Abstract
Purpose: :
Fatty acid transport protein 1 (FATP1) is a member of the long chain and very long chain acyl-CoA synthetase family. We (Guignard et al., JBC, 2010) previously showed the expression of FATP1 protein in the retinal pigment epithelium and its physical interaction with RPE65. This protein-protein interaction caused the inhibition of the isomerase activity of RPE65. Here we evaluated in vitro the inhibitory effect of peptides derived from the FATP1 interacting domain and analysed the visual function of transgenic mice overexpressing the human FATP1 protein in the retinal pigment epithelium (RPE).
Methods: :
Nucleotide sequences encoding peptides from the 307 C-terminal aa residues of FAPT1 were used in yeast two hybrid systems to quantify the interaction with the human RPE65. Peptides were transiently co-expressed with RPE65 in 293 cells stably expressing human LRAT and CRALBP proteins. Transfected cells were incubated with all-trans-retinol and retinoid extracted to measure 11-cis-retinol production. We engineered transgenic mice with RPE-specific overexpression of human FATP1 using the VMD2 promoter. Q-PCR was used to determine the level of transgene expression. RPE and neuroretina from F1 and F2 transgenic generations were isolated and used for western blot analysis. For adaptation-ERG, mice were subjected to seven repetitions of a 1.59 cd.s-1.m-2 blue flash; the seven b-wave amplitudes were averaged.
Results: :
The 307 C-terminal polypeptide of FATP1 was as effective as the whole protein at inhibiting 11-cis retinol production. Among five overlapping peptides of 102 aa covering the C-terminal, three showed a strong interaction that defined a more specific region. These peptides are currently being evaluated for inhibition of RPE65 isomerase. Two independent lines of transgenic mice showed expression of the human FATP1 cDNA specifically in RPE. One line displayed a delayed recovery of the b-wave amplitude after bleaching.
Conclusions: :
FATP1 could inhibit the retinoid isomerase RPE65 both in vitro and in vivo by protein-protein physical interactions, which could be restricted to the polypeptide domain. This could allow the design of small peptides capable of slowing the visual cycle as a therapeutic tool for Stargardt’s disease.
Keywords: retinoids/retinoid binding proteins • transgenics/knock-outs • retinal pigment epithelium