Biomedical subjects

Gennadiy Moiseyev

Publications and source records attributed to Gennadiy Moiseyev.

14 recordsLinked to original sources

PubMed2007

Light induces programmed cell death by activating multiple independent proteases in a cone photoreceptor cell line.

PURPOSE: Although the apoptotic death of photoreceptor cells in retinal degenerative disorders is well documented, the molecular mechanism is not understood. The objective of this study was to determine the molecular events leading to the death of photoreceptor cells. METHODS: An assay was developed wherein 661W cells, a cone photoreceptor cell line, were stressed with light and percentage of surviving cells was determined. The degree of cell death was established using the MTT assay. Western blot analysis was used to confirm the activation of multiple proteases. Amounts of retinaldehydes were determined by extraction and HPLC. RESULTS: 661W cells were more susceptible to light stress only in the presence of the chromophore 9-cis retinal for 4 hours. On exposure to light, 9-cis retinal was converted to all-trans retinal, which was found to be toxic to cells in the presence of light. However, all-trans retinol, which is the product of action by the enzyme retinol dehydrogenase on all-trans retinal, was not toxic. The sensitivity to light increased with serum deprivation. Light stress activated caspases, calpain 2, and cathepsin D independently and led to the demise of the cell. The mitochondria-dependent apoptotic pathway was also activated after the truncation of Bid, the pre-proapoptotic protein. Truncation of Bid led to the release of cytochrome c from the mitochondria and the activation of caspase 9. CONCLUSIONS: The activation of multiple proteases by light-induced stress is a relevant finding for studies conducted to investigate the use of pharmaceutical agents to retard or cure the loss of cone photoreceptors observed in age-related macular degeneration and other degenerative retinal diseases.

Animals↗

PubMed2006

Impacts of two point mutations of RPE65 from Leber's congenital amaurosis on the stability, subcellular localization and isomerohydrolase activity of RPE65.

RPE65, a membrane-associated protein in the retinal pigment epithelium, is the isomerohydrolase essential for regenerating 11-cis retinal, the chromophore for visual pigments. RPE65 mutations are associated with inherited retinal dystrophies. Here we report that single point mutations of RPE65, Y144D and P363T, identified in patients with Leber's congenital amaurosis (LCA), significantly decreased the stability of RPE65. Moreover, these mutations altered subcellular localization of RPE65 and abolished its isomerohydrolase activity. These observations suggest that the decreased protein stability and altered subcellular localization of RPE65 may represent a mechanism for these mutations to lead to vision loss in LCA patients.

Amino Acid Substitution↗

PubMed2006

Two point mutations of RPE65 from patients with retinal dystrophies decrease the stability of RPE65 protein and abolish its isomerohydrolase activity.

RPE65 is the isomerohydrolase in the retinoid visual cycle essential for recycling of 11-cis retinal, the chromophore for visual pigments in both rod and cone photoreceptors. Mutations in the RPE65 gene are associated with inherited retinal dystrophies with unknown mechanisms. Here we show that two point mutations of RPE65, R91W and Y368H, identified in patients with retinal dystrophies both abolished the isomerohydrolase activity of RPE65 after a subretinal injection into the Rpe65-/- mice and in the in vitro isomerohydrolase activity assay, independent of their protein levels. Further, the R91W and Y368H mutants showed significantly decreased protein levels but unchanged mRNA levels when compared with the wild-type RPE65 (wtRPE65). Protein stability analysis showed that wtRPE65 is a fairly stable protein, with an apparent half-life longer than 10 h, when expressed in 293A cells. Under the same conditions, mutants R91W and Y368H both showed substantially decreased protein stabilities, with half-lives less than 2 and 6 h, respectively. Subcellular fractionation and Western blot analysis demonstrated that wtRPE65 predominantly exists in the membrane fraction, while both of the mutants are primarily distributed in the cytosolic fraction, suggesting that these mutations disrupt the membrane association of RPE65. However, palmitoylation assay showed that wtRPE65 and both of the mutants were palmitoylated. These results suggest that these mutations may result in critical structural alterations of RPE65 protein, disrupt its membrane association, and consequently impair its isomerohydrolase activity, leading to retinal degeneration.

Animals↗

PubMed2006

RPE65 gene delivery restores isomerohydrolase activity and prevents early cone loss in Rpe65-/- mice.

PURPOSE: Recent in vitro evidence has shown that RPE65 is the isomerohydrolase that converts all-trans retinyl ester to 11-cis retinal, the chromophore for visual pigments in vertebrates. Homozygous RPE65 knockout (Rpe65-/-) mice lack 11-cis retinoids and have early cone degeneration. The purpose of this study is to determine whether RPE65 gene delivery restores the isomerohydrolase activity and normal profile of endogenous retinoids in Rpe65-/- mice. METHODS: Adenovirus-expressing RPE65 (Ad-RPE65) was injected into the subretinal space of Rpe65-/- mice. The expression of RPE65 was determined by immunohistochemistry and Western blot analysis. The isomerohydrolase activity was measured in vitro in eyecup homogenates. Endogenous retinoid profile in the eyecups was analyzed by high-performance liquid chromatography (HPLC). Photoreceptor-specific gene expression was determined with real-time RT-PCR. Cone degeneration was determined by cone-specific staining and counting cones in flatmounted retina. RESULTS: High levels of RPE65 expression from the Ad-RPE65 injection generated robust isomerohydrolase activity in the eyecup of Rpe65-/- mice, at levels comparable to those in wild-type (wt) mice. Consequently, the RPE65 gene delivery resulted in substantial amounts of 11-cis retinal in Rpe65-/- mice. The RPE65 gene delivery prevented the downregulation of cone-specific genes, including both cone opsins and cone tranducin alpha subunit in Rpe65-/- mice. Moreover, the Ad-RPE65 injection also prevented massive cone degeneration at early ages of Rpe65-/- mice. CONCLUSIONS: RPE65 gene delivery generates isomerohydrolase activity and restores retinoid profile in Rpe65-/- mice. Regeneration of 11-cis retinal is essential for survival of cone photoreceptors.

Adenoviridae↗

PubMed2006

The roles of three palmitoylation sites of RPE65 in its membrane association and isomerohydrolase activity.

PURPOSE: RPE65, a membrane-associated protein predominantly expressed in the retinal pigment epithelium, is the isomerohydrolase in the retinoid visual cycle. Three cysteine (Cys) residues, Cys231, Cys329, and Cys330, in RPE65 have been shown to be palmitoylated and have been suggested to be responsible for its membrane association. The purpose of this study was to define the role of these Cys residues in palmitoylation, membrane association, and isomerohydrolase activity of RPE65. METHODS: The three Cys residues in RPE65 were replaced by Alanine (Ala) with site-directed mutagenesis. The mutant protein levels and subcellular localizations were determined by Western blot analysis and subcellular fractionation, respectively. Their enzymatic activities were evaluated with the in vitro isomerohydrolase activity assay. Palmitoylation of the mutants was examined by labeling of the protein with [3H]-labeled palmitic acid. RESULTS: Mutation of any single residue of these three Cys significantly reduced protein levels of RPE65. Similar to wild-type RPE65, however, all three single Cys mutants were predominantly present in the membrane fraction. Mutations of any one or two of these Cys substantially weakened the isomerohydrolase activity of RPE65, whereas mutations of all three Cys (triple mutant) completely abolished the enzymatic activity. However, this triple Cys mutant was still palmitoylated and associated with the membrane, although at a reduced level. CONCLUSIONS: There are additional yet to be identified palmitoylation sites in RPE65. The structural distortions induced by the Cys mutations may be responsible for the mislocalization and decreased isomerohydrolase activities of RPE65.

Alanine↗

PubMed2005

RPE65 is an iron(II)-dependent isomerohydrolase in the retinoid visual cycle.

The isomerization of all-trans-retinyl ester to 11-cis-retinol in the retinal pigment epithelium (RPE) is a critical step in the visual cycle and is essential for normal vision. Recently, we have established that protein RPE65 is the isomerohydrolase catalyzing this reaction. The present study investigated if metal ions are required for the isomerohydrolase activity of RPE65. The conversion of all-trans-[3H]retinol to 11-cis-[3H]retinol was used as the measure for isomerohydrolase activity. Metal chelators 2,2'-bipyridine and 1,10-phenanthroline both showed dose-dependent inhibitions of the isomerohydrolase activity in bovine RPE microsomes, with IC50 values of 0.5 and 0.2 mm, respectively. In the same reaction systems, however, lecithin-retinol acyltransferase (LRAT) activity was not affected by these metal chelators. The isomerohydrolase activity inhibited by the metal chelators was restored by FeSO4 but not by CuSO4, ZnCl2, or MgCl2. Moreover, addition of Fe(III) citrate or FeCl3 did not restore the activity, indicating that Fe2+ is the metal ion essential for the isomerohydrolase activity. To confirm this result in recombinant RPE65, we expressed RPE65 in a 293A cell line stably expressing LRAT. In vitro activity assay showed that both metal chelators inhibited isomerohydrolase activity of recombinant RPE65. The addition of FeSO4 restored the enzymatic activity of the recombinant RPE65. Further, two specific iron-staining methods showed that purified RPE65 contains endogenous iron. Inductively coupled plasma mass spectrometry measurements showed that bovine RPE65 binds iron ion with a stoichiometry of 0.8 +/- 0.1. These results indicate that RPE65 is an iron-dependent isomerohydrolase in the visual cycle.

Animals↗

PubMed2005

Identification of conserved histidines and glutamic acid as key residues for isomerohydrolase activity of RPE65, an enzyme of the visual cycle in the retinal pigment epithelium.

We have recently reported that RPE65 from the retinal pigment epithelium is the isomerohydrolase, a critical enzyme in the visual cycle for regeneration of 11-cis retinal, the chromophore for visual pigments. Here, we demonstrated that mutation of any one of the absolutely conserved four histidine and one glutamic acid residues to alanine in RPE65 abolished its isomerohydrolase activity. Substitution of the conserved glutamic acid with glutamine also resulted in loss of the activity. Moreover, these mutations significantly reduced protein stability of RPE65. These results indicate that these conserved residues are essential for the isomerohydrolase activity of RPE65 and its stability.

Carrier Proteins↗

PubMed2005

RPE65 is the isomerohydrolase in the retinoid visual cycle.

RPE65 is an abundant protein in the retinal pigment epithelium. Mutations in RPE65 are associated with inherited retinal dystrophies. Although it is known that RPE65 is critical for regeneration of 11-cis retinol in the visual cycle, the function of RPE65 is elusive. Here we show that recombinant RPE65, when expressed in QBI-293A and COS-1 cells, has robust enzymatic activity of the previous unidentified isomerohydrolase, an enzyme converting all-trans retinyl ester to 11-cis retinol in the visual cycle. The initial rate for the reaction is 2.9 pmol/min per mg of RPE65 expressed in 293A cells. The isomerohydrolase activity of RPE65 requires coexpression of lecithin retinol acyltransferase in the same cell to provide its substrate. This enzymatic activity is linearly dependent on the expression levels of RPE65. This study demonstrates that RPE65 is the long-sought isomerohydrolase and fills a major gap in our understanding of the visual cycle. Identification of the function of RPE65 will contribute to the understanding of the pathogenesis for retinal dystrophies associated with RPE65 mutations.

Acyltransferases↗

PubMed2004

Identification of RDH10, an All-trans Retinol Dehydrogenase, in Retinal Muller Cells.

PURPOSE: To investigate the expression of RDH10, an all-trans retinol dehydrogenase identified in the retinal pigment epithelium (RPE), in retinal Muller cells. METHODS: The RDH10 protein levels in mouse eyecups and bovine tissues were examined by Western blot analysis using a polyclonal antibody against RDH10. The cellular localization in the retina was determined by immunohistochemistry. Expression of RDH10 in rMC-1, a cell line derived from rat Muller cells, was determined by RT-PCR and Western blot analysis. All-trans retinol dehydrogenase activity assays were performed using lysates from rMC-1 cells. The generation of all-trans retinal from tritiated all-trans retinol was analyzed by HPLC. RESULTS: RDH10, retinal G protein-coupled receptor (RGR), and RPE65 all had higher expression levels in the eyecups of BALB/c than in C57Bl/6 mice. In addition to the RPE, RDH10 was also detected at lower levels in the retina and liver. Immunohistochemistry showed that RDH10 was localized in Muller cells in retinal sections. RDH10 was detected in rMC-1 cells, at both the RNA and protein levels. The rat RDH10 cDNA containing the full-length coding region was cloned from rMC-1 cells. The rat RDH10 cDNA encodes a protein of 341 amino acids and shares 99% sequence identity with human, bovine, and mouse RDH10 at the amino acid level. In rMC-1 cells, all-trans retinol dehydrogenase activity was detected in the microsomal fraction. NADP was shown to be the preferred cofactor, which is identical with the cofactor preference of the recombinant RDH10. CONCLUSIONS: RDH10 was expressed in retinal Muller cells, in addition to the RPE. RDH10 generates all-trans retinal, which is the substrate for the photoisomerase RGR in Muller cells.

Alcohol Oxidoreductases↗

PubMed2003

Collagen XVIII/endostatin is essential for vision and retinal pigment epithelial function.

Age-related macular degeneration (ARMD) with abnormal deposit formation under the retinal pigment epithelium (RPE) is the major cause of blindness in the Western world. basal laminar deposits are found in early ARMD and are composed of excess basement membrane material produced by the RPE. Here, we demonstrate that mice lacking the basement membrane component collagen XVIII/endostatin have massive accumulation of sub-RPE deposits with striking similarities to basal laminar deposits, abnormal RPE, and age-dependent loss of vision. The progressive attenuation of visual function results from decreased retinal rhodopsin content as a consequence of abnormal vitamin A metabolism in the RPE. In addition, aged mutant mice show photoreceptor abnormalities and increased expression of glial fibrillary acidic protein in the neural retina. Our data demonstrate that collagen XVIII/endostatin is essential for RPE function, and suggest an important role of this collagen in Bruch's membrane. Consistent with such a role, the ultrastructural organization of collagen XVIII/endostatin in basement membranes, including Bruch's membrane, shows that it is part of basement membrane molecular networks.

Aging↗

PubMed2003

Isorhodopsin rather than rhodopsin mediates rod function in RPE65 knock-out mice.

The chromophore of visual pigments is 11-cis-retinal and, thus, in its absence, opsin is not photosensitive and no visual function exists. However, in the RPE65 knockout (Rpe65-/-) mouse, where synthesis of 11-cis-retinal does not occur, a minimal visual response from rod photoreceptors is obtained. We have examined if an alternative pathway exists for cis-retinoid generation in the absence of RPE65. Cyclic-light-reared, 2-month-old Rpe65-/- mice were placed in complete darkness. No exogenous retinoids were administered. After 4 weeks, enhanced a- and b-wave amplitudes were obtained, increasing >10-fold for the a-wave and >3-fold for the b-wave as compared with cyclic-light-reared Rpe65-/- mice. Visual-pigment levels increased to approximately 10 pmol per retina, compared with no measurable pigment for cyclic-light-reared Rpe65-/- mice. The lambdamax of the isolated pigment was 487 nm, characteristic for isorhodopsin. Retinoid extractions confirmed the presence of 9-cis-retinal and the absence of 11-cis-retinal. Once the Rpe65-/- mice were returned to cyclic light, within 48 h the electroretinogram function returned to levels found in Rpe65-/- mice maintained in cyclic light. This dark-mediated pathway is also operational in older animals, because 13-month-old Rpe65-/- mice kept in prolonged darkness (12 weeks) had increased isorhodopsin levels and electroretinogram a- and b-wave amplitudes. These studies demonstrate that a pathway exists in the eye for the generation of 9-cis-retinal that is independent of RPE65 and light.

Animals↗

PubMed2003

Retinyl esters are the substrate for isomerohydrolase.

Regeneration of 11-cis retinal from all-trans retinol in the retinal pigment epithelium (RPE) is a critical step in the visual cycle. The enzyme(s) involved in this isomerization process has not been identified and both all-trans retinol and all-trans retinyl esters have been proposed as the substrate. This study is to determine the substrate of the isomerase enzyme or enzymatic complex. Incubation of bovine RPE microsomes with all-trans [(3)H]-retinol generated both retinyl esters and 11-cis retinol. Inhibition of lecithin retinol acyltransferase (LRAT) with 10-N-acetamidodecyl chloromethyl ketone (AcDCMK) or cellular retinol-binding protein I (CRBP) diminished the generation of both retinyl esters and 11-cis retinol from all-trans retinol. The 11-cis retinol production correlated with the retinyl ester levels, but not with the all-trans retinol levels in the reaction mixture. When retinyl esters were allowed to form prior to the addition of the LRAT inhibitors, a significant amount of isomerization product was generated. Incubation of all-trans [(3)H]-retinyl palmitate with RPE microsomes generated 11-cis retinol without any detectable production of all-trans retinol. The RPE65 knockout (Rpe65(-/-)) mouse eyecup lacks the isomerase activity, but LRAT activity remains the same as that in the wild-type (WT) mice. Retinyl esters in WT mice plateau at 8 weeks-of-age, but Rpe65(-/-) mice continue to accumulate retinyl esters with age (e.g., at 36 weeks, the levels are 20x that of WT). Our data indicate that the retinyl esters are the substrate of the isomerization reaction.

Acyltransferases↗

PubMed2003

Visual cycle retinoid processing proteins are present in HEK293S cells.

In HEK293S cells expressing opsin, rhodopsin regenerates on addition of all-trans retinol. This study was to determine if key proteins in the retinal pigment epithelium (RPE) are present in these cells. Cellular retinoid binding protein, cellular retinoic-acid binding protein, RPE65, caveolin-1-alpha- and -beta-isoforms, interphotoreceptor retinoid binding protein, and 11-cis retinol dehydrogenase, but not lecithin:retinol acyltransferase (LRAT), were identified by Western blot analysis. LRAT transcripts were found by RT-PCR and Southern blot analysis. Small interference RNA specific to LRAT reduced ester formation, confirming that the enzyme is present. Therefore, HEK293S cells contain the functional components of the retinoid cycle found in the RPE.

Acyltransferases↗

PubMed2002

Identification of the RPE65 protein in mammalian cone photoreceptors.

PURPOSE: The protein RPE65 plays a critical role in retinoid processing in the retinal pigment epithelium (RPE). Previous studies have identified the RPE65 mRNA in salamander cones, but not in rods. The purpose of the present study was to determine whether RPE65 is expressed at the protein level in mammalian cones, as well as in those of amphibians. METHODS: The specificity of the anti-RPE65 antibody was demonstrated by Western blot analysis. RPE65 cellular localization was determined using immunohistochemistry on flatmounted retinas and retinal sections. RESULTS: RPE65 protein was detected in cones in flatmounted retinas of the mouse, rabbit, and cow, in addition to Xenopus laevis. The morphology and location of labeled cones in the retina were confirmed by double staining of mouse retina sections with the anti-RPE65 antibody and peanut agglutinin (PNA) lectin, which is known to label both types of cones in mouse. The double staining in the flatmounted retinas demonstrated that RPE65 was expressed in both types of the cones in the mouse retina. Under the same double-labeling conditions, however, cones in homozygous RPE65-knockout mouse were labeled by PNA lectin, but not by the anti-RPE65 antibody, indicating that the protein recognized by the anti-RPE65 antibody is encoded by the RPE65 gene rather than by another homologous gene. No RPE65 was detected in rods of any of the species tested. CONCLUSIONS: RPE65 is expressed in mammalian cones, but not in rods. These results provide further support for physiological observations that cones may have an alternative retinoid cycle.

Animals↗