Tropomyosin in the antiangiogenic activity of HKa

原肌球蛋白在 HKa 的抗血管生成活性中的作用

• 批准号: 7217280
• 负责人: Keith R. McCrae
• 金额: $ 32.72万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7217280
• 关键词: Acids; Address; Affinity; Affinity Chromatography; Angiogenesis Inhibitors; Antibodies; Apoptosis; Binding; Binding Sites; Biological Process; Biology; Blocking Antibodies; Bradykinin; Cell physiology; Cell surface; Cleaved cell; Clinical Research; Coagulation Process; Confocal Microscopy; Cytoskeletal Proteins; Development; Diabetic Retinopathy; Disease; Disruption; Endostatins; Endothelial Cells; Endothelium; Experimental Neoplasms; Generations; Glycoproteins; Goals; Growth; High-Molecular-Weight Kininogen; Human; Immunoprecipitation; In Vitro; Kallikrein-Kinin System; Macular degeneration; Measurement; Mediating; Membrane Proteins; Nature; Neoplasms; Pan Genus; Pathway interactions; Peptides; Plasma; Plasma Kallikrein; Play; Proliferating; Protein Fragment; Protein Isoforms; Proteins; Random Peptide Libraries; Recombinants; Regulation; Reporting; Role; Sepharose; Surface; System; Tropomyosin; angiogenesis; base; chymotrypsin; extracellular; in vivo; inhibitor/antagonist; interest; member; molecular modeling; prevent; receptor

DESCRIPTION (provided by applicant): High molecular weight kininogen (HK) is an abundant plasma glycoprotein that plays a central role in the kallikrein-kinin system. Cleavage of HK by plasma kallikrein results in release of bradykinin and generation of two-chain high molecular weight kininogen (HKa). We have reported that HKa and recombinant HKa domain 5 (which is exposed following HK cleavage) induce selective apoptosis of proliferating endothelial cells in a Zn2+-dependent manner, and inhibit angiogenesis. Based on molecular modeling studies suggesting that HKa domain 5 has structural homology to endostatin, and a report suggesting that endostatin bound to endothelial cells through interactions with tropomyosin, we determined whether tropomyosin was involved in the antiangiogenic activity of HKa. We observed that an anti-tropomyosin antibody blocked HKa-induced endothelial cell apoptosis, as well as the binding of HKa to proliferating endothelial cells. This antibody also blocked the antiangiogenic effects of HKa in vivo, and additional antitropomyosin antibodies shared these effects. Endothelial cells express at least 5 isoforms of tropomyosin, and studies employing confocal microscopy, immunoprecipitation of biotinylated endothelial cell surface proteins and acid elution approaches suggest that tropomyosin is exposed on the surface of proliferating endothelial cells. Direct measurement of the binding of HKa to tropomyosin demonstrated high affinity binding to all tropomyosin isoforms studied, suggesting that HKa bound to a homologous region within these proteins. Finally, affinity purification of chymotrypsin-digested tropomyosin on HKa-sepharose, as w ell as panning of a cyclic random peptide Library on HKa, led to tentative identification of HKa binding regions within tropomyosin. In this application, we propose to 1) compare the expression and subcellular distribution of different tropomyosin isoforms by subconfluent, proliferating and confluent endothelial cells, 2) determine whether tropomyosin is exposed on the surface of angiogenic endothelial cells in vivo, and whether it serves as a binding site for HKa in this setting, and 3) define the HKa binding site in tropomyosin, and assess its functional importance. These studies challenge the paradigm in which cytoskeletal components are considered inaccessible to the extracellular milieu, and should provide new information concerning the biology of endothelial tropomyosin, and its roles in angiogenesis and the antiangiogenic activity of HKa.

描述（由申请人提供）：高分子量激肽原（HK）是一种丰富的血浆糖蛋白，在激肽释放酶-激肽系统中发挥核心作用。 HK 被血浆激肽释放酶裂解，导致缓激肽释放并产生双链高分子量激肽原 (HKa)。我们报道了 HKa 和重组 HKa 结构域 5（HK 裂解后暴露）以 Zn2+ 依赖性方式诱导增殖内皮细胞选择性凋亡，并抑制血管生成。基于分子模型研究表明 HKa 结构域 5 与内皮抑素具有结构同源性，并且有报告表明内皮抑素通过与原肌球蛋白相互作用与内皮细胞结合，我们确定了原肌球蛋白是否参与了 ​​HKa 的抗血管生成活性。我们观察到抗原肌球蛋白抗体可阻断 HKa 诱导的内皮细胞凋亡以及 HKa 与增殖内皮细胞的结合。该抗体还在体内阻断了 HKa 的抗血管生成作用，并且其他抗原肌球蛋白抗体也具有这些作用。内皮细胞表达至少 5 种原肌球蛋白亚型，采用共聚焦显微镜、生物素化内皮细胞表面蛋白免疫沉淀和酸洗脱方法的研究表明，原肌球蛋白暴露在增殖内皮细胞的表面。对 HKa 与原肌球蛋白结合的直接测量表明，HKa 与所研究的所有原肌球蛋白亚型具有高亲和力结合，表明 HKa 与这些蛋白质内的同源区域结合。最后，在 HKa 琼脂糖上亲和纯化胰凝乳蛋白酶消化的原肌球蛋白，以及在 HKa 上淘选环状随机肽文库，初步鉴定了原肌球蛋白内的 HKa 结合区域。在此应用中，我们建议 1) 比较亚汇合、增殖和汇合内皮细胞不同原肌球蛋白亚型的表达和亚细胞分布，2) 确定原肌球蛋白是否暴露在体内血管生成内皮细胞的表面，以及在这种情况下它是否作为 HKa 的结合位点，以及 3) 定义 HKa 结合位点 原肌球蛋白，并评估其功能重要性。这些研究挑战了细胞骨架成分被认为无法进入细胞外环境的范式，并且应该提供有关内皮原肌球蛋白的生物学及其在血管生成中的作用和 HKa 的抗血管生成活性的新信息。