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: 7029677
• 负责人: Keith R. McCrae
• 金额: $ 33.7万
• 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 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7029677
• 关键词: angiogenesis inhibitors; apoptosis; athymic mouse; binding sites; cell proliferation; clinical research; enzyme activity; human tissue; kininogens; molecular weight; protein isoforms; protein localization; protein structure function; tropomyosin; vascular endothelium

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（由申请方提供）：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。血浆激肽释放酶切割HK导致缓激肽释放和双链高分子量激肽原（HKa）的产生。我们已经报道了HKa和重组HKa结构域5（其在HK切割后暴露）以Zn 2+依赖的方式诱导增殖内皮细胞的选择性凋亡，并抑制血管生成。基于分子模拟研究表明HKa结构域5与内皮抑素具有结构同源性，以及一份报告表明内皮抑素通过与原肌球蛋白的相互作用与内皮细胞结合，我们确定原肌球蛋白是否参与HKa的抗血管生成活性。我们观察到，抗原肌球蛋白抗体阻断HKA诱导的内皮细胞凋亡，以及HKA与增殖内皮细胞的结合。该抗体还阻断了HKA在体内的抗血管生成作用，另外的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的免疫沉淀和酸洗脱方法的研究表明，原肌球蛋白暴露于增殖的内皮细胞表面。直接测量HKa与原肌球蛋白的结合表现出与所有研究的原肌球蛋白亚型的高亲和力结合，表明HKa与这些蛋白质内的同源区域结合。最后，胰凝乳蛋白酶消化的原肌球蛋白在HKa-琼脂糖凝胶上的亲和纯化，以及在HKa上的环状随机肽文库的淘选，导致初步鉴定原肌球蛋白内的HKa结合区域。在本申请中，我们建议1）比较亚融合、增殖和融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于体内血管生成内皮细胞表面，以及在这种情况下是否作为HKa的结合位点，3）定义原肌球蛋白中的HKa结合位点，并评估其功能重要性。这些研究挑战的范例中，细胞骨架成分被认为是无法访问的细胞外环境，并应提供新的信息，内皮原肌球蛋白的生物学，其在血管生成和抗血管生成活性的HKA的作用。