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（由申请人提供）：高分子量吉诺原（HK）是一种丰富的血浆糖蛋白，在Kallikrein-Kinin系统中起着核心作用。血浆kallikrein裂解HK会导致缓激肽的释放并产生两链高分子量吉诺原（HKA）。我们报道说，HKA和重组HKA结构域5（在HK裂解后暴露）以Zn2+依赖性方式诱导增生的内皮细胞的选择性凋亡，并抑制血管生成。基于分子建模研究，表明HKA结构域5具有结构性同源性，并表明，通过与tropomyosin的相互作用，内皮替汀与内皮细胞结合，我们确定了tropomyosin是否参与了HKA的抗基因生成活性。我们观察到，抗冰霉素抗体阻断了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体内HKA的抗血管生成作用，而其他抗抗霉素抗体具有这些作用。内皮细胞至少表达对真菌菌素的至少5种同工型，并且采用共聚焦显微镜，生物素基化内皮细胞表面蛋白的免疫沉淀和酸性洗脱方法的研究表明，tropomyosin暴露于增殖的内皮细胞的表面上。直接测量HKA与肌球蛋白的结合表现出与所研究的所有肌动蛋白同工型的高亲和力结合，这表明HKA与这些蛋白质中的同源区域结合。最后，在HKA-Sepharose上的胰凝乳蛋白酶消化的thropomyosin的亲和力纯化，就像在HKA上的环状随机肽文库平移一样，导致了对肌动蛋白内HKA结合区域的暂时鉴定。在此应用中，我们建议1）通过亚汇合，增殖和汇合的内皮细胞比较不同的肌动蛋白同工型的表达和亚细胞分布，2）确定对tropomyosin在体内血管生成细胞上是否在体内的血管生成细胞表面暴露在体内，以及它是否适用于该固定位点，并在此固定位置中固定在HKA中，以及3）在hka中的固定位置，以及3）在设置中，以及3）在设置中，以及3）inte ins in n n in n.bka ins in n n in n.bka ins in n in n.bka ins in n n in n. 3）ank ink ink in n in n in n. 3） tropomyosin，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访问的细胞骨架成分，并且应提供有关内皮斜霉素生物学及其在血管生成和HKA的抗血管生成中的作用的新信息。