Inhibition of RPE65 retinol isomerase activity by inhibitors of lipid metabolism

Monday, September 22, 2014 — Poster Session II
4:00 p.m. – 6:00 p.m.	FAES Academic Center	NEI	MOLBIO-5

Authors

• A EROGLU
• S Gentleman
• E Poliakov
• TM Redmond

Abstract

RPE65 is the key isomerase in the RPE visual cycle that catalyzes the conversion of all-trans retinyl ester (ATRE) into 11-cis retinol. We are interested in potential parallels between the RPE65 catalytic mechanism and that of lipid metabolism enzymes. We tested inhibitors of lipid metabolism to determine their effect on RPE65 isomerase activity. HEK 293-F cells were transiently transfected with expression vectors for visual cycle proteins (RPE65, LRAT, CRALBP and RDH5) and isomerase activity was measured in cellulo in the presence of added substrate and inhibitors. Membrane preparations of transfected cells were used to test the effect of additives on isomerase activity in vitro. To determine if these fatty acid analogs compete with the ATRE substrate specifically, we incubated membranes prepared from transfected cells with liposomes containing a range of ATRE concentrations. We have identified fatty acid analogs used in studies of lipid metabolism as potent inhibitors of RPE65 and that compete with the ATRE substrate of RPE65 for binding, thereby inhibiting its isomerase activity. The effects of these inhibitors provide further insight into the catalytic mechanism of RPE65 retinol isomerase.

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