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: 8556813
• 负责人: SOFIA P BECERRA
• 金额: $ 75.49万
• 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/8556813
• 关键词: ATP Synthesis Pathway; Affect; Affinity; Affinity Chromatography; Alanine; Amines; Angiogenesis Inhibitors; Animal Model; Anions; Antibodies; Apoptosis; Area; Attenuated; Binding; Biochemical; Biological; Biological Assay; Biological Markers; Biotin; Biotinylation; Breast Cancer Cell; Cancer cell line; Cations; Cause of Death; Cell Differentiation process; Cell Fraction; Cell Line; Cell Nucleus; Cell Survival; Cell membrane; Cell surface; Cells; Cessation of life; Charge; Choroidal Neovascularization; Circular Dichroism; Collaborations; Column Chromatography; Corneal Neovascularization; Development; Diffuse; Docking; Endothelial Cells; Epitope Mapping; Epitopes; Evaluation; Exhibits; Fatty Acids; Fluorescein; Fluorescein-5-isothiocyanate; Heparin; Image; Immunoblotting; In Vitro; Inherited; Ionic Strengths; Isoelectric Focusing; Label; Length; Life; Ligands; Light; Link; Longevity; Maintenance; Malignant Epithelial Cell; Malignant neoplasm of urinary bladder; Mammals; Maps; Measures; Mediating; Membrane; Membrane Proteins; Microscopic; Modeling; Molecular; Molecular Weight; Monitor; Neoplasm Metastasis; Neuronal Differentiation; Pattern; Peptides; Phospholipase; Phospholipase A2; Phospholipids; Photoreceptors; Piceatannol; Plant Resins; Property; Protein Isoforms; Proteins; Proteolysis; Pyroxylin; Reagent; Recombinants; Relative (related person); Research; Retina; Retinal; Retinal Degeneration; Retinal Ganglion Cells; Retinal Neovascularization; Reverse Transcriptase Polymerase Chain Reaction; Role; Scanning; Series; Serpin Superfamily; Serum; Small Interfering RNA; Sodium Dodecyl Sulfate-PAGE; Staining method; Stains; Starvation; Streptavidin; Structural Models; Structure; Structure of retinal pigment epithelium; Structure-Activity Relationship; Sulfhydryl Compounds; Surface; Surface Plasmon Resonance; System; Testing; Time; Transcript; Tube; Tumor Cell Line; Western Blotting; Work; Wound Healing; axon growth; bladder Carcinoma; cell motility; cytotoxic; design; electric impedance; extracellular; immunoreactivity; inhibitor/antagonist; interest; member; neoplastic cell; neuronal survival; novel; pigment epithelium-derived factor; pigment epithelium-derived factor receptor; polypeptide; protein expression; protein structure; receptor; receptor expression; relating to nervous system; research study; retinal ischemia; sensor; synthetic peptide; tumor; tumorigenesis; vector

PEDF is a member of the serpin superfamily. It has high affinity for pigment epithelium-derived factor receptor (PEDF-R), a patatin-like phospholipase domain-containing 2 (PNPLA2) protein identified in the retina. PEDF-R is a membrane-linked phospholipase, and PEDF binding stimulates its enzymatic phospholipase A2 activity, which catalyzes the cleavage of fatty acids from phospholipids. A study on the identification of epitopes of an antibody to PEDF-R was completed. We mapped the epitopes using recombinant PEDF-R polypeptides fragments and synthetic peptides, and we identified peptides that specifically block its immunoreactivity. We continued working on the characterization of PEDF-R. His-tagged PEDF-R, and His-tagged C-terminally and internally truncated PEDF-R polypeptides (PEDFR203-232, PEDFR4 and PEDFR4203-232) were prepared with an in vitro protein expression system. We performed His-tag pull-down assays and found that His-tagged PEDF-R and His-tagged PEDF-R4 bound fluorescein conjugated PEDF, but His-tagged PEDFR203-232 and His-tagged PEDF-R4193-232 did not. We also performed cell surface biotinylation of retina R28 cells using a membrane impermeable, thiol cleavable, amine reactive sulfo-N-hydroxysuccinimide-SS-biotin reagent. Membrane proteins were isolated and subsequently the biotinylated proteins were fractionated using strepavidin-affinity resins. Western blot of the biotinylated proteins detected PEDF-R among the membrane R28 proteins, demonstrating that PEDF-R was present in the surface of R28 cells. Furthermore, we tested the capacity of PEDF-R siRNAs to decrease the expression of PEDF-R in R28 cells. RT-PCR was performed and revealed a significant decrease in the PEDF-R transcript levels in cells transfected with the PEDF-R siRNA. Western blots of membrane protein fractions were performed and showed that the PEDF-R protein levels decreased with the silencing vectors in the membrane of R28 cells. We also evaluated cell survival of serum-starved R28 cells using intracellular ATP as a biomarker for live cells. We found that PEDF did not increase the viability in cells in which PEDF-R had been silenced relative to the untransfected or scrambled siRNA-transfected cells. Using TUNNEL staining, we evaluated the apoptosis in R28 cells and found that PEDF did not decrease the TUNNEL positive nuclei number when the PEDF-R expression had been silenced relative to untransfected or scrambled siRNA-transfected cells. PEDF can induce the expression of Bcl2 in R28 cells. Bcl2 expression levels were also measured by RT-PCR in untransfected and siRNA-transfected cells to silence PEDF-R, and without and with PEDF in the presence of PEDF-R derived peptides as blocking peptides. PEDF was assayed for axon growth and survival of retinal ganglion cells, in collaboration with Dr. Yuqin Yin. PEDF protected retina cells against death and promoted axon growth of retinal ganglion cells, and a PEDF-binding peptide corresponding to the PEDF binding region of PEDF-R was an effective blocking agent for the PEDF-mediated axon growth activities. A study to identify PEDF isoforms was completed. Recombinant PEDF proteins were purified by cation- and anion-exchange column chromatography, and subjected to SDS-polyacrylamide gel electrophoresis, isoelectric focusing, deglycosylation, heparin affinity chromatography, and limited proteolysis. Cell viability, real-time electrical impedance of cells, and wound healing assays were performed using bladder and breast cancer cell lines, and retinal R28, and ARPE-19 cells. We found two distinct PEDF protein peaks after anion-exchange column chromatography: PEDF-1 eluting with lower ionic strength than PEDF-2. PEDF-1 protein had higher pI value and lower apparent molecular weight than PEDF-2 protein. Both PEDF forms were glycosylated, bound to heparin, and had identical patterns by limited proteolysis. However, PEDF-2 emerged as being highly potent in lowering cell viability in all tumor cell lines tested, and in inhibiting tumor and ARPE-19 cell migration. In contrast, PEDF-1 minimally affected tumor cell viability and cell migration but protected R28 cells against death caused by serum starvation. We concluded that the identification of two distinct biochemical forms of PEDF varying in overall charge and with distinct biological effects on tumor cell viability and migration may explain the multimodal functionality of the PEDF protein. We also completed a study on the inhibition of tumor cell surface ATP synthesis by PEDF. Incubation of urinary bladder carcinoma cell lines in media containing recombinant PEDF protein for 48-96 h dramatically decreased cell viability in a concentration-dependent fashion as monitored by real-time cell impedance with a microelectronic system, microscopic imaging and biomarkers of live cells. Intact tumor cells exhibited cell surface ATP synthesis activity, which was inhibited by piceatannol, a specific inhibitor of F1/F0-ATP synthase. Immunoblotting revealed that the subunit of F1-ATP synthase was present in plasma membrane fractions of these cells. Pre-incubation of tumor cells with PEDF inhibited the activity of cell surface ATP synthase in a concentration-dependent fashion. The PEDF-derived peptide 34-mer decreased tumor cell viability and inhibited extracellular ATP synthesis to the same extent as full-length PEDF. Moreover, ATP additions attenuated both the PEDF-mediated decrease in tumor cell viability and the inhibition of endothelial cell tube formation. The findings imply that PEDF is a novel inhibitor of tumor cell surface ATP synthase activity that exhibits a cytotoxic effect on tumor cells, and that the structural determinants for these properties are within the peptide region 34-mer of the PEDF polypeptide. Two biologically active regions have been identified in PEDF: an antiangiogenic 34-mer peptide and a neurotrophic 44-mer peptide. We began studies toward mapping the region in PEDF that binds to PEDF-R. The structure of a peptide P1 containing the PEDF binding region of PEDF-R was predicted in an ab initio model, and the resultant structure was then used in a global docking search using Rosetta, with a PEDF structural model. Evaluation of P1 by circular dichroism, indicated that P1 is composed of -helical structure. Peptides from PEDF neurotrophic and antiangiogenic regions, and P1 were chemically synthesized and purified. Binding was determined by ligand blotting of PEDF-R peptides immobilized on nitrocellulose membrane with FITC labeled PEDF ligands. Ligand blot of immobilized P1 to Fl-PEDF, FITC-conjugated-34-mer and FITC-44-mer revealed that PEDF bound to soluble and immobilized P1, and that the neuroprotective 44-mer, but not the antiangiogenic 34-mer bound to P1. Surface plasmon resonance with P1 sensor chips showed that only the 44-mer bound to P1 in real time like PEDF. Pull-down experiments with in vitro synthesized His-tagged PEDF-R revealed that the 44-mer and not the 34-mer bound to PEDF-R polypeptides. A smaller peptide derived from the central region of the 44-mer, namely a 17-mer, shares neurotrophic activity with PEDF. We performed ligand blot of P1 to FITC-17-mer peptide and found that the smaller 17-mer peptide is sufficient for binding PEDF-R. We designed a series of peptides from the 17-mer by alanine scanning in which one by one residue was substituted with an alanine. We observed that some residues of 17-mer were more critical for binding P1, and others enhanced P1 binding when altered to alanine. The results imply that the neuroprotective region of PEDF binds to PEDF-R and support the role of PEDF-R as a neurotrophic receptor.

PEDF是Serpin超家族的成员。 它对色素上皮衍生的因子受体（PEDF-R）具有高亲和力，这是一种含有patatin的含磷脂酶结构域2（PNPLA2）蛋白，在视网膜中鉴定出来。 PEDF-R是一种膜连接的磷脂酶，PEDF结合刺激其酶促磷脂酶A2活性，可促进脂肪酸从磷脂的裂解。 完成了一项关于鉴定PEDF-R抗体表位的研究。 我们使用重组PEDF-R多肽片段和合成肽映射了表位，并确定了特异性阻断其免疫反应性的肽。 我们继续研究PEDF-R的表征。 用体外蛋白质表达系统制备了他的标记为PEDF-R，以及他标记的C端和内部截短的PEDF-R多肽（PEDFR203-232，PEDFR4和PEDFR4和PEDFR4203-232）。 我们进行了HIS标签下拉测定法，发现他标记的PEDF-R和他标记的PEDF-R4结合荧光素结合的PEDF，但是他标记的PEDFR203-232和他的标签PEDF-R4193-232没有。 我们还使用膜不渗透，可裂解的，可裂解的胺反应性硫酸-N-羟基糖酰亚胺 - 二酰亚胺 - 二酰亚胺 - 生物素试剂对视网膜R28细胞进行了细胞表面生物素化。 分离膜蛋白，随后使用片状质素亲和力树脂分离生物素化蛋白。 生物素化蛋白的蛋白质印迹在膜R28蛋白中检测到PEDF-R，表明PEDF-R存在于R28细胞表面。 此外，我们测试了PEDF-R siRNAs降低R28细胞中PEDF-R表达的能力。 进行了RT-PCR，并揭示了用PEDF-R siRNA转染的细胞中PEDF-R转录水平的显着降低。 进行了膜蛋白分数的蛋白质印迹，并表明PEDF-R蛋白水平随R28细胞膜中的沉默载体而降低。 我们还使用细胞内ATP作为活细胞的生物标志物评估了血清饥饿的R28细胞的细胞存活。 我们发现，相对于未转染或加扰的siRNA转染的细胞，PEDF并没有增加pEDF-R被沉默的细胞活力。 使用隧道染色，我们评估了R28细胞中的凋亡，发现当PEDF-R表达相对于未转染或炒作的siRNA转染细胞时，PEDF并没有减少隧道正核数。 PEDF可以诱导R28细胞中BCL2的表达。 还通过RT-PCR在未转染和siRNA转染的细胞中通过RT-PCR测量BCl2表达水平，以使PEDF-R沉默，并且在存在PEDF-R衍生的肽存在的情况下，没有PEDF作为阻断肽。 与Yuqin Yin博士合作，对PEDF进行了视网膜神经节细胞的轴突生长和存活。 PEDF保护的视网膜细胞免受死亡并促进视网膜神经节细胞的轴突生长，而与PEDF-R的PEDF结合区域相对应的PEDF结合肽是PEDF介导的轴突生长活性的有效阻断剂。 一项识别PEDF同工型的研究完成了。 重组PEDF蛋白通过阳离子和阴离子 - 交换柱色谱法纯化，并经过SDS-聚丙烯酰胺凝胶电泳，等电聚焦，脱胶糖基化，肝素亲和力色谱和有限的蛋白质解。 细胞活力，细胞的实时电阻抗以及使用膀胱和乳腺癌细胞系以及视网膜R28和ARPE-19细胞进行伤口愈合测定。 我们在阴离子交换柱色谱法之后发现了两个不同的PEDF蛋白峰：与PEDF-2相比，离子强度低的PEDF-1洗脱。 PEDF-1蛋白具有比PEDF-2蛋白更高的PI值和明显的分子量。 两种PEDF形式都是糖基化的，与肝素结合，并具有有限的蛋白水解的相同模式。 然而，PEDF-2在所有测试的肿瘤细胞系以及抑制肿瘤和ARPE-19细胞迁移方面的降低细胞活力方面具有很高的能力。 相反，PEDF-1最小影响肿瘤细胞活力和细胞迁移，但保护R28细胞免受血清饥饿引起的死亡。 我们得出的结论是，鉴定PEDF的两种不同的生化形式在总体电荷上不同，并且对肿瘤细胞活力和迁移的生物学作用有明显的作用，这可能解释了PEDF蛋白的多峰功能。 我们还完成了一项有关PEDF抑制肿瘤细胞表面ATP合成的研究。 在含有重组PEDF蛋白的培养基中孵育膀胱癌细胞系48-96小时，以浓度依赖性的方式急剧降低细胞活力，并通过微电池系统，微观成像和活细胞的生物标志物通过实时细胞阻抗监测。 完整的肿瘤细胞表现出细胞表面ATP合成活性，该合成活性受到F1/F0-ATP合酶的特异性抑制剂Piceatannol抑制。 免疫印迹表明，F1-ATP合酶的亚基存在于这些细胞的质膜部分中。 肿瘤细胞与PEDF的预孵育以浓度依赖性方式抑制细胞表面ATP合酶的活性。 PEDF衍生的肽34-mer降低了肿瘤细胞活力，并抑制了细胞外ATP合成，其程度与全长PEDF相同。 此外，ATP的添加减弱了PEDF介导的肿瘤细胞活力的降低和内皮细胞管形成的抑制。 研究结果表明，PEDF是一种新型的肿瘤细胞表面ATP合酶活性的抑制剂，对肿瘤细胞表现出细胞毒性作用，并且这些特性的结构决定因素在PEDF多肽的肽区域内34-MER内。 在PEDF中已经确定了两个具有生物活性的区域：一种抗血管生成的34-mer肽和神经营养44-mer肽。 我们开始研究与PEDF-R结合的PEDF中的区域。 从头算模型中预测了含有PEDF-R的PEDF结合区域的肽P1的结构，然后在使用Rosetta的全局对接搜索中使用了所得的结构，并带有PEDF结构模型。 通过圆形二科主义评估P1，表明P1由螺旋结构组成。 来自PEDF神经营养和抗血管生成区域以及P1的肽是化学合成和纯化的。 结合是通过用FITC标记的PEDF配体在硝酸纤维素膜上的PEDF-R肽的配体印迹确定的。 固定的P1的配体印迹与FL-PEDF，FITC偶联-34-mer和FITC-44-MER表明，PEDF与可溶性和固定的P1结合，而神经保护性44-mer，但没有与P1结合的抗雄激素34-MER。 带有P1传感器芯片的表面等离子体共振显示，只有44-mer像PEDF一样实时与P1结合。 体外合成的HIS标记的PEDF-R进行了下拉实验表明，与PEDF-R多肽结合的44-mer而不是34-mer。 源自44-mer的中央区域的较小肽，即17-mer，与PEDF具有神经营养活性。 我们对P1的配体印迹对FITC-17-MER肽进行了，发现较小的17-Mer肽足以结合PEDF-R。 我们通过丙氨酸扫描从17-MER设计了一系列肽，其中一个残基用丙氨酸取代。 我们观察到，某些17-MER的残基对于结合P1更为重要，而另一些残基在改变与丙氨酸时会增强P1结合。 结果表明，PEDF的神经保护区与PEDF-R结合并支持PEDF-R作为神经营养受体的作用。