Protein Interactions in Retina Development and Function

视网膜发育和功能中的蛋白质相互作用

• 批准号: 7139185
• 负责人: SOFIA P BECERRA
• 金额: --
• 依托单位: NATIONAL EYE INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7139185
• 关键词: Primates; angiogenesis; angiogenesis inhibitors; cell surface receptors; choroid uvea; confocal scanning microscopy; enzyme activity; erythropoietin; eye circulation; fluorimetry; growth factor receptors; histogenesis; hypoxia; laboratory mouse; laboratory rat; neurotrophic factors; protease inhibitor; protein protein interaction; protein structure function; receptor binding; retina; retinal pigment epithelium; serine proteinases; tissue /cell culture; vascular endothelial growth factors; visual pigments; yeast two hybrid system

Work in this research group is aimed at elucidating the mechanisms that regulate the activities of pigment epithelium-derived factor (PEDF), an extracellular serpin with neurotrophic and antiangiogenic activities. We continued working on the characterization of a putative receptor for PEDF, PEDF-R, and demonstrated that this novel protein had binding affinity for PEDF and phospholipase activity. We compared the affinities of PEDF and other serpins to PEDF-R, engineered cells overexpressing the PEDF-R gene, constructed mutants and deletion versions of PEDF-R, and developed antibodies to this interesting protein. We determined its subcellular localization to the cell-surface and cytosol of mammalian cells using antibodies to predicted extracellular and intracellular loops. We exposed live cells to flourescein-conjugated PEDF (Fl-PEDF) and showed fluorescent staining on cell-surfaces in a dose-dependent and specific fashion. We examined the effects of PEDF on extracellular ATP synthesis activity of endothelial cells and compared them to those by angiostatin and ATP synthase inhibitors, and found that all of them inhibited significantly the extracellular ATP synthesis activity. We examined the direct binding of PEDF to highly purified yeast F1-ATP synthase with a His-tagged a-subunit. Size-exclusion ultrafiltration, Ni-NTA pull-down and Surface Plasmon Resonance assays showed that PEDF bound to F1 with similar affinities as those between PEDF and intact endothelial cells. We continued to develop and optimize a method for evaluating and quantifying chroroidal neovascularization (CNV) in rodents. CNV lesions were induced with laser and the morphology and volume of the lesions evaluated in choroid/RPE flat mounts by confocal microscopy using 3-D reconstructions. We studied the structure-function relationships of PEDF?s antiangiogenic activity. We compared the efficacy of PEDF peptides, other antiangiogenic factors and serpins using tube formation, an ex vivo chick aortic arch and the in vivo CNV assay. We found that PEDF and 34-mer, a small PEDF peptide towards the amino-end region, inhibited angiogenesis in these assays. We examined the direct binding of PEDF to hyaluronan by coprecipitation with cetylpyridinium chloride. We prepared site-specific mutants encoding modified PEDF at a putative hyaluronan binding region and found that single point alterations reduced >70% the binding affinity relative to the unmodified protein, while cumulative modifications abolished the hyaluronan-binding activity of PEDF. All the modified proteins retained binding affinity to collagen similar to that of the unmodified protein. These results showed that the homologous hyaluronan-binding motif is a functional region in this serpin. In the spatial PEDF structure it is located between helix F and strand s3A, and is distinct from the previously reported collagen- and heparin-binding regions.

该研究小组的工作旨在阐明调节色素上皮衍生因子活性（PEDF）的机制，这是一种具有神经营养性和抗血管生成活性的细胞外SERPIN。 我们继续研究PEDF，PEDF-R的推定受体的表征，并证明了这种新型蛋白质对PEDF和磷脂酶活性具有结合亲和力。我们将PEDF和其他SERPINS的亲和力与PEDF-R的亲和力，过表达PEDF-R基因的工程细胞，构建的突变体和PEDF-R的缺失版本，并开发了对这种有趣蛋白质的抗体。我们使用抗体预测细胞外和细胞内环的抗体确定了其在哺乳动物细胞的细胞表面和细胞质中的亚细胞定位。我们将活细胞暴露于粉状偶联的PEDF（FL-PEDF）中，并以剂量​​依赖性和特定的方式显示在细胞表面上的荧光染色。 我们检查了PEDF对内皮细胞细胞外ATP合成活性的影响，并将其与Angiostatin和ATP合酶抑制剂进行了比较，并发现所有这些抑制剂都显着抑制了细胞外ATP合成活性。我们检查了PEDF与高度纯化的酵母F1-ATP合酶的直接结合，并使用了His标签的A-Subunit。尺寸 - 排斥超滤，Ni-NTA下拉和表面等离子体共振分析表明，PEDF与F1结合，与PEDF和完整的内皮细胞之间的亲密关系相似。 我们继续开发和优化一种评估和量化啮齿动物中成熟成成熟的新生血管形成（CNV）的方法。用激光诱导CNV病变，并使用3D重建通过共聚焦显微镜在脉络/RPE扁平架子中评估的病变的形态和体积。我们研究了PEDF的抗血管生成活性的结构功能关系。我们比较了PEDF肽，其他抗血管生成因子和使用管形成，离体鸡主动脉弓和体内CNV测定法的疗效。我们发现，PEDF和34-mer是朝氨基末端区域的小PEDF肽，在这些测定中抑制了血管生成。 我们通过与氯吡啶丁基共沉淀PEDF与透明质酸的直接结合。我们制备了在推定的透明质酸结合区域编码修饰PEDF的位点特异性突变体，发现单点改变降低了> 70％的结合亲和力相对于未修饰的蛋白质，而累积修饰消除了PEDF的透明质酸结合活性。所有改性蛋白保留了与胶原蛋白相似的胶原蛋白的结合亲和力。这些结果表明，同源的透明质酸结合基序是该SERPIN中的一个功能区域。在空间PEDF结构中，它位于螺旋F和Strand S3A之间，与先前报道的胶原蛋白和肝素结合区域不同。