title: Inhibition of RPE65 Retinol Isomerase Activity by Inhibitors of Lipid Metabolism.
abstract: RPE65 is the isomerase catalyzing conversion of all-trans-retinyl ester (atRE) into 11-cis-retinol in the retinal visual cycle. Crystal structures of RPE65 and site-directed mutagenesis reveal aspects of its catalytic mechanism, especially retinyl moiety isomerization, but other aspects remain to be determined. To investigate potential interactions between RPE65 and lipid metabolism enzymes, HEK293-F cells were transfected with expression vectors for visual cycle proteins and co-transfected with either fatty acyl:CoA ligases (ACSLs) 1, 3, or 6 or the SLC27A family fatty acyl-CoA synthase FATP2/SLCA27A2 to test their effect on isomerase activity. These experiments showed that RPE65 activity was reduced by co-expression of ACSLs or FATP2. Surprisingly, however, in attempting to relieve the ACSL-mediated inhibition, we discovered that triacsin C, an inhibitor of ACSLs, also potently inhibited RPE65 isomerase activity in cellulo. We found triacsin C to be a competitive inhibitor of RPE65 (IC50 = 500 nm). We confirmed that triacsin C competes directly with atRE by incubating membranes prepared from chicken RPE65-transfected cells with liposomes containing 0-1 μM atRE. Other inhibitors of ACSLs had modest inhibitory effects compared with triascin C. In conclusion, we have identified an inhibitor of ACSLs as a potent inhibitor of RPE65 that competes with the atRE substrate of RPE65 for binding. Triacsin C, with an alkenyl chain resembling but not identical to either acyl or retinyl chains, may compete with binding of the acyl moiety of atRE via the alkenyl moiety. Its inhibitory effect, however, may reside in its nitrosohydrazone/triazene moiety.
authors.affiliation: From the Laboratory of Retinal Cell & Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Abdulkerim
authors.initials: A
authors.lastname: Eroglu
authors.affiliation: From the Laboratory of Retinal Cell & Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Susan
authors.initials: S
authors.lastname: Gentleman
authors.affiliation: From the Laboratory of Retinal Cell & Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Eugenia
authors.initials: E
authors.lastname: Poliakov
authors.affiliation: From the Laboratory of Retinal Cell & Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892 redmondd@helix.nih.gov.
authors.forename: T Michael
authors.initials: TM
authors.lastname: Redmond
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D003066:Coenzyme A Ligases / Q000037:antagonists & inhibitors; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding; D014226:Triazenes / Q000494:pharmacology*; D019745:cis-trans-Isomerases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000099178:Retinoid Isomerohydrolase
chemical_list: D003066:Coenzyme A Ligases; D004791:Enzyme Inhibitors; D014226:Triazenes; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase; C034613:triacsin C
keywords: RPE65; enzyme inhibitor; fatty acyl:CoA ligase; liposome; retinal metabolism; retinoid; triacsin C; vision; visual cycle