Protein Interactions and Conformational Changes in Fibronectin Fibril Formation

 描述(申请人提供)：致命性肿块的产生需要肿瘤细胞招募并维持自身的血液供应。在缺乏血液供应的情况下，肿瘤可以作为休眠的显微病变持续存在，但它们不会超过1-2毫米的大小。因此，抑制新血管生长(血管生成)为根除肿瘤或将癌症转变为一种慢性可控疾病提供了巨大的希望。此外，抑制血管生成为治疗其他一系列依赖血管生成的疾病提供了巨大的希望，包括老年性黄斑变性、糖尿病视网膜病变、类风湿性关节炎和动脉粥样硬化。该应用的重点是Anastellin，一种细胞外基质蛋白纤维连接蛋白的片段，在小鼠模型中抑制血管生成、肿瘤生长和转移。Anastellin在体内的抗血管生成活性需要内源性纤维连接蛋白，在体外它与纤维连接蛋白结合，将可溶性蛋白质转化为不溶性纤维。我们的长期目标是阐明Anastellin与纤维连接蛋白的相互作用如何导致纤维聚集的形成，以及纤维连接蛋白的结构如何决定其功能。这项应用的目的是确定Anastellin和FN之间相互作用的分子基础。我们建议用二硫键交联、核磁共振和X射线结晶学的方法研究Anastellin与其靶FN3结构域之间的络合物的结构和动力学。此外，我们建议用顺磁松弛增强(PRE)和单分子Förster共振能量转移(FRET)来研究纤维连接蛋白中Anastellin结合区的构象。识别导致Anastellin活性的结构特征将提供对这种血管生成抑制物如何在分子水平上发挥作用的详细了解，并可能使新的抗癌药物的开发成为可能。拟议的结构研究还将深入了解可溶性纤维连接蛋白转化为纤维基质形式所涉及的分子相互作用和重排。

 DESCRIPTION (provided by applicant): The generation of a lethal tumor mass requires that the tumor cells recruit and sustain their own blood supply. In the absence of blood supply, tumors can persist as dormant microscopic lesions but they do not expand beyond the size of 1-2 mm. Hence, inhibition of new blood vessel growth (angiogenesis) offers great promise to eradicate tumors or to convert cancer to a chronic manageable disease. In addition, inhibition of angiogenesis offers great promise to treat a range of other diseases that depend on angiogenesis, including age-related macular degeneration, diabetic retinopathy, rheumatoid arthritis and atherosclerosis. The focus of this application is anastellin, a fragment of the extracellular matrix protein fibronectin that inhibits angiogenesis, tumor growth and metastasis in mouse models. Anastellin requires endogenous fibronectin for its in vivo anti- angiogenic activity, and it binds to fibronectin in vitro and converts the soluble protein to insoluble fibril. Our long term goal is to elucidate how the interaction of anastellin with fibronectin leads to formation of fibrillar aggregates, and in general how the structure of fibronectin determines its function. The goal of this application is to identify the molecular basis of the interaction betwee anastellin and fibronectin. We propose to investigate the structure and dynamics of the complexes between anastellin and its target fibronectin type III (FN3) domains by disulfide crosslinking, nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography. In addition, we propose to investigate the conformation of the anastellin binding region in fibronectin using paramagnetic relaxation enhancement (PRE) and single molecule Förster resonance energy transfer (FRET). Identification of structural features that are responsible for the activity of anastellin will provide detailed understanding of how this inhibitor of angiogenesi acts at the molecular level and may enable development of new anticancer drugs. The proposed structural studies will also provide insight into the molecular interactions and rearrangements that are involved in conversion of soluble fibronectin to the fibrillar matrix form.