Role of Protein Interactions in Retina Development and Function

蛋白质相互作用在视网膜发育和功能中的作用

• 批准号: 7594058
• 负责人: SOFIA P BECERRA
• 金额: $ 129.81万
• 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/7594058
• 关键词: ATP Synthesis Pathway; Active Sites; Affinity; Amino Acids; Angiogenesis Inhibitors; Angiostatins; Animal Model; Anterior; Antibodies; Apoptosis; Area; Behavior; Binding; Binding Sites; Biological Assay; Bos taurus; C-terminal; Cattle; Cell Cycle Kinetics; Cell Differentiation process; Cell Line; Cell Survival; Cell membrane; Cell model; Cells; Chemicals; Collaborations; Collagen; Corneal Neovascularization; Cultured Cells; Development; Dexamethasone; Diffuse; Electronics; Endothelial Cells; Engineering; Enzymes; Exhibits; F1-ATPase; Fatty Acids; Gelatin; Human; Human Activities; Hyaluronan; Immunohistochemistry; Inherited; Label; Life; Longevity; Maintenance; Mammals; Membrane Proteins; Metalloproteases; Modification; Molecular; Monitor; Neuronal Differentiation; Organ; Oxidative Stress; Peptides; Phospholipase; Phospholipases A; Photoreceptors; Plasmids; Point Mutation; Protein Overexpression; Proteins; Recombinants; Regulation; Reporting; Research; Retina; Retinal; Retinal Degeneration; Retinal Ganglion Cells; Role; Serum; Site-Directed Mutagenesis; Solutions; Starvation; Structure; Structure of retinal pigment epithelium; Structure-Activity Relationship; Surface; Surface Plasmon Resonance; System; Technology; Time; Trabecular meshwork structure; Transcript; Transfection; Work; Yeasts; design; extracellular; genetically modified cells; indexing; interest; pigment epithelium-derived factor; pigment epithelium-derived factor receptor; polypeptide; protein structure; relating to nervous system; research study; response; retinal ischemia; sensor; synthetic peptide; uptake; vector

We continued working on the characterization of PEDF-R protein, a receptor for PEDF with binding affinity for PEDF and phospholipase activity. We constructed several plasmids with deletion versions of PEDF-R and a point mutation at a putative phospholipase A active site, and expressed them heterologously. The resulting recombinant polypeptides were assayed for binding to PEDF and for phospholipase activity. The C-terminal truncated PEDF-R polypeptides exhibited phospholipase A activity and had binding affinity for PEDF. PEDF-R with an altered amino acid at the putative phospholipase A active site bound PEDF but did not exhibit phospholipase A activity. Synthetic peptides were designed from a putative extracellular loop. PEDF binding assays were performed and showed that a peptide from the central region of the loop had binding affinity for PEDF. Immunohistochemistry of bovine retina and RPE showed distribution of PEDF-R the RPE, in the inner segments of photoreceptors and inner layers of the retina. Surface Plasmon Resonance experiments showed that bovine retina plasma membrane proteins contained a PEDF binding component that can be captured with antibodies to PEDF-R. The PEDF-R protein was also immunodetected in westerns of plasma membranes of retina, retinal pigment epithelium, and R28, RGC-5 and ARPE-19 cells. PEDF-R derived from ARPE-19 cells was an active phospholipase enzyme, which PEDF stimulated to release fatty acids. We genetically engineered ARPE-19, retina R28 and retinal ganglion cell RCG-5 lines to overexpress or silence the PEDF-R expression. Plasma membrane extracts from genetically modified cells overexpressing PEDF-R transcripts had higher phospholipase activity than those from untransfected cells. In contrast, transfection of silencing vectors lowered the levels of PEDF-R transcripts and those for plasma membrane phospholipase activity. The effects of these modified cell lines on oxidative-stress-induced apoptosis cell model were also studied using a label-free real-time assay using electronic cell sensor technology system. The results showed a differential behavior in the uptake of the oxidative agent and the response in cell kinetics upon PEDF addition. Living retina R28 and RCG-5 cells were also monitored by the electronic cell sensor system and cell viability assays. Cells were induced to apoptosis by serum starvation. Treatments with PEDF of increased the electronic index and viability of the cells. We continued examining the interactions between PEDF and ectopic ATP synthase. Plasma membrane fractions from human microvascular endothelial cell (HMVEC) and bovine retina contained a component with affinity for PEDF on SPR surface chips, which were captured with specific antibodies to beta subunit of F1 ATP synthase. Direct binding to highly purified yeast F1-ATPase with a His-tagged beta-subunit showed PEDF binding affinities similar to those with intact cells. PEDF competed with angiostatin K1-5 for F1 binding. PEDF inhibited significantly the extracellular ATP synthesis activity of HMVEC endothelial cells. We completed studies on the interactions of PEDF with hyaluronan. The hyaluronan-binding region was examined by chemical modification and site-directed mutagenesis. In the spatial PEDF structure the hyaluronan-binding motifs are located in areas distinct from previously reported neurotrophic and antiangiogenic active regions, and from the collagen-binding site. The regulation of PEDF by dexamethasone in trabecular meshwork continued to be examined (in collaboration with Terete Borras). We investigated the effect of DEX on metalloproteinases by gelatin solution assays and zymography. While dexamethasone can increase levels of PEDF transcripts and PEDF protein, it decreased the gelatinolytic activities of trabecular meshwork in anterior segments organs and cell cultures.

我们继续研究PEDF-R蛋白的表征，PEDF-R蛋白是PEDF的受体，具有对PEDF和磷脂酶活性的结合亲和力。我们在假定的磷脂酶A活性位点构建了几种具有缺失版本的PEDF-R和点突变的质粒，并将其异源表达。 测定所得的重组多肽与PEDF和磷脂酶活性结合。 C末端截短的PEDF-R多肽表现出磷脂酶A活性，并且对PEDF具有结合亲和力。 PEDF-R用改变的氨基酸在推定的磷脂酶A活性位点结合的PEDF，但没有表现出磷脂酶A活性。 合成肽是根据推定的细胞外环设计的。 进行了PEDF结合测定，并表明来自环路中央区域的肽对PEDF具有结合亲和力。 牛视网膜和RPE的免疫组织化学显示了PEDF-R RPE的分布，在感光体和视网膜的内层的内部段中。表面等离子体共振实验表明，牛视网膜质膜蛋白包含PEDF结合成分，可以用抗PEDF-R抗体捕获。 在视网膜，视网膜色素上皮和R28，RGC-5和ARPE-19细胞的质膜西部，PEDF-R蛋白也被免疫实现。 源自ARPE-19细胞的PEDF-R是一种活跃的磷脂酶，PEDF刺激释放脂肪酸。我们通过基因设计的ARPE-19，视网膜R28和视网膜神经节细胞RCG-5线过表达或使PEDF-R表达保持沉默。 来自过表达PEDF-R转录物的遗传修饰细胞中的质膜提取物的磷脂酶活性高于未转染细胞的磷脂酶活性。相反，沉默载体的转染降低了PEDF-R转录物的水平和质膜磷脂酶活性的水平。还使用电子细胞传感器技术系统研究了这些改性细胞系对氧化应激诱导的细胞凋亡模型模型的影响。结果表明，氧化剂摄取的差异行为以及添加PEDF时细胞动力学的反应。 还通过电子细胞传感器系统和细胞活力测定法监测活性视网膜R28和RCG-5细胞。 通过血清饥饿诱导细胞凋亡。用PEDF治疗细胞的电子指数和生存能力增加。 我们继续研究PEDF和异位ATP合酶之间的相互作用。 来自人类微血管内皮细胞（HMVEC）和牛视网膜的质膜级分包含对PEDF的SPR表面芯片的成分，这些成分在SPR表面芯片上具有与F1 ATP合酶的β亚基的特定抗体捕获。与高度纯化的酵母F1-ATPase与具有HIS标签的β-亚基的直接结合显示出与完整细胞相似的PEDF结合亲和力。 PEDF与Angiostatin K1-5竞争F1结合。 PEDF显着抑制了HMVEC内皮细胞的细胞外ATP合成活性。 我们完成了有关PEDF与透明质酸相互作用的研究。通过化学修饰和定分诱变检查了透明质酸结合区域。在空间PEDF结构中，透明质酸结合基序位于不同于先前报道的神经营养和抗血管生成的区域，以及胶原结合位点。 继续检查小梁网中PEDF的调节（与Terete Borras合作）。我们通过明胶溶液分析和Zymography研究了DEX对金属蛋白酶酶的影响。 虽然地塞米松可以增加PEDF转录物和PEDF蛋白的水平，但它降低了小梁网状器官和细胞培养物中小梁网的明胶活性。