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: 9155554
• 负责人: SOFIA P BECERRA
• 金额: $ 83.68万
• 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/9155554
• 关键词: Affinity; Affinity Chromatography; Age related macular degeneration; Air; Alanine; Angiogenesis Inhibitors; Animal Model; Anions; Antibodies; Arachidonic Acids; Area; Binding; Binding Sites; Biological; Biological Assay; Biological Markers; Blood Vessels; Brain; Buffers; Cations; Cattle; Cell Count; Cell Culture Techniques; Cell Death; Cell Nucleus; Cell Survival; Cells; Characteristics; Choroidal Neovascularization; Chronic lung disease; Circular Dichroism Spectroscopy; Clinical; Collaborations; Column Chromatography; Conditioned Culture Media; Corneal Neovascularization; Cytolysis; Defect; Development; Developmental Bone Diseases; Dissection; Docking; Dysplasia; Enzyme-Linked Immunosorbent Assay; Enzymes; Exhibits; Extracellular Matrix; Eye; Family; Family suidae; Fatty Acids; Fluorescein-5-isothiocyanate; Fluorescence; Fracture; Generations; Genes; Growth; Human; Hydrolysis; Hyperoxia; Immunoprecipitation; In Situ; In Situ Nick-End Labeling; In Vitro; Inherited; Interest Group; LOX gene; Label; Length; Leukotriene B4; Life; Ligand Binding; Ligation; Light; Link; Lipoxygenase; Lipoxygenase Inhibitors; Longevity; Lung; Lysophospholipids; Maintenance; Mammals; Measures; Mediating; Membrane; Messenger RNA; Metabolism; Molecular; Mus; Mutation; N-terminal; Neonatal; Neoplasm Metastasis; Neuronal Differentiation; Osteogenesis Imperfecta; Oxidative Stress; Patients; Peptide Hydrolases; Peptides; Phospholipase; Phospholipase A2; Phospholipids; Photoreceptors; Physiologic calcification; Polyunsaturated Fatty Acids; Positioning Attribute; Predisposition; Premature Infant; Proteins; Protocols documentation; R peptide; RNA; Rattus; Reactive Oxygen Species; Recombinant Proteins; Recombinants; Relative (related person); Research; Retina; Retinal; Retinal Degeneration; Retinal Neovascularization; Reverse Transcriptase Polymerase Chain Reaction; Role; Sampling; Serpin Superfamily; Serum; Structure; Structure of retinal pigment epithelium; Structure-Activity Relationship; Testing; Transcript; Western Blotting; Work; angiogenesis; bone; cell fixing; design; expression vector; fetal; genetic manipulation; his6 tag; hypoxia neonatorum; in vivo; inhibitor/antagonist; interest; intravitreal injection; member; mouse model; mutant; neuronal survival; nexin; novel; null mutation; overexpression; pigment epithelium-derived factor; pigment epithelium-derived factor receptor; protective effect; protein protein interaction; receptor; research study; retinal ischemia; tumorigenesis; vasculogenesis

We completed studies on the characterization of the PEDF area that interacts with a distinct ectodomain on PEDF-R to promote photoreceptor survival. Molecular docking studies suggested that the ligand binding site of PEDF-R interacts with the neurotrophic region of PEDF (44mer, positions 78-121). Binding assays demonstrated that PEDF-R bound the 44mer peptide. Peptide P1 from the PEDF-R ectodomain had affinity for the 44mer and a shorter fragment within it, 17mer (positions 98-114). Single residue substitutions to alanine along the 17mer sequence were designed and tested for binding and biological activity. Altered 17merR99A did not bind to the P1 peptide, while 17merH105A had higher affinity than the unmodified 17mer. Peptides 17mer, 17merH105A and 44mer, exhibited cytoprotective effects in cultured retina R28 cells. Intravitreal injections of these peptides and PEDF in the rd1 mouse model of retinal degeneration decreased the numbers of dying photoreceptors, 17merH105A being most effective (in collaboration with Dr. V. Marigo). The blocking peptide P1 hindered their protective effects both in retina cells and in vivo. In addition to demonstrating that the region comprised of positions 98-114 of PEDF contains critical residues for PEDF-R interaction that mediates survival effects, the findings reveal distinct small PEDF fragments with neurotrophic effects on photoreceptors. The cytoprotective activity of PEDF peptides 17mer and 17merH105A was evaluated in the presence of selective and competitive catalytic inhibitor of PEDF-R, atglistatin. No decrease in the TUNEL positive cells was observed with PEDF or peptides in the presence of atglistatin, demonstrating that PLA activity of PEDF-R is critical for PEDF peptide mediated antiapoptotic effect. Live R28 cells were labeled with FITC-PEDF peptides in increasing concentrations. Cells were lysed in RIPA buffer and samples were collected and the fluorescence intensity was measured using SpectraMAX plus. Expression vectors for full-length human PEDF with single alterations at R99A and H105A and a FLAG-tag at their N-end were constructed and transfected into BHK cells. The recombinant proteins were purified from conditioned media by a two-step cation- and anion-exchange column chromatography protocol. PEDF was followed by SDS-PAGE, western blots and ELISA, and purity reached >90% pure. The purified proteins were analyzed by circular dichroism spectroscopy to compare secondary structure and the mutant proteins share similar fold. They were tested for binding to the PEDF-R peptides P1 and E5b, and for biological activity in cell cultures and in vivo. FLAG-PEDFH105A had cytoprotective activity on R28 cells and on photoreceptors of the rd1 mouse model (collaboration with Dr. V Marigo), while PEDFR99A did not. We continued the studies on a novel inhibitor of lipoxygenase discovered from a PEDF-R region, namely P1 peptide, which we showed previously binds to 5-LOX and protects ARPE-19 cells from cell death due to oxidative stress. 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. The effect of PNPLA2 genetic manipulation under oxidative stress was evaluated. Commercially purchased human PNPLA2 and 5-LOX siRNAs were tested in ARPE-19 cells. RT-PCR and western blots were performed to confirm decrease in the expression of PNPLA2 and 5-LOX genes and PEDF-R and 5-LOX proteins in transfected cells. An expression vector designed in our lab was used to transfect ARPE-19 and overexpression was confirmed by RT-PCR and westerns of ARPE-19 cells. Relative cell numbers were quantified using two different biomarkers for live and dead cells, intracellular ATP content and dead cell protease activity in cells subject to oxidative stress. The results from above experiments showed that both PEDF-R and peptide P1 increased the viability and decreased the number of dead cells under oxidative stress. Relative dead cell numbers were also quantitated in oxidative stress assay in the presence of scrambled peptide P1, obtained by random rearrangement of peptide P1. The effect of chemically synthesized small peptides spanning the PEDF-R ectodomain Leu159-Met325 on LOX activity was assessed. The scrambled peptide P1 was used as a control and P1 peptide was the only one with inhibitory activity against LOX. Binding of the mammalian full-length PEDF-R to 5-LOX was tested using pull-down experiments with differentially tagged proteins. Full-length PEDF-R tagged with N-terminal V5 tag and 5-LOX with N-terminal FLAG tag were co-expressed in ARPE-19 cells. Immunoprecipitation with FLAG antibody followed by probing with V5 antibody and vice versa were performed. In situ proximity ligation assay was performed to visualize protein-protein interactions in fixed cells. Given that LTB4 is a product of the 5-LOX enzymatic activity, the concentration of the secreted LTB4 was measured in the media from cells (ARPE-19 and pig RPE) by ELISA as a way of determining the 5-LOX activity in cells. We started to study another member of the serpin superfamily, protease nexin-1 (PN-1). PN-1 is encoded by the SERPINE2 gene and has neurotrophic effects in the brain and inhibits angiogenesis in the retina, resembling the activities of PEDF. The principal objective of this study is to compare PN-1 and PEDF as potential neuroprotectors for the retina. Murine RPE and retinal RNA was obtained from dissected C57BL/6N Crb1rd8 mouse eyes. Bovine protein lysates were obtained from bovine eye dissection. Serpine2 transcript and PN-1 protein levels were evaluated by RT-PCR and Western blotting, respectively. Recombinant human PN-1 and PEDF proteins were used. Biological activity in rat-derived retinal precursor (R28) cells was assessed with TUNEL cell death assays. PEDF-R was synthesized in vitro. Binding was evaluated through pulldown assays with full-length His6 Tag-PEDF-R and peptide affinity chromatography to peptide E5b derived from PEDF-R. Our results revealed that PN-1 is analogous in primary and tertiary structure to PEDF, and contains a region that shares homology with the neurotrophic active region of PEDF. SERPINE2 mRNA was detected in ARPE-19 cells, native human fetal RPE and retina, and murine RPE and retina. PN-1 protein was detected in ARPE-19 lysate, and extracted from the extracellular matrix (ECM), particularly bovine interphotoreceptor matrix (IPM), with increased NaCl. Low concentrations of PN-1 reduced TUNEL-positive nuclei in serum-starved R28 cells relative to untreated cells. 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 (bone-restricted ifitm-like protein), cause type V osteogenesis imperfecta. We continued the collaborative study investigating the role of PEDF in type V OI with clinical characteristics of type VI OI. RT-PCR and westerns of ARPE-19 cells, dissected mouse RPE and retina were performed and the results showed that BRIL is present in the RPE and the retina. Broncopulmonary dysplasia is a chronic lung disease of preterm infants characterized by arrested microvascularization and alveolarization. We completed the 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.

我们完成了有关PEDF区域的表征的研究，该研究与PEDF-R上的独特外生域相互作用以促进感光器的存活率。分子对接研究表明，PEDF-R的配体结合位点与PEDF的神经营养区域相互作用（44mer，位置78-121）。结合测定表明PEDF-R结合了44mer肽。来自PEDF-R外生域的肽P1对44mer和较短的片段具有亲和力，17mer（位置98-114）。设计并测试了沿17mer序列的丙氨酸的单个残基取代，以进行结合和生物学活性。改变的17merr99a与P1肽没有结合，而17merh105a的亲和力高于未修饰的17mer。肽17mer，17merh105a和44mer在培养的视网膜R28细胞中表现出细胞保护作用。在视网膜变性的RD1小鼠模型中对这些肽和PEDF的玻璃体内注射减少了垂死的光感受器的数量，17merh105a是最有效的（与V. Marigo博士合作）。阻塞肽P1阻碍了视网膜细胞和体内的保护作用。除了证明由PEDF的98-114位置组成的区域还包含介导生存效应的PEDF-R相互作用的关键残基外，这些发现揭示了具有神经营养效应对感光体的独特小PEDF碎片。 在PEDF-R Atglistatin的选择性和竞争性催化抑制剂的存在下，评估了PEDF肽17mer和17merh105a的细胞保护活性。在Atglistatin存在下，用PEDF或肽观察到TUNEL阳性细胞的降低，这表明PEDF-R的PLA活性对于PEDF肽介导的抗凋亡效应至关重要。 Live R28细胞用FITC-PEDF肽标记为浓度越来越高。将细胞裂解在RIPA缓冲液中，并收集样品，并使用Spectramax Plus测量荧光强度。 在R99a和H105a处进行单个变化的全长人PEDF的表达向量以及在其N端的标志标签被构造并转染到BHK细胞中。通过两步阳离子和阴离子 - 交换柱色谱方案从条件培养基中纯化重组蛋白。 PEDF之后是SDS-PAGE，Western印迹和ELISA，纯度达到了90％。通过圆形二色性光谱分析纯化的蛋白质，以比较二级结构，而突变蛋白具有相似的折叠。测试了它们与PEDF-R肽P1和E5B的结合，以及细胞培养物和体内的生物学活性。 FLAG-PEDFH105A在R28细胞和RD1小鼠模型的光感受器上具有细胞保护活性（与V Marigo博士的协作），而PEDFR99A则没有。 我们继续对从PEDF-R区域发现的新型脂氧酶抑制剂（即P1肽）进行研究，即我们先前与5-lox结合，并保护ARPE-19细胞由于氧化应激引起的细胞死亡。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。评估了氧化应激下PNPLA2遗传操作的影响。在ARPE-19细胞中测试了商业购买的人类PNPLA2和5-lox siRNA。进行了RT-PCR和Western印迹，以确认转染细胞中PNPLA2和5-LOX基因的表达和PEDF-R和5-LOX蛋白的表达降低。在我们实验室中设计的表达载体用于转染ARPE-19，并通过RT-PCR和ARPE-19细胞的西方证实过表达。使用两个不同的生物标志物，用于活细胞和死细胞，细胞内ATP含量以及受氧化应激的细胞中的死细胞蛋白酶活性来定量相对细胞数。上述实验的结果表明，PEDF-R和肽P1均增加了活力，并减少了氧化应激下的死细胞数量。在存在加毒性肽P1的情况下，在氧化应激测定中还定量相对死细胞数，并通过随机重排肽P1获得。 评估了跨越PEDF-R外生域LEU159-MET325化学合成的小肽对LOX活性的影响。加扰的肽P1用作对照，P1肽是唯一具有抑制活性的对照。使用具有差异标记蛋白的下拉实验测试了哺乳动物全长PEDF-R与5-Lox的结合。在ARPE-19细胞中共表达了用N末端V5 TAG和5-LOX标记的全长PEDF-R。用FLAG抗体进行免疫沉淀，然后用V5抗体进行探测，反之亦然。进行原位接近连接测定法以可视化固定细胞中的蛋白质 - 蛋白质相互作用。鉴于LTB4是5-lox酶活性的产物，因此通过ELISA从细胞（ARPE-19和Pig RPE）中测量了分泌的LTB4的浓度，作为确定细胞中5-LOX活性的一种方式。 我们开始研究Serpin超家族蛋白酶Nexin-1（PN-1）的另一个成员。 PN-1由SERPINE2基因编码，在大脑中具有神经营养作用，并抑制视网膜中的血管生成，类似于PEDF的活性。这项研究的主要目的是将PN-1和PEDF作为视网膜的潜在神经保护剂。从解剖的C57BL/6N CRB1RD8小鼠眼睛获得鼠RPE和视网膜RNA。牛蛋白裂解物是从牛眼剖分获得的。通过RT-PCR和Western印迹评估SERPINE2转录本和PN-1蛋白水平。使用重组人PN-1和PEDF蛋白。用TUNEL细胞死亡测定法评估了大鼠衍生的视网膜前体（R28）细胞中的生物学活性。 PEDF-R在体外合成。通过具有全长His6 tag-PEDF-R和肽亲和色谱与源自PEDF-R的肽E5B的肽亲和色谱法的下拉测定法评估结合。我们的结果表明，PN-1在PEDF的主要和第三纪结构中是类似的，并且包含一个与PEDF的神经营养活性区共有同源性的区域。在ARPE-19细胞，人类胎儿RPE和视网膜以及鼠RPE和视网膜中检测到Serpine2 mRNA。在ARPE-19裂解物中检测到PN-1蛋白，并从细胞外基质（ECM）中提取，尤其是牛型中的牛间基质基质（IPM），NaCl增加。低浓度的PN-1相对于未处理的细胞，在血清饥饿的R28细胞中降低了TUNEL阳性核。 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，编码黑质（骨限制IFITM样蛋白），导致型肌生成型不完美。 我们继续进行协作研究，研究了PEDF在V型OI中具有VI型临床特征的作用。进行了RT-PCR和ARPE-19细胞的西部，解剖的小鼠RPE和视网膜，结果表明RPE和视网膜中存在黑色。 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。我们完成了协作研究，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部相比，PEDF水平升高。 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。