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: 8938298
• 负责人: SOFIA P BECERRA
• 金额: $ 68.86万
• 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/8938298
• 关键词: 9-cis-retinal; Affinity; Affinity Chromatography; Age related macular degeneration; Air; Alanine; Alternative Splicing; Amino Acids; Angiogenesis Inhibitors; Animal Model; Animals; Antibodies; Apoptotic; Arachidonate 5-Lipoxygenase; Arachidonic Acids; Area; Attenuated; Binding; Biological Assay; Blood Vessels; Cell Death; Cell Line; Cell Survival; Cell membrane; Cells; Characteristics; Choroidal Neovascularization; Chronic lung disease; Clinical; Complementary DNA; Conditioned Culture Media; Corneal Neovascularization; Culture Media; Data; Databases; Defect; Detection; Development; Developmental Bone Diseases; Dysplasia; Enzyme-Linked Immunosorbent Assay; Enzymes; Exclusion; Exons; Expressed Sequence Tags; Eye; Family; Family suidae; Fatty Acids; Fracture; Gel; Generations; Genes; Genetic Transcription; Growth; Human; Hydrogen Peroxide; Hydrolysis; Hydroxyeicosatetraenoic Acids; Hyperoxia; Immunoblotting; In Situ Nick-End Labeling; In Vitro; Inflammatory; Inherited; Interest Group; LOX gene; Length; Leukotriene B4; Ligation; Light; Link; Lipoxygenase; Lipoxygenase Inhibitors; Longevity; Lung; Lysophospholipids; Maintenance; Mammalian Cell; Mammals; Mass Spectrum Analysis; Measures; Mediating; Membrane; Metabolism; Methods; Modeling; Molecular; Molecular Weight; Mus; Mutate; Mutation; Neonatal; Neoplasm Metastasis; Neuronal Differentiation; Nuclear; Osteoblasts; Osteogenesis Imperfecta; Oxidative Stress; Pathway interactions; Patients; Peptides; Phospholipase; Phospholipase A2; Phospholipids; Photoreceptors; Physiologic calcification; Plasmids; Polymerase Chain Reaction; Polyunsaturated Fatty Acids; Predisposition; Premature Infant; Protein Isoforms; Proteins; Protocols documentation; RNA Splicing; Rattus; Reaction; Reactive Oxygen Species; Recombinant Proteins; Recombinants; Relative (related person); Research; Retina; Retinal Cone; Retinal Degeneration; Retinal Neovascularization; Role; Scanning; Serum; Site; Solvents; Soybeans; Starvation; Structure of retinal pigment epithelium; Structure-Activity Relationship; System; Techniques; Time; Transcript; Translations; Variant; Vascular Endothelial Growth Factors; Western Blotting; Work; design; design and construction; expression vector; hypoxia neonatorum; in vivo; interest; liquid chromatography mass spectrometry; member; migration; mouse model; neuronal survival; novel; null mutation; overexpression; oxidative damage; photoreceptor degeneration; pigment epithelium-derived factor; pigment epithelium-derived factor receptor; polyacrylamide; prevent; proband; receptor; research study; retinal ischemia; tumorigenesis; vasculogenesis; vector

We continued studies on the structure-function relationship of PEDF to identify additional structural components necessary for its antiapoptotic activity. PEDF has two biologically active regions: an antiangiogenic 34-mer peptide and a neurotrophic 44-mer peptide. A smaller peptide derived from the solvent exposed region of the 44-mer, a particular 17-mer, shares neurotrophic activity with PEDF and binds P1, a peptide derived from the PEDF binding region of PEDF-R. P1 blocks both the PEDF-PEDF-R interactions and the antiapoptotic activity of PEDF. A set of alanine scanning peptides derived from the 17mer region had varying affinities for P1 and antiapoptotic effects on retina cells. The blocking effects of P1 inhibiting the cytoprotective activities of these 17mer peptides were assessed in retina cells under serum starvation in culture as well as in in the inherited mouse model of retina degeneration rd1/rd1 in vivo. PEDF reduced the number of TUNEL positive photoreceptors in the outer nuclear layer and the blocking P1 peptide attenuated the antiapoptotic effect. To evaluate the PEDF-R binding affinities of full length PEDF protein modified as in the 17mer peptides, expression plasmids containing the SERPINF1 gene with mutations altering single amino acids within the 17-mer region of PEDF were individually designed and constructed. BHK cells were transfected with these plasmids to overexpress the mutated SERPINF1 and obtain recombinant proteins. Purification protocols were developed for the recombinant altered PEDF proteins from the conditioned media of the stably transfected BHK cells containing PEDF expression vectors. We completed a study evaluating the effects of PEDF in Ccl2-/-/Cx3cr1-/- on C57BL/6N Crb1rd8 (DKO rd8) mice, a model for progressive focal retinal degeneration. Serpinf1 transcripts in the RPE and retina, and PEDF protein levels in the culturing media of RPE cells from these animals were significantly reduced, respectively. PEDF administration significantly attenuated focal photoreceptor degeneration associated with apoptotic and inflammatory pathways, as well as lowered vascular endothelial growth factor (VEGF) expression in DKO rd8 eyes. We also completed a study investigating PEDF survival activity on photoreceptors using 661W cells, a cell line derived from mouse cone photoreceptors. PEDF increased the number of 661W cells exposed to light in the presence of 9-cis retinal. PEDF-R was immunodetected in 661W plasma membrane fractions. PEDF increased the phosphorylated Akt to total Akt ratio in these cells. Another study searching for alternatively Pnpla2 spliced variants was completed. Ensembl and other expressed sequence tag databases reveal putative alternative splice variants in mouse and rat for Pnpla2. To obtain experimental evidence for Pnpla2 splice variants polymerase chain reaction (PCR) primer pairs were designed to flank the putative splice sites. Exon exclusion real time PCR was used to reduce amplification of the full-length Pnpla2 transcript and enhance amplification of low abundant splice variants. Recombinant plasmids with human full-length PNPLA2 or PNPLA2 cDNAs lacking exon 5b were used to validate the techniques. PCR products for Pnpla2 transcripts resolved into a single band following amplification with multiple primer pairs. Simultaneous amplification of two PNPLA2 cDNAs at various molar ratios prevented the detection of lower abundant transcripts. Even when the cDNA for the full-length Pnpla2 transcript was significantly excluded using the exon exclusion method, no bands corresponding to Pnpla2 splice variants were detectable. Nonetheless, immunoblots of 661W cells revealed PEDF-R isoforms. The data provide evidence for the existence of a single, full-length Pnpla2 transcript for PEDF-R likely regulated at the posttranslational level. We continued the studies on a novel inhibitor of lipoxygenase discovered fromm a PEDF-R region. Lipoxygenases are enzymes responsible for the metabolism of arachidonic acid and other polyunsaturated fatty acids, thereby contributing to the generation of reactive oxygen species under oxidative stress. Two peptides from the PEDF-binding region of PEDF-R protected ARPE-19 cells from oxidative damage and specifically bound and inhibited soybean lipoxygenase V activity. Human 5-lipoxygenase also bound to P1 peptide and PEDF-R by affinity chromatography and pull-down assays, respectively. Proximity ligation assays were also performed to evaluate the interactions between PEDF-R and 5-lipoxygenase in ARPE-19 cells. Downstream effects of P1 on human 5-lipoxygenase activity in oxidative damaged RPE cells were followed by measuring the 5-lipoxygenase products (LTB4 and 5-HETE) by ELISA and LC/MS. Expression of the PNPLA2 and 5-LOX genes was measured in RPE cells under oxidative stress with hydrogen peroxide treatments by RT-PCR. Primary cultures of RPE cells from pig eyes were developed and optimized. The cytoprotective effects of peptides from the PEDF-R ectodomain on these cells under oxidative stress were evaluated. Silencing vectors were used to ablate the PNPLA2 and 5-LOX expression in ARPE-19 cells. A PNPLA2 expression vector was used to increase the levels of PEDF-R in ARPE-19 cells. RT-PCR and western blots were performed to confirm the changes in the levels of each gene. The effects of silencing and overexpression were then evaluated and compared in the cells undergoing oxidative stress by cell death and viability assays. Experiments to establish the precise apparent molecular weight of PEDF-R were performed. Expression vectors with the human and mouse PNPLA2 cDNAs were constructed. The recombinant proteins were obtained in bacterial transcription/translation systems and mammalian cells transfected with the plasmids. Proteins from in vitro transcription/translation reactions and total lysates of transfected cells were analyzed by western blotting with specific antibodies using gels with three different concentrations of polyacrylamide. The apparent molecular weights of the recombinant proteins were interpolated from a plot generated from logarithm of molecular weight as a function of relative migration of known unlabeled protein molecular weight standards. We continued investigating the downstream effects of PEDF-R interactions in cells. The enzymatic stimulation of PEDF-R by PEDF was quantified by determining fatty acid levels in PEDF-treated R28 cells by mass spectrometry. Recessive null mutations in SERPINF1 cause type VI osteogenesis imperfecta, a heritable bone dysplasia characterized by high susceptibility to fracture, growth deficiency and defects in bone mineralization. Serum levels of PEDF are significantly decreased in type VI OI patients. Dominant mutations in IFITM5, encoding BRIL, cause type V osteogenesis imperfecta. In a collaborative study investigating the role of PEDF in type V OI with clinical characteristics of type VI OI, we found that although its serum levels were not decreased in patients, SERPINF1 expression was decreased in proband osteoblasts, and PEDF protein was barely secreted from proband cells in culture. The data suggested that BRIL can regulate PEDF in osteoblasts. The expression of IFITM5 in human, pig and mouse RPE cells was evaluated by RT-PCR. Broncopulmonary dysplasia is a chronic lung disease of preterm infants characterized by arrested microvascularization and alveolarization. In a collaborative study evaluating the role of PEDF in lung vascular development in neonatal hypoxia, it was found that PEDF levels increase in hyperoxia compared to room air-exposed lungs. The levels of PEDF were positively correlated with reduced vasculogenesis and alveolarization in neonatal hyperoxia, implying that PEDF mediates impaired lung vascular development in neonatal hyperoxia.

我们继续研究PEDF的结构功能关系，以确定其抗凋亡活性所需的其他结构成分。 PEDF有两个具有生物活性的区域：抗血管生成34-mer肽和神经营养44-mer肽。 源自44-mer的溶剂暴露区域（一种特定17-mer）的较小肽与PEDF共享神经营养活性，并结合P1，这是源自PEDF-R的PEDF结合区域的肽。 P1阻止了PEDF-PEDF-R相互作用和PEDF的抗凋亡活性。 衍生自17mer区域的一组丙氨酸扫描肽对视网膜细胞的P1和抗凋亡作用具有不同的亲和力。 在培养物以及体内的视网膜变性RD1/RD1的遗传小鼠模型中，评估了在视网膜细胞中评估这些17Mer肽的P1的阻断作用。 PEDF减少了外核层中TUNEL阳性光感受器的数量，而阻塞P1肽减少了抗凋亡效应。 为了评估所修饰的全长PEDF蛋白的PEDF-R结合亲和力，如17mer肽中的表达质粒，这些质粒含有serpinf1基因，其突变改变了PEDF区域内17-Mer区域内的单个氨基酸的突变，是单独设计和构造的。 用这些质粒转染BHK细胞，以过表达突变的SERPINF1并获得重组蛋白。 为重组改变的PEDF蛋白从稳定转染的含有PEDF表达载体的条件培养基中开发了纯化方案。 我们完成了一项研究，评估了PEDF对CCL2 - / - /CX3CR1 - / - 对C57BL/6N CRB1RD8（DKO RD8）小鼠的影响，这是一种进行渐进的局灶性视网膜变性模型。 RPE和视网膜中的SERPINF1转录本，以及这些动物RPE细胞培养培养基中的PEDF蛋白水平分别显着降低。 PEDF给药可显着减弱与凋亡和炎症途径相关的局灶性光感受器变性，以及DKO RD8眼中的血管内皮生长因子（VEGF）表达降低。 我们还完成了一项研究，研究了使用661W细胞（源自小鼠锥光受体的细胞系）对PEDF生存活性的研究。 PEDF增加了在9-CIS视网膜存在下暴露于光线的661W细胞的数量。 PEDF-R在661W质膜级分中进行免疫进行。 PEDF增加了这些细胞中磷酸化的AKT与总AKT比。 另一项研究搜索pnpla2剪接变体的研究完成了。 Enembl和其他表达的序列标签数据库揭示了鼠标和PNPLA2大鼠中的推定替代剪接变体。为了获得PNPLA2剪接变体的实验证据，将聚合酶链反应（PCR）引物对设计用于侧翼。外显子排除实时PCR用于减少全长PNPLA2转录物的扩增，并增强低丰富的剪接变体的扩增。具有人类全长PNPLA2或PNPLA2 cDNA缺乏外显子5B的重组质粒用于验证技术。 PNPLA2转录本的PCR产物通过多个引物对放大后分解为单个带。同时在各种摩尔比下同时扩增两个PNPLA2 cDNA，阻止了较低丰富的转录本的检测。即使使用外显子排除法显着排除了全长PNPLA2转录本的cDNA，也没有检测到与PNPLA2剪接变体相对应的频段。但是，661W细胞的免疫印迹显示PEDF-R同工型。该数据提供了证据证明可能在翻译后水平受到调节的PEDF-R单一的全长PNPLA2转录本。 我们继续研究了一种从PEDF-R区域发现的新型脂氧合酶抑制剂。脂氧酶是负责花生四烯酸和其他多不饱和脂肪酸代谢的酶，从而有助于在氧化应激下产生活性氧。 来自PEDF-R的PEDF结合区域的两种肽免受氧化损伤的保护ARPE-19细胞，并特别结合并抑制大豆脂氧合酶V活性。 人类5-脂氧酶也分别通过亲和力色谱和下拉测定法与P1肽和PEDF-R结合。 还进行了接近连接测定，以评估ARPE-19细胞中PEDF-R和5-脂氧酶之间的相互作用。 通过通过ELISA和LC/MS测量5-脂氧合酶产物（LTB4和5-HETE），P1对氧化损坏的RPE细胞中人类5-脂氧酶活性的下游影响之后。 PNPLA2和5-lox基因的表达在RT-PCR氧化应激下在RPE细胞中测量。 开发并优化了来自猪眼的RPE细胞的一级培养。 评估了在氧化应激下对这些细胞的肽对这些细胞的细胞保护作用。沉默载体用于在ARPE-19细胞中消除PNPLA2和5-LOX表达。 使用PNPLA2表达载体来增加ARPE-19细胞中PEDF-R的水平。进行RT-PCR和Western印迹以确认每个基因水平的变化。然后，通过细胞死亡和生存力测定法评估并比较沉默和过表达的影响。 进行了PEDF-R的精确表观分子量的实验。 构建了与人和小鼠PNPLA2 cDNA的表达向量。 重组蛋白是在细菌转录/翻译系统中获得的，并用质粒转染的哺乳动物细胞。 通过使用特定抗体的特定抗体，使用具有三种不同浓度的聚丙烯酰胺的凝胶，通过蛋白质印迹分析了来自体外转录/翻译反应的蛋白质和转染细胞的总裂解物。 重组蛋白的明显分子量是从分子量对数产生的图中插值的，这是已知的未标记蛋白分子量标准品的相对迁移的函数。 我们继续研究细胞中PEDF-R相互作用的下游效应。 通过通过质谱法确定PEDF处理的R28细胞中的脂肪酸水平来量化PEDF-R的酶促刺激。 SERPINF1中隐性零突变导致VI型成骨的不完美，这是一种可遗传的骨骼发育异常，其特征是骨折，生长缺乏症和骨矿化缺陷的敏感性高。 VI型患者的PEDF的血清水平显着降低。 IFITM5中的主要突变，编码新的，导致v型成骨的不完美。 在一项协作研究中，研究了PEDF在V型中具有VI型临床特征的作用，我们发现，尽管患者的血清水平并未降低，但在探针和成骨细胞中，SERPINF1表达降低，并且PEDF蛋白在培养中几乎不属于概率细胞。数据表明，布里尔可以在成骨细胞中调节PEDF。 通过RT-PCR评估了人，猪和小鼠RPE细胞中IFITM5的表达。 肺肺发育不良是一种以幼虫为特征的早产儿的慢性肺部疾病，其特征是微血管造成的和肺泡化。在一项合作研究中，评估了PEDF在新生儿缺氧中肺血管发育中的作用，发现与房间暴露的肺相比，PEDF水平增加了高氧。 PEDF的水平与新生儿高氧的血管生成和肺泡化的降低呈正相关，这意味着PEDF介导了新生儿高氧中的肺血管发育受损。