STRUCTURAL ANALYSIS OF CELL GROWTH REGULATORS

细胞生长调节剂的结构分析

• 批准号: 6311527
• 负责人: JOSEPH NOEL
• 金额: $ 27.95万
• 依托单位: SALK INSTITUTE FOR BIOLOGICAL STUDIES
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-05-01 至 2001-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6311527
• 关键词: X ray crystallography; biological signal transduction; cell growth regulation; chemical kinetics; molecular cloning; nuclear factor kappa beta; optics; phosphorylation; protein engineering; protein structure function; protein tyrosine phosphatase; protooncogene; recombinant proteins; surface plasmon resonance; thermodynamics; transcription factor

How a cell responds to an extra-cellular signal and mounts an intracellular response plays a pivotal role in the life cycle of every cell. Implicit in this statement is the fact that such signalling mechanisms regulate the growth and differentiation fate of cells. In particular, abherrent signalling cascades or mutations within the proteins comprising such cascades can lead to disease states such as cancer. Much of the information that is conveyed throughout the cell in various signalling cascades propagates in the form of conformational changes in the proteins involved or in changes in the phosphorylation state of signalling proteins. Given that premise, our goal is to provide a stereochemical foundation for the biological roles played by receptor tyrosine phosphatases and other regulators of cell growth, proliferation, and differentiation. Our major tool for conducting these studies is macromolecular x-ray crystallography. We will characterize the structural features of the tandem catalytic domains of receptor tyrosine phosphatase alpha both alone and when complexed with physiologically relevant phosphotyrosine containing substrates. This list includes the inhibitory C-terminal tail of the proto-oncogene cSrc and the C-terminal tail of the second catalytic domain of receptor tyrosine phosphatase alpha involved in the regulation of signalling through the adapter protein Grb2. In addition, we will provide a structural foundation for all receptor tyrosine phosphatases possessing twin catalytic domains by undertaking the crystallization and structural elucidation of the intracellular portion of receptor tyrosine phosphatase alpha possessing two catalytically distinct domains. Given the importance of transcriptional regulation in growth control, we have begun a collaborative effort aimed at determining the three dimensional structure of the transcriptional regulator, IkappaBalpha (human MAD3), both alone and complexed with the p65 domain of NF-kappaB. Given the importance of regulatory phosphorylation of IkappaBalpha we hope that over the long-term we can provide a structural explanation for the effect of phosphorylation on IkappaBalpha. We will employ a two tiered approach in all the proposed work involving a combination of macromolecular x-ray crystallography for high resolution structural analysis and surface plasmon resonance (BIAcore) for the kinetic and thermodynamic analysis of receptor tyrosine phosphatase alpha/phosphopeptide complexes and NF-kappaB/IkappaBalpha complexes. In this regard, we currently have solved the structures of both catalytic domains of receptor tyrosine phosphatase alpha in an uncomplexed state. In terms of the IkappaBalpha project, we now have overexpression and purification systems for IkappaBalpha and NF-kappaB p65 from baculovirus.

细胞如何响应细胞外信号并安装 细胞内反应在每个生物的生命周期中起着举足轻重的作用 手机。这一声明隐含着这样一个事实，即这种信号 机制调节细胞的生长和分化的命运。在……里面 具体而言，异常的信号级联或内部突变 包含这种级联反应的蛋白质可以导致疾病状态，如 癌症。在整个细胞中传递的大部分信息都是 各种信号级联以构象的形式传播 涉及的蛋白质的变化或磷酸化的变化 信号蛋白的状态。在这一前提下，我们的目标是提供 受体发挥生物学作用的立体化学基础 酪氨酸磷酸酶和其他细胞生长、增殖的调节剂， 和差异化。我们进行这些研究的主要工具是 大分子X射线结晶学。我们将描述 酪氨酸受体串联催化域的结构特征 单独和生理上与磷酸酶结合时的α-磷酸酶 相关的含磷酸酪氨酸底物。该列表包括 原癌基因CSRC的抑制性C末端和C末端 受体酪氨酸磷酸酶第二催化结构域的尾部 通过适配器调节信令所涉及的阿尔法 Grb2蛋白。此外，我们还将为以下项目提供结构性基础 所有具有双催化区的受体酪氨酸磷酸酶 承担结晶和结构鉴定工作 受体酪氨酸磷酸酶α胞内部分具有 两个催化截然不同的结构域。 鉴于转录调控在生长控制中的重要性，我们 已经开始了一项旨在确定这三个 转录调控因子IkappaBalpha的空间结构 (人MAD3)，单独或与核因子-kappaB的p65结构域复合。 鉴于IkappaBalpha调控磷酸化的重要性 希望从长远来看，我们可以提供一个结构性的解释 磷酸化对IkappaBalpha的影响。 我们会在所有拟议的工作中采用两层模式，包括 用于高分辨率的大分子X射线结晶学组合 结构分析和表面等离子体共振(Biacore) 受体酪氨酸磷酸酶的动力学和热力学分析 α/磷肽复合体和核因子-kappaB/IkappaBalpha复合体。在……里面 在这方面，我们目前已经解决了两种催化剂的结构 受体酪氨酸磷酸酶α结构域的非络合状态。 在IkappaBalpha项目方面，我们现在有过度表达和 杆状病毒IkappaBalpha和NF-kappaB p65的纯化系统。