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Molecular mechanisms of SHP2 signaling dissected with designer binding proteins

使用设计结合蛋白剖析 SHP2 信号传导的分子机制

基本信息

批准号：
9889907
负责人：
SHOHEI KOIDE
金额：
$ 37.35万
依托单位：
NEW YORK UNIVERSITY SCHOOL OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-04-15 至 2022-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9889907
关键词：
Active Sites Acute Address Affect Affinity Antibodies Architecture Behavior Binding Binding Proteins Binding Sites Biochemical Biological Biology C-terminal Cell physiology Cells Chemicals Closure by clamp Cytokine Signaling Cytoplasm Dependence Development Directed Molecular Evolution Disease Epitopes Equilibrium Event Exhibits Generations Genetic Goals Growth Factor Human Human Genome Individual Knock-out Libraries Ligands Link Mediating Methodology Methods Molecular Molecular Conformation Monitor Mutation Mutation Analysis N-terminal Nature Oxidation-Reduction Oxides PTPN11 gene Pathogenesis Performance Phage Display Phosphoric Monoester Hydrolases Phosphotransferases Play Protein Engineering Protein Family Proteins Proteomics Publishing Reader Regulation Research Resolution Role Signal Pathway Signal Transduction Site Son of Sevenless Proteins Specificity Structure Structure-Activity Relationship Surface System Technology Time Tyrosine Phosphorylation biochemical tools cell growth regulation defined contribution developmental disease drug discovery genetic regulatory protein growth factor receptor-bound protein 2 human disease inhibitor/antagonist innovation knock-down member novel oxidation protein phosphatase inhibitor-2 public health relevance sensor small molecule inhibitor src Homology Region 2 Domain success synthetic protein temporal measurement tool tumorigenesis

项目摘要

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.

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，促进对含SH 2结构域的磷酸酶2（SHP 2）的细胞作用的高分辨率理解，并阐明其调控的分子机制。SHP 2由PTPN 11基因编码，在正常信号传导、肿瘤发生和发育疾病中具有重要作用。SHP 2具有模块化结构，并且SHP 2的至少四个不同区域可以单独充当“信号传导节点”：N-末端SH 2结构域、C-末端SH 2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。SHP 2可以通过至少四种不同的机制起作用：（i）作为靶向PTP，其从与SHP 2 SH 2结构域或与其C-末端pY基序相互作用的其他分子中去除pY;（ii）作为将含pY的蛋白质连接到GRB 2/SOS的衔接子;（iii）作为pY基序与其他SH 2结构域相互作用的竞争性抑制剂;以及（iv）潜在地作为氧化还原传感器。这些机制的作用似乎是信号/途径依赖性的。由于SHP 2中节点的功能重叠作用和整合行为，定义这些机制对特定信号传导事件的贡献以及这些机制在疾病中如何改变一直具有挑战性。攻击SHP 2生物学和发病机制中这些基本问题的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，产生高性能的结合蛋白，以SHP 2信号节点，可以遗传编码的细胞内使用。我们将利用设计师结合蛋白平台，称为“monobodies”和“pY-clamp”，这是我们在过去十年中开创和完善的。与常规抗体及其片段不同，这些经设计的结合蛋白在细胞质的还原条件下容易折叠成其功能形式。我们已经产生了单抗体和pY-钳，它们以精确的特异性和高效力识别SHP 2信号传导节点。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，用于生化控制细胞中SHP 2的功能。使用这些工具，我们将（i）建立SHP 2调控的结构-功能关系的定量理解，（ii）定义SHP 2功能机制在不同信号传导背景和肿瘤发生中的细胞作用，（iii）确定SHP 2 PTP的直接底物及其在信号传导中的作用。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研究。了解磷酸酶如何影响细胞生理学和疾病发病机制，并告知针对这类重要调节蛋白的药物发现工作。