EXTRACELLULAR MATRIX AND CELL ATTACHMENT PROTEINS

细胞外基质和细胞附着蛋白

• 批准号: 2837626
• 负责人: HAROLD P ERICKSON
• 金额: $ 30.14万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-05-01 至 2000-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2837626
• 关键词: Caenorhabditis elegans; Drosophilidae; X ray crystallography; affinity chromatography; binding proteins; cell adhesion molecules; embryogenesis; enzyme linked immunosorbent assay; extracellular matrix; gene expression; genetic library; genetically modified animals; immunochemistry; laboratory mouse; laminin; membrane proteins; neoplastic growth; northern blottings; polymerase chain reaction; protein structure function; tissue /cell culture; western blottings; wound healing

Continued studies will focus primarily on tenascin - the giant hexabrachion molecule in the extracellular matrix of tumors, healing wounds and specific embryonic tissues. We are already making transgenic mice that overexpress tenascin. We propose to make others that will be defective in hexabrachion assembly (monobrachion mice), and mice with depletion in tenascin secretion. We will determine the effects of overexpression, underexpression or defective hexabrachions on embryonic development, tumors and wound healing. A second project is a search for the tenascin gene in Drosophila and C. elegans. These species have highly developed genetic maps and large numbers of mutants, offering a powerful new approach to understanding the functions of tenascin. We will continue our studies of the cell biology and biochemistry of tenascin, to identify and characterize cell surface receptors for different domains of tenascin. A key tool for this project is the library of bacterial expression proteins that we have recently developed, providing large quantities of defined segments of the hexabrachion arm. We will use a similar approach to identify ECM molecules that bind to tenascin. Another continuing project is to characterize a new form of laminin that we identified during the purification of tenascin from cell culture supernatant. This new protein appears to have a variant A chain, a unique tissue distribution, and a cell adhesion activity quite different from any known laminin. Finally, we propose to expand our collaborative projects on the x-ray crystallography of tenascin. Our approach is to crystallize one domain at a time, using our PCR approach to make precisely defined bacterial expression proteins. The two FN-III domains that we have tried so far, the RGD domains from tenascin and fibronectin, have given excellent crystals, and high resolution x-ray diffraction studies are in progress. Additional domains are now proposed for future studies. An atomic resolution structure of the entire hexabrachion is a feasible goal.

持续的研究将主要集中在生腱蛋白——巨人 肿瘤细胞外基质中的 hexabrachion 分子，愈合 伤口和特定的胚胎组织。我们已经在进行转基因 过度表达生腱蛋白的小鼠。我们建议让其他人 六臂体组装缺陷（单臂体小鼠），以及具有 生腱蛋白分泌减少。我们将确定影响 胚胎上的六臂体过度表达、表达不足或有缺陷 发育、肿瘤和伤口愈合。第二个项目是寻找 果蝇和线虫中的腱蛋白基因。这些物种具有高度 开发了遗传图谱和大量突变体，提供了强大的 了解生腱蛋白功能的新方法。我们将继续 我们对生腱蛋白的细胞生物学和生物化学的研究，以确定 并表征不同域的细胞表面受体 生腱蛋白。该项目的一个关键工具是细菌库 我们最近开发的表达蛋白，提供大量 六边形臂的定义片段的数量。我们将使用一个 类似的方法来识别与生腱蛋白结合的 ECM 分子。其他 持续进行的项目是表征我们研究的一种新形式的层粘连蛋白 在从细胞培养物中纯化生腱蛋白过程中鉴定出 上清液。这种新蛋白质似乎有一个变体 A 链，这是一种独特的 组织分布和细胞粘附活性与 任何已知的层粘连蛋白。最后，我们建议扩大我们的合作 腱蛋白的 X 射线晶体学项目。我们的方法是 使用我们的 PCR 方法一次结晶一个结构域 精确定义的细菌表达蛋白。两个 FN-III 结构域 到目前为止我们已经尝试过，来自生腱蛋白和纤连蛋白的 RGD 结构域， 提供了优良的晶体和高分辨率的X射线衍射 研究正在进行中。现在建议将来添加更多域 研究。整个六面体的原子分辨率结构是 可行的目标。