ABSTRACT
The isomerization of all-trans retinol (vitamin A) to 11-cis retinol in the retinal pigment epithelium (RPE) is a key step in the visual process for the regeneration of the visual pigment chromophore, 11-cis retinal. LRAT and RPE65 are recognized as the minimal isomerase catalytic components. However, regulators of this rate-limiting step are not fully identified and could account for the phenotypic variability associated with inherited retinal degeneration (RD) caused by mutations in the RPE65 gene. To identify new RPE65 partners, we screened a porcine RPE mRNA library using a yeast two-hybrid assay with full-length human RPE65. One identified clone (here named FATP1c), containing the cytosolic C-terminal sequence from the fatty acid transport protein 1 (FATP1 or SLC27A1, solute carrier family 27 member 1), was demonstrated to interact dose-dependently with the native RPE65 and with LRAT. Furthermore, these interacting proteins colocalize in the RPE. Cellular reconstitution of human interacting proteins shows that FATP1 markedly inhibits 11-cis retinol production by acting on the production of all-trans retinyl esters and the isomerase activity of RPE65. The identification of this new visual cycle inhibitory component in RPE may contribute to further understanding of retinal pathogenesis.
Subject(s)
Acyltransferases/metabolism , Carrier Proteins/metabolism , Eye Proteins/metabolism , Fatty Acid Transport Proteins/metabolism , Vitamin A/antagonists & inhibitors , Animals , Glutathione Transferase/metabolism , Humans , Insecta , Mice , Phenotype , Retina/metabolism , Subcellular Fractions/metabolism , Swine , Time Factors , Two-Hybrid System Techniques , Vitamin A/chemistry , cis-trans-IsomerasesABSTRACT
In mammals, the permanence of hearing loss is due mostly to the incapacity of the cochlea to replace lost mechano-receptor cells (i.e., hair cells [HCs]). The generation of new HCs from a renewable source of progenitors is a principal requirement for developing a cell therapy within this sensory organ. A subset of stem cells, termed side population (SP), has been identified in several tissues of mammals. The ATP-binding cassette transporter Abcg2/Bcrp1 contributes to the specification of the SP phenotype and is proposed as a universal marker for stem/progenitor cells. A defining character of these SP cells is a high efflux capacity for Hoechst dye. Here, we demonstrate that Abcg2 transporter is expressed with two other stem/progenitor cell markers (i.e., Nestin and Musashi1) in distinct and overlapping domains of the supporting cells within the postnatal cochlea. We have developed and describe a fluorescence-activated cell sorting (FACS) technique that enables the purification of a discrete subpopulation of SP-supporting cells from the early postnatal mouse cochlea based on their ability to exclude Hoechst dye. These FACS-isolated cells can divide and express markers of stem/progenitor cells such as Abcg2, a determinant of the SP phenotype, and Musashi1, a neural stem/progenitor cell marker. These markers can differentiate cells expressing markers of HCs and supporting cells in vitro. Our observation that these SP cells are capable of differentiating into HC-like cells implies a possible use for such cells (i.e., the replacement of lost auditory HCs within damaged cochlea).
