STRUCTURAL ANALYSIS OF CELL GROWTH REGULATORS

细胞生长调节剂的结构分析

• 批准号: 6296024
• 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: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6296024
• 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.

细胞如何对细胞外信号作出反应， 细胞内反应在每一个细胞的生命周期中起着关键作用。 cell. 这句话中隐含的事实是， 这些机制调节细胞的生长和分化命运。 在 特别的，不相容的信号级联或突变， 包含这种级联的蛋白质可导致疾病状态， 癌 细胞内传递的大部分信息 各种信号级联以构象的形式传播， 参与蛋白质的变化或磷酸化的变化 信号蛋白的状态。 在这个前提下，我们的目标是提供 受体生物学作用的立体化学基础 酪氨酸磷酸酶和其它细胞生长，增殖， 和差异化。 我们进行这些研究的主要工具是 大分子X射线晶体学 我们将描述 受体酪氨酸串联催化结构域的结构特征 磷酸酶α单独和当与生理上的磷酸酶α复合时， 相关的含磷酸酪氨酸的底物。 此列表包括 原癌基因cSrc的抑制性C末端尾部和C末端 受体酪氨酸磷酸酶第二催化结构域尾部 α参与调节通过衔接子的信号传导 Grb 2蛋白。 此外，我们将提供一个结构基础， 所有具有双催化结构域的受体酪氨酸磷酸酶 进行结晶和结构解析， 具有受体酪氨酸磷酸酶α的细胞内部分 两个催化不同的域。 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 转录调节因子IkappaB α的三维结构 （人MAD 3），单独和与NF-κ B的p65结构域复合。 考虑到调节IkappaB α磷酸化的重要性， 我希望从长远来看，我们可以提供一个结构性解释， 磷酸化对I κ B α的影响。 我们会采用两个层次的方法进行所有建议的工作， 结合高分子X射线晶体学的高分辨率 结构分析和表面等离子体共振（BIAcore）的 受体酪氨酸磷酸酶动力学和热力学分析 α/磷酸肽复合物和NF-κ B/I κ B α复合物。 在 在这方面，我们目前已经解决了催化剂的结构， 未复合状态的受体酪氨酸磷酸酶α的结构域。 就IkappaB α项目而言，我们现在有了过度表达， 从杆状病毒中纯化IkappaB α和NF-κ B p65的纯化系统。