Metabolic Basis of Visual Cycle Inhibition by Retinoid and Nonretinoid Compounds in the Vertebrate Retina

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Background: Cellular uptake of retinol bound to its serum binding protein depends on a cell surface receptor. Results: Functional coupling of this receptor with lecithin:retinol acyltransferase is required for the regulated cellular uptake of retinol. Conclusion:The lecithin: retinol acyltransferase is critical for retinol uptake and homeostasis. Significance: Blood retinol homeostasis is associated with blinding retinopathies and diabetes.

Section: Discussionmentioning

confidence: 99%

Section: Animals-twelve-week-old Female Lratmentioning

confidence: 99%

Lecithin:Retinol Acyltransferase Is Critical for Cellular Uptake of Vitamin A from Serum Retinol-binding Protein

Amengual

Golczak

Palczewski

et al. 2012

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“…A slight slowing of the life-long molecular mechanisms responsible for macular degeneration might postpone the onset of the disease for decades. Because A2E is believed to contribute to declining retinal health, it is often used as an early marker for evaluating the efficacy of therapeutic interventions to treat AMD in murine models (20,21,23,49). A2E levels are often taken as a quantitative measure of ocular lipofuscin (50,51) as the levels of A2E coincide with granular lipofuscin deposits in animal models, in elderly humans and in individuals with certain genetic eye disorders (1, 2, 52, 53).…”

Section: Discussionmentioning

confidence: 99%

Deuterium Enrichment of Vitamin A at the C20 Position Slows the Formation of Detrimental Vitamin A Dimers in Wild-type Rodents

Kaufman

Washington

2011

Degenerative eye diseases are the most common causes of untreatable blindness. Accumulation of lipofuscin (granular deposits) in the retinal pigment epithelium (RPE) is a hallmark of major degenerative eye diseases such as Stargardt disease, Best disease, and age-related macular degeneration. The intrinsic reactivity of vitamin A leads to its dimerization and to the formation of pigments such as A2E, and is believed to play a key role in the formation of ocular lipofuscin. We sought a clinically pragmatic method to slow vitamin A dimerization as a means to elucidate the pathogenesis of macular degenerations and to develop a therapeutic intervention. We prepared vitamin A enriched with the stable isotope deuterium at carbon twenty (C20-D 3 -vitamin A). Results showed that dimerization of deuterium-enriched vitamin A was considerably slower than that of vitamin A at natural abundance as measured in vitro. Administration of C20-D 3 -vitamin A to wild-type rodents with no obvious genetic defects in vitamin A processing, slowed A2E biosynthesis. This study elucidates the mechanism of A2E biosynthesis and suggests that administration of C20-D 3 -vitamin A may be a viable, long-term approach to retard vitamin A dimerization and by extension, may slow lipofuscin deposition and the progression of common degenerative eye diseases.

“…Generation and Epitope Mapping of Anti-RPE65 Monoclonal Antibody-Anti-RPE65 monoclonal antibody was raised against full-length recombinant human RPE65 protein expressed in Sf9 cells and purified as described in Golczak et al (15). The purified protein was used to immunize mice as described previously (16).…”

Section: Methodsmentioning

confidence: 99%

Importance of Membrane Structural Integrity for RPE65 Retinoid Isomerization Activity

Golczak

Kiser

Lodowski

et al. 2010

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Regeneration of visual chromophore in the vertebrate visual cycle involves the retinal pigment epithelium-specific protein RPE65, the key enzyme catalyzing the cleavage and isomerization of all-trans-retinyl fatty acid esters to 11-cis-retinol. Although RPE65 has no predicted membrane spanning domains, this protein predominantly associates with microsomal fractions isolated from bovine retinal pigment epithelium (RPE). We have re-examined the nature of RPE65 interactions with native microsomal membranes by using extraction and phase separation experiments. We observe that hydrophobic interactions are the dominant forces that promote RPE65 association with these membranes. These results are consistent with the crystallographic model of RPE65, which features a large lipophilic surface that surrounds the entrance to the catalytic site of this enzyme and likely interacts with the hydrophobic core of the endoplasmic reticulum membrane. Moreover, we report a critical role for phospholipid membranes in preserving the retinoid isomerization activity and physical properties of RPE65. Isomerase activity measured in bovine RPE was highly sensitive to phospholipase A 2 treatment, but the observed decline in 11-cis-retinol production did not directly reflect inhibition by products of lipid hydrolysis. Instead, a direct correlation between the kinetics of phospholipid hydrolysis and retinoid isomerization suggests that the lipid membrane structure is critical for RPE65 enzymatic activity. We also provide evidence that RPE65 operates in a multiprotein complex with retinol dehydrogenase 5 and retinal G protein-coupled receptor in RPE microsomes. Modifications in the phospholipid environment affecting interactions with these protein components may be responsible for the alterations in retinoid metabolism observed in phospholipid-depleted RPE microsomes. Thus, our results indicate that the enzymatic activity of native RPE65 strongly depends on its membrane binding and phospholipid environment.In vertebrates, the perception of light is preceded by a quantum event in which a photon of light hits the 11-cis-retinylidene chromophore of rhodopsin in the retina. Photoisomerization of this chromophore to its all-trans isomer results in activation of rhodopsin triggering a cascade of signal transduction events (summarized in Ref. 1). This process simultaneously leads to loss of photoreceptor light sensitivity making continuous regeneration of 11-cis-retinal a requirement to sustain vision. The visual (retinoid) cycle is the fundamental series of reactions responsible for regeneration of the visual chromophore that occurs in photoreceptors and the retinal pigment epithelium (RPE) 4 (summarized in Ref.2). The key component of this enzymatic cycle is the RPE-specific microsomal membrane protein known as RPE65 (3-5). The importance of RPE65 in the retinoid cycle is underscored by the metabolic dysfunction found in Rpe65 Ϫ/Ϫ mice, which is characterized by complete lack of 11-cis-retinoid production accompanied by accumulation of the all-tr...