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Abdulkerim EROGLU, Susan Gentleman, Eugenia Poliakov, T Michael Redmond; Inhibition of RPE65 retinol isomerase activity by inhibitors of lipid metabolism. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3474.
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© ARVO (1962-2015); The Authors (2016-present)
RPE65 is the key isomerase in the RPE visual cycle that catalyzes the conversion of all-trans retinyl ester (ATRE) into 11-cis retinol. Recent crystal structures of RPE65 and site-directed mutagenesis studies have revealed aspects of its catalytic mechanism, especially with respect to retinyl moiety isomerization, but other aspects remains to be determined. We are particularly 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. Isomerase activity was determined by normal phase HPLC of isomeric retinols extracted from these cells or membranes.
In experiments to determine potential interactions between RPE65 and acyl-CoA synthases, we discovered that fatty acyl analog inhibitors of fatty acid CoA ligases (ACSLs), also potently inhibited RPE65 isomerase activity in HEK293-F cells transfected with RPE65, LRAT, CRALBP and RDH5. LRAT activity was not affected. RPE65 protein expression was not affected by any concentration of these inhibitors. Testing with a range of ATROL substrate concentrations revealed the mode of inhibition to be generally competitive in nature. 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. Our results also indicated a competitive nature of inhibition.
We have identified inhibitors of ACSLs 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 mode of inhibition of these agents likely depends directly on competition with the fatty acyl moiety of the retinyl ester in binding to the enzyme. The effects of this class of inhibitors provide further insight into the catalytic mechanism of RPE65 retinol isomerase.
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