protein engineering technologies. Encoded by the gene PTPN11, SHP2 has important roles in normal signaling, oncogenesis and developmental disease. SHP2 has a modular architecture, and at least four distinct regions of SHP2 individually could serve as "signaling nodes": the N-terminal SH2 domain, C- terminal SH2 domain, phosphatase (PTP) domain, and pY motifs in the unstructured C-terminal region. SHP2 can act via at least four different mechanisms: (i) as a targeted PTP that removes pY from other molecules that interact with SHP2 SH2 domains or with its C-terminal pY motifs; (ii) as an adaptor that links pY-containing proteins to GRB2/SOS; (iii) as a competitive inhibitor for the interaction of pY motifs with other SH2 domains; and (iv) potentially as a redox sensor. The role of these mechanisms appears to be signal/pathway-dependent. Because of the functionally overlapping roles and integrated behavior of the nodes in SHP2, it has been challenging to define contributions of these mechanisms to specific signaling events and how these mechanisms are altered in diseases. A major obstacle to attacking these fundamental questions in SHP2 biology and pathogenesis has been the absence of selective and potent inhibitors. Genetic knockdown does not provide a node-level resolution or rapid temporal resolution required. This project will utilize innovative protein engineering technologies to generate high-performance binding proteins to SHP2 signaling nodes that can be genetically encoded for intracellular use. We will utilize the designer binding protein platforms, termed "monobodies" and "pY-clamps", that we have pioneered and refined over the last decade. These designer binding proteins, unlike conventional antibodies and their fragments, readily fold into their functional form under reducing conditions of the cytoplasm. We have already generated monobodies and pY-clamps that recognize SHP2 signaling nodes with exquisite specificity and high potency. We will extend these initial successes to generating a comprehensive set of genetically encoded tools for biochemically controlling SHP2 function in cells. Using these tools, we will (i) establish a quantitative understanding of the structure-function relationship of SHP2 regulation, (ii) define the cellular roles of the mechanisms of SHP2 function in diverse signaling contexts and in oncogenesis, and (iii) identify direct substrates of SHP2 PTP and their roles in signaling. Potent and selective molecular tools that can probe the role of a specific node of a single phosphatase in specific signaling pathways will greatly aid our understanding of how phosphatases influence cellular physiology and disease pathogenesis, and inform drug discovery effort directed to this important class of regulatory proteins.