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T. Guignard, A. Weber, B. Ayoub, C. Hamel, P. Brabet; Energetic Metabolism Proteins Interact With RPE65 in vitro . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2030.
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RPE65 activity is critical for the visual cycle and is crucial for the normal vision, producing 11–cis retinol from all–trans retinylesters in the RPE. Regulation of this key reaction is not yet fully identified and could explain phenotypic variability in inherited retinal dystrophies. This study aims at identifying RPE65 partners likely to be involved in the RPE physiology and vision mechanisms and accounting for potential therapeutic targets in patients.
Through a yeast two hybrid approach, we have screened a porcine RPE mRNA library using full length human RPE65. Positives and specific clones have been tested with fragments of RPE65 and blasted for identification. GST pull–down approach was performed to corroborate the yeast interactions; we fused the positive clones to Glutathion–S–Transferase (GST) and incubated them with a radiolabeled in vitro translated hRPE65. Immunohistology of BALB/c mice retina was achieved using antibody directed against the orthologous proteins. To visualize cellular colocalization, we have performed immunocytochemistry on HEK293 cells transfected with an EGFP–RPE65 vector and expressing human orthologous proteins corresponding to positive clones.
We found several potential partners of RPE65, notably three energetic metabolism proteins –Aldolase A, PKM2, FATP1– and several unknown clones. Using RT–PCR, we confirmed the mRNA expression of these 3 proteins in the RPE (strong for FATP1) and also in other tissues, as well as for 4 unknown clones that are preferentially expressed in the porcine RPE. GST pull–down experiments indicate that clones fused to GST interact with RPE65 independently of GST. Immunohistological studies suggest that the clones and RPE65 are evenly expressed in the RPE layer. Immunocytological studies show that subcellular localization patterns of RPE65 and clones are overlapping.
This work has led to the identification of RPE–expressed proteins that interact in vitro with RPE65 and could be potential regulators of RPE65 activity, visual cycle and RPE physiology.
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