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: 10706097
• 负责人: SOFIA P BECERRA
• 金额: $ 72.81万
• 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/10706097
• 关键词: ANXA5 gene; Ablation; Acetylation; Age related macular degeneration; Age-Months; Aging; Amino Acids; Angiogenesis Inhibitors; Animal Model; Apical; Apoptosis; BCL-2 Protein; BODIPY; Bax protein; Binding; Biological Assay; Bromodeoxyuridine; CDKN1A gene; Cations; Cattle; Cell Aging; Cell Culture Techniques; Cell Death; Cell Nucleus; Cell Survival; Cells; Cessation of life; Chemicals; Choroid; Choroidal Neovascularization; Chromatin; Code; Collaborations; Colorado; Column Chromatography; Complementary DNA; Confocal Microscopy; Corneal Neovascularization; Crystallization; Culture Media; Development; Developmental Process; Enzyme-Linked Immunosorbent Assay; Escherichia coli; Evaluation; Exposure to; Eye; Family; Flow Cytometry; Fluorescent Probes; Formazans; Functional disorder; GLB1 gene; Galactosidase; Genes; Genetic Transcription; H2AFX gene; Human; Hydrolysis; Hydroxybutyrates; Immunofluorescence Immunologic; In Situ Nick-End Labeling; Inclusion Bodies; Incubated; Inflammation; Inherited; Interest Group; Intervention; Ion Exchange; Knockout Mice; Laboratories; Lactate Dehydrogenase; Length; Ligands; Light; Link; Lipase; Lipids; Longevity; Maintenance; Mammals; Measures; Mediating; Membrane; Microscopy; Mitochondria; Molecular; Morphology; Mus; Mutate; National Institute of Diabetes and Digestive and Kidney Diseases; Neoplasm Metastasis; Neurites; Neuronal Differentiation; Neurons; Nuclear; Opsin; Oxidative Stress; Peptide Fragments; Peptides; Phagocytes; Phagocytosis; Phosphatidylserines; Phospholipase; Phospholipids; Photoreceptors; Positioning Attribute; Production; Propidium Diiodide; Proteins; RNA; Rattus; Recombinants; Regulation; Research; Retina; Retinal Degeneration; Retinal Neovascularization; Retinal Pigments; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin; Role; Serpin Superfamily; Serum-Free Culture Media; Stains; Structure of retinal pigment epithelium; TNF gene; Triglycerides; Urea; Variant; Western Blotting; Work; albino rat; blocking factor; coronavirus disease; cytokine; cytokine release syndrome; cytotoxicity; design; expression vector; genetic regulatory protein; in vitro Model; inhibitor; member; milligram; monolayer; neuronal survival; neurotrophic factor; pigment epithelium-derived factor; pigment epithelium-derived factor receptor; potential biomarker; preservation; prevent; receptor; response; retina outer nuclear layer; retinal damage; retinal ischemia; retinal neuron; screening; senescence; synthetic peptide; tumorigenesis; vector; zaprinast

A study to evaluate the neurotrophic effects of PEDF and its fragments in an in vitro model of cultured primary retinal neurons that die spontaneously in the absence of trophic factors was completed. We used Wistar albino rats. Cell death was assayed by immunofluorescence and flow cytometry using TUNEL, BrDU, propidium iodide, mitotracker, and annexin V. Immunofluorescence of cells for visualizing rhodopsin, CRX, and antisyntaxin under confocal microscopy was performed. Neurite outgrowth was also quantified. Results show that PEDF protected photoreceptor precursors from apoptosis, preserved mitochondrial function and promoted polarization of opsin enhancing their developmental process, as well as induced neurite outgrowth in amacrine neurons. These effects were abolished by an inhibitor of the PEDF receptor or receptor-derived peptides that block ligand/receptor interactions. While all the activities were specifically conferred by short peptide fragments (17 amino acid residues) derived from the PEDF neurotrophic domain, no effects were triggered by peptides from the PEDF antiangiogenic region. The observed effects on retinal neurons imply a specific activation of the PEDF receptor by a small neurotrophic region of PEDF. In addition, cytotoxicity assay was performed measuring the formazan formation due to lactate dehydrogenase activity released to the media by cells. Bcl2 and Bax proteins were also examined by western blotting of cell lysates and mitochondrial fractions of the rat primary cultures treated with and without the effectors. A study on expression and production of recombinant human PEDF and PEDF-R variants by mammalian and bacterial cells was completed. To obtain large amounts of recombinant PEDF proteins, we subcloned the coding sequence of human SERPINF1 mutated versions into the pCEP4 vector and generated stably transfected HEK.Ebna cells. Recombinant PEDF protein production and secretion was detected by SDS-PAGE and western blotting of the culturing media. The recombinant PEDF proteins were purified by ion-exchange column chromatography and milligram amounts of highly purified protein were recovered. Recombinant PEDF-R truncated versions were obtained from Escherichia coli containing expression vectors with human PNPLA2 cDNAs with 3'end deletions and by induction with isopropyl -d-1-thiogalactopyranoside. The bacterially derived PEDF-R proteins in insoluble inclusion bodies were solubilized with urea and purified by cation-exchange column chromatography. PEDF binding assays of the recombinant PEDF-R versions to bind PEDF were performed. Cells expressing for a full-length human PEDF version with a single point alteration at H105A were cultured at large scale, and the proteins secreted from the cells into the serum-free media were collected, concentrated, and dialyzed (in collaboration with the Y Shiloach laboratory). The proteins were purified using a two-step ion-exchange column chromatography. The purified PEDF protein combined with a chemically synthesized peptide fragment of the ectodomain of PEDF-R was used in high-throughput crystallization screening (in collaboration with V Sagar in the Wistow laboratory). Expression and production of recombinant human PEDF-R versions in E. coli cells were performed. Conditions for overproduction, solubilization and purification were optimized. A study to examine the effects of PEDF deletion on the induction of cellular senescence in RPE cells was completed. We determined the expression of senescence-associated genes in the RPE of 3-month-old mice that lack the Serpinf1 gene by RT-PCR and found that Serpinf1 expression induced H2ax for histone H2AX protein, Cdkn1a for p21 protein, and Glb1 gene for -galactosidase. Senescence-associated -galactosidase activity increased in the Serpinf1 null RPE when compared with wild-type RPE in RPE/choroid flatmounts. Evaluation of the RPE subcellular morphology showed that ablation of Serpinf1 increased the volume and the number of the nuclei of RPE cells, implying chromatin reorganization. Given that the RPE phagocytic function declines with aging, we assessed the expression of the Pnpla2 gene by RT-PCR and PEDF-R protein levels by immunofluorescence and found that both declined with the Serpinf1 gene ablation. Determination of rhodopsin and lipid accumulation by immunofluorescence and BODIPY staining in the RPE flatmounts was performed. The levels of both rhodopsin and lipids in the RPE of the Serpinf1 null mice accumulated compared to littermate controls, implying an association of PEDF deficiency with RPE phagocytosis dysfunction. The findings established PEDF loss as a cause of senescence-like changes in the RPE, highlighting PEDF as both a retinoprotective and a regulatory protein of aging-like changes associated with defective degradation of the photoreceptor outer segment in the RPE. We examined the polarization of PEDF secretion and functionality of induced-primary RPE (ipRPE) monolayers of cells in transwells cultured for 15-50 days (collaboration with the laboratory of Valeria Canto-Soler, U Colorado). The PEDF concentration was measured in the apical and basal compartments of the transwells by ELISA and western blotting. The capacity of ipRPE monolayers to internalize and degrade photoreceptor outer segments during phagocytosis was assessed. We determined the amount of rhodopsin in cell lysates and the amount of hydroxybutyrate secreted to the culturing media of cells that had been exposed to bovine outer segments. We continued investigating the regulation of CRX by PEDF during photoreceptor survival. Serpinf1-/-, Serpinf1+/- and Serpinf1+/+ mice at 3 months of age were used. We prepared cultures of mouse retinal explants and treated them with recombinant human PEDF protein. The transcriptional levels of Crx, Pde6a, Opn1mw and Opn1sw were assessed using RT-PCR. Subcellular distribution of CRX, PDE6A and pan-acetylation in the outer nuclear layer of the retinal explants from Serpinf1-/- and Serpinf1+/+ mice was detected by immunofluorescence. Zaprinast was added to the retinal explant cultures to induce photoreceptor death. A fluorescent probe that targets anionic phospholipids such as phosphatidylserine, was used to identify dying photoreceptor cells in the retinal explants from Serpinf1-/-, and Serpinf1+/+ mice treated with combinations of zaprinast and PEDF. Subcellular distribution of CRX was visualized and evaluated by immunofluorescence in the outer nuclear layer of Serpinf1-/- and Serpinf1+/+ retinal explants treated with combinations of zaprinast and PEDF. To study the effects of PEDF to inhibit the cytokine induction ARPE-19 cell cultures were incubated with recombinant human tumor necrosis factor alpha (TNF-) to induce interlukin-6 (IL-6). Cell viability was determined and followed by microscopy. The culture media was collected to determine the IL-6 protein concentration secreted by the cells. Cells were collected to isolate RNA for RT-PCR. To challenge the stimulation of IL-6, we added recombinant human PEDF protein to the cultures. Concentration-response relationships were assessed for TNF--mediated induction of IL-6 and for the PEDF blocking effects. We used synthetic peptides designed from the pro-death 34-mer (positions 44-77), and the pro-survival 44-mer (78-121) and 17-merH105A (98-114) of PEDF to challenge the IL-6 overproduction. Serpinf1-/- mice were bred for a study of PEDF as potential biomarker for intervention in COVID-16 during cytokine storm (collaboration with R Star and P Yuen, NIDDK).

一项研究旨在评估PEDF及其片段在没有营养因子的情况下自发死亡的培养的原发性神经元的体外模型中的神经营养作用。我们使用了Wistar白化大鼠。使用TUNEL，BRDU，碘化丙啶，Mitotracker和Annexin V通过免疫荧光和流式细胞仪测定细胞死亡。在共摄影显微镜下可视化细胞的免疫荧光，以可视化Rhodopsin，crx和抗异糖素。神经突生长也被定量。结果表明，PEDF保护的光感受器前体免受凋亡的影响，保留了线粒体功能，并促进了Opsin增强其发育过程的极化，以及氨基氨甲藻神经元中诱导的神经突生长。 PEDF受体或受体衍生的肽的抑制剂消除了这些作用，该抑制剂阻断了配体/受体相互作用。尽管所有活性都是由源自PEDF神经营养域的短肽片段（17个氨基酸残基）专门赋予的，但PEDF抗血管生成区域的肽不会触发任何效果。观察到的对视网膜神经元的作用暗示了PEDF的小神经营养区域对PEDF受体的特异性激活。此外，对细胞毒性测定进行测量，测量甲酸脱氢酶的活性，测量细胞释放给培养基的乳酸脱氢酶活性。还通过有或没有效应子处理的大鼠原发性培养物的细胞裂解物和线粒体馏分的蛋白质印迹检查了BCL2和BAX蛋白。 哺乳动物和细菌细胞的重组人PEDF和PEDF-R变体的表达和生产的研究完成了。为了获得大量的重组PEDF蛋白，我们将人serpinf1突变版本的编码序列取代到PCEP4载体中，并产生了稳定转染的HEK.EBNA细胞。通过SDS-PAGE和培养基的蛋白质印迹检测到重组PEDF蛋白的产生和分泌。通过离子交换柱色谱纯化重组PEDF蛋白，并回收了高度纯化的蛋白质。重组PEDF-R截断的版本是从含有3'End缺失的人PNPLA2 cDNA的表达载体的大肠杆菌中获得的，并通过异丙基-D-1- thiogalactopyranoside诱导。用尿素溶解了细菌衍生的PEDF-R蛋白，并通过阳离子交换柱色谱纯化。进行了重组PEDF-R版本以结合PEDF的PEDF结合测定。大规模培养了针对H105a培养具有单点改变的全长人PEDF版本的细胞，并收集，浓缩和透析（与Y Shiloach实验室合作），从细胞分泌到无血清的培养基中。使用两步离子 - 交换柱色谱法纯化蛋白质。纯化的PEDF蛋白与PEDF-R外生域的化学合成肽片段结合使用，用于高通量结晶筛选（与Wistow实验室中的V Sagar合作）。进行了大肠杆菌细胞中重组人PEDF-R版本的表达和产生。优化了过度生产，溶解和纯化的条件。 一项研究完成了PEDF缺失对RPE细胞细胞衰老诱导的影响的研究。我们确定了3个月大的小鼠的RPE中与衰老相关基因的表达，这些小鼠缺乏RT-PCR的SERPINF1基因，发现SERPINF1的表达诱导了P21蛋白和GLB1基因的组蛋白H2AX蛋白，CDKN1A的H2AX诱导H2AX。与RPE/Choroid Flatmounts中的野生型RPE相比，SERPINF1 NULL RPE的衰老相关 - 半乳糖苷酶活性增加。对RPE亚细胞形态的评估表明，serpinf1的消融增加了RPE细胞核的体积和数量，这意味着染色质的重组。鉴于RPE吞噬功能随老化而下降，我们通过免疫荧光评估了RT-PCR和PEDF-R蛋白水平的PNPLA2基因的表达，发现这两者都随SERPINF1基因烧蚀而下降。通过在RPE Flatmounts中通过免疫荧光和BODIPY染色来测定Rhodopsin和脂质的积累。与同窝对照组相比，SERPINF1 NULL小鼠RPE中的Rhodopsin和脂质的水平都意味着PEDF缺乏症与RPE吞噬作用障碍的关联。这些发现确立了PEDF损失是导致RPE衰老变化的原因，强调了PEDF是视网膜保护性和调节性蛋白质的衰老样变化的蛋白质，与RPE中光感受器外部段的降解有缺陷相关。 我们检查了培养的Transwells中诱导主要RPE（IPRPE）单层的PEDF分泌和功能的极化（与科罗拉多州瓦莱里亚·坎托·塞勒的实验室合作）。通过ELISA和Western印迹在Transwells的顶端和基底室中测量了PEDF浓度。评估了IPRPE单层在吞噬作用期间内化和降解光感受器外部段的能力。我们确定了细胞裂解物中的视紫红素的量以及分泌给牛外部片段的细胞的培养基的羟基丁酸含量。 我们继续研究感光器存活期间PEDF对CRX的调节。 Serpinf1 - / - ，使用3个月大时的Serpinf1 +/-和Serpinf1+/+小鼠。我们制备了小鼠视网膜外植体的培养物，并用重组人PEDF蛋白治疗它们。使用RT-PCR评估CRX，PDE6A，OPN1MW和OPN1SW的转录水平。通过免疫荧光检测到来自SERPINF1 - / - 和SERPINF1+/+小鼠的视网膜外植体的CRX，PDE6A和泛乙酰化的亚细胞分布。将Zaprinast添加到视网膜外植体培养物中，以诱导感光器死亡。靶向阴离子磷脂（如磷脂酰丝氨酸）的荧光探针被用来鉴定来自Serpinf1 - / - 的视网膜外植体中的垂死的感光细胞，以及用Zaprinast和Pedf组合处理的SERPINF1+/+小鼠。 CRX的亚细胞分布可视化并通过免疫荧光在Serpinf1 - / - 和Serpinf1+/+视网膜外植体的外部核层中进行评估，并用Zaprinast和Pedf组合处理。 为了研究PEDF抑制细胞因子诱导的作用，将ARPE-19细胞培养物与重组人肿瘤坏死因子α（TNF-）孵育，以诱导Interlukin-6（IL-6）。确定细胞活力，然后进行显微镜检查。收集培养基以确定细胞分泌的IL-6蛋白浓度。将细胞收集以分离RT-PCR的RNA。为了挑战IL-6的刺激，我们在培养物中添加了重组人PEDF蛋白。评估了浓度 - 响应关系的TNF介导IL-6和PEDF阻断效应的诱导。我们使用了由Pro-Death 34-Mer（位置44-77）设计的合成肽，以及PEDF的PRO-SURVIVAL 44-MER（78-121）和17-MERH105A（98-114）的合成肽来挑战IL-6的过量生产。培养了Serpinf1 - / - 小鼠，以研究PEDF作为细胞因子风暴期间COVID-16的潜在生物标志物（与R Star和P Yuen，NIDDK的合作）。