September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Acyl-CoA synthetase and N-terminal regions of fatty acid transport protein 4 (FATP4) are important for interacting with and inhibiting RPE65 isomerase of the visual cycle
Author Affiliations & Notes
  • Minghao Jin
    Department of Ophthalmology, LSU School of Medicine, New Orleans, Louisiana, United States
  • John F Green
    Department of Ophthalmology, LSU School of Medicine, New Orleans, Louisiana, United States
  • Sonia Adtani
    Department of Ophthalmology, LSU School of Medicine, New Orleans, Louisiana, United States
  • Songhua Li
    Department of Ophthalmology, LSU School of Medicine, New Orleans, Louisiana, United States
  • Footnotes
    Commercial Relationships   Minghao Jin, None; John Green, None; Sonia Adtani, None; Songhua Li, None
  • Footnotes
    Support  NIH Grand EY021208 and Research to Prevent Blindness
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 3626. doi:
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      Minghao Jin, John F Green, Sonia Adtani, Songhua Li; Acyl-CoA synthetase and N-terminal regions of fatty acid transport protein 4 (FATP4) are important for interacting with and inhibiting RPE65 isomerase of the visual cycle. Invest. Ophthalmol. Vis. Sci. 2016;57(12):3626.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : RPE65 catalyzes synthesis of 11-cis-retinol from all-trans retinyl fatty acid esters in the retinal pigment epithelium. FATP4 is a recently identified negative regulator of RPE65. Mutations that eliminate the acyl-CoA synthetase activity of FATP4 are associated with ichthyosis prematurity syndrome (IPS). The purpose of this study was to determine whether IPS-associated FATP4 mutants inhibit synthesis of 11-cis-retinol and what domains of FATP4 interact with and inhibit RPE65.

Methods : The IPS-associated point mutations and several N-terminal and C-terminal deletion mutations were introduced into pFATP4 by PCR using a site-directed mutagenesis kit. All deletion mutants were C-terminally fused with a FLAG tag. RPE65 was co-expressed with wild-type (WT) or mutant FATP4 in 293T-LC or 293T-C cells stably expressing lecithin-retinol acyltransferase (LRAT) and/or CRALBP. Association of RPE65 with FATP4 was determined by immunoprecipitation and immunocytochemistry. The inhibitory effects of WT and mutant FATP4s on the synthesis of 11-cis-retinol were determined by isomerase assays using all-trans-retinol or all-trans retinyl palmitate as substrate. LRAT activity was determined by monitoring synthesis of all-trans retinyl esters from all-trans-retinol. Retinoids were analyzed by high performance liquid chromatography.

Results : The LRAT-catalyzed synthesis of all-trans retinyl esters was not inhibited by FATP4 whereas the RPE65-catalyzed synthesis of 11-cis-retinol was inhibited at least 50% by WT FATP4 in both in vitro and in vivo (living cell) isomerase assays. All IPS-associated FATP4 mutants also inhibited synthesis of 11-cis-retinol. However, their inhibitory efficiencies were reduced by 10-30%, as compared to WT FATP4. Under the same experimental conditions, the C-terminal region of FATP4 had no inhibitory effect on the synthesis of 11-cis-retinol. The C-terminal region was not associated with RPE65 in immunoprecipitation. Most of other FATP4 mutants were colocalized with RPE65 in immunocytochemistry and were associated with RPE65 as evidenced by co-immunoprecipitation.

Conclusions : The very long-chain fatty acid-CoA synthetase activity of FATP4 partially contributes to the FATP4 inhibition of 11-cis-retinol synthesis catalyzed by RPE65. The N-terminal region of FATP4 is important for interacting with and inhibiting RPE65.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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