Extracellular Matrix and Cell Attachment Proteins

细胞外基质和细胞附着蛋白

• 批准号: 7624695
• 负责人: HAROLD P ERICKSON
• 金额: $ 31.36万
• 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 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7624695
• 关键词: Affinity; Analytical Centrifugation; Bacteria; Bacterial Adhesins; Binding; Biochemical; Cell Adhesion; Cell Culture Techniques; Cells; Collaborations; Crystallization; Crystallography; Cytoskeleton; Dimerization; Disulfides; Elasticity; Embryonic Development; Engineering; Enzyme-Linked Immunosorbent Assay; Extracellular Matrix; Fibronectins; Fluorescence Resonance Energy Transfer; Goals; In Vitro; Integrins; Length; Location; Maps; Mediating; Modeling; Molecular; Molecular Conformation; N-terminal; Nature; Peptides; Physiological; Principal Investigator; Recombinants; Resolution; Rest; Site; Stretching; Structure; Testing; Time; Work; Wound Healing; X-Ray Crystallography; anastellin; base; cell attachment protein; crosslink; deoxycholate; design; dimer; extracellular; fluorophore; in vivo; macromolecular assembly; monomer; novel; prevent; programs; sedimentation equilibrium; sensor; stoichiometry; tool

DESCRIPTION (provided by applicant): Fibronectin (FN) forms the primordial extracellular matrix in embryonic development and wound healing. Remarkably, the assembly of FN matrix fibrils is one of the least understood macromolecular assemblies. Our major goal in the next five years is to determine the nature of the intermolecular bonds that form the fibrils. We propose that there are two steps in fibril assembly. The first step, already understood qualitatively, is association of molecules by reversible bonds, primarily involving the N-terminal 70k fragment. The second step, still completely unknown, is formation of strong, irreversible bonds. We propose to study the first step quantitatively, using Biacore and analytical centrifugation to determine the stoichiometry and Kd of the reversible bonds. We then propose a novel hypothesis for the second step - that the strong bonds are formed by a domain swapping mechanism. Specifically we propose that FN-III domains open up and swap beta strands with domains on another molecule. We propose to test this by engineering "disulfide locks" in the suspected FN-III domains, and testing if this blocks the ability to form fibrils. We have recently engineered a disulfide lock that inhibited assembly of "super fibronectin," and are now ready to test this for assembly of recombinant FN fragments and matrix fibrils in cell culture. We also propose x-ray crystallography of FN-III domains 1-2, which are key players in assembly of sFN and matrix fibrils. We have shown that FN matrix fibrils are very elastic, and are mostly stretched 2-5 times rest length. A second project is to investigate the mechanism of elasticity, and to distinguish between two proposed mechanisms. One mechanism proposes that FN-III domains unfold upon stretching, and the other that stretching is achieved by a compact-to-extended conformation change of the whole molecule, without domain unfolding. We propose to design intramolecular force sensors, which will tell us whether the force is sufficient to unfold FN-III domains. The sensors will be based on FRET, with CFP and YFP separated by entropic springs of different strength. After our initial applications and testing in FN, these sensors should be widely applicable to determine the tension in other ECM and cytoskeletal systems.

描述（由申请人提供）：纤连蛋白（FN）在胚胎发育和伤口愈合中形成原始细胞外基质。值得注意的是，FN基质原纤维的组装是最知名的大分子组件之一。我们未来五年的主要目标是确定形成原纤维的分子间键的性质。我们建议在原纤维组件中有两个步骤。第一步已经定性地理解，它是通过可逆键通过可逆键的结合，主要涉及N末端70K片段。第二步，仍然完全未知，是形成强，不可逆的键。我们建议使用BIACORE和分析离心定量研究第一步，以确定可逆键的化学计量和KD。然后，我们提出了第二步的新假设 - 强键是由域交换机制形成的。具体而言，我们建议FN-III结构域打开并将Beta链与另一个分子上的域交换。我们建议通过在可疑的FN-III域进行工程“二硫键”来测试这一点，并测试是否会阻止形成原纤维的能力。我们最近设计了一个二硫键锁，该硫化物锁定抑制了“超级纤连蛋白”的组装，现在准备测试细胞培养中重组FN片段和基质原纤维的组装。我们还提出了FN-III域1-2的X射线晶体学，这是SFN和基质原纤维组装中的关键参与者。我们已经证明了FN基质原纤维非常弹性，并且大部分时间延伸了2-5倍的休息时间。第二个项目是研究弹性的机制，并区分两个提出的机制。一种机制提出，伸展时FN-III结构域展开，另一种是通过紧凑的整个分子的紧密构象变化来实现伸展，而没有域展开。我们建议设计分子内力传感器，这将告诉我们力是否足以展开FN-III域。传感器将基于FRET，CFP和YFP通过不同强度的熵弹簧隔开。在我们在FN中进行初始应用和测试之后，这些传感器应广泛适用于确定其他ECM和细胞骨架系统中的张力。