Role of Protein Interactions in Retina Development and F

National Eye Institute

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Abstract

Work in this research group is aimed at elucidating the mechanisms that regulate the activities of pigment epithelium-derived factor (PEDF), an extracellular serpin with neurotrophic and antiangiogenic activities.[unreadable] [unreadable] We continued working on the characterization of a putative receptor for PEDF, PEDF-R, with binding affinity for PEDF and phospholipase activity. We demonstrated that this novel protein is found in plasma membrane extracts of retina, retinal pigment epithelium and ARPE-19 cells, as an active phospholipase enzyme, which can be stimulated by PEDF ligands. We engineered cells overexpressing and silencing the PEDF-R gene, and constructed deletion versions of PEDF-R. We obtained additional evidence for its subcellular localization to the cell-surface of ARPE-19 cells by surface-biotinylation (in collaboration with Peggy Zelenka?s laboratory, NEI). [unreadable] [unreadable] We investigated the antiapoptotic and cell survival effects of PEDF on RPE and retina cell lines. Living cells were monitored by a label-free real-time assay using electronic cell sensor technology. Preincubation with PEDF increased the viability of cells induced to apoptosis by H2O2 exposure and/or serum starvation. Cell viability was also determined by MTS and by Cell-Titer Glo viability assays.[unreadable] [unreadable] We continued examining the interactions between PEDF and ectopic ATP synthase. We demonstrated that plasma membrane fractions from human microvascular endothelial cell (HMVEC) contained a component with affinity for PEDF, which reacted with ATP synthase beta subunit antibodies by surface plasmon resonance and western assays. Direct binding to highly purified yeast F1-ATP synthase with a His-tagged beta-subunit showed PEDF binding affinities similar to those with intact cells, which were higher than F1:angiostatin K1-5 interactions. We performed competition for F1 binding assays with angiostatin, which showed that PEDF efficiently competed with K1-5 for F1 binding. We performed ATP synthesis activity assays on HMVECs and found that PEDF inhibited significantly the extracellular ATP synthesis activity of endothelial cells as did angiostatin and piceatannol. [unreadable] [unreadable] We developed a high throughput method for quantification of PEDF in mouse ocular tissues that is sensitive, efficient and accurate. The method uses macrospin ion-exchange column chromatography and ELISA steps, and was applied to quantify PEDF in small tissue samples of retina disease-related mouse models.