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.     

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.     

Negative charge of the glutamic acid 417 residue is crucial for isomerohydrolase activity of RPE65

RPE65 is the isomerohydrolase essential for regeneration of 11-cis retinal, the chromophore of visual pigments. Here we compared the impacts of two mutations in RPE65, E417Q identified in patients with Leber congenital amaurosis (LCA), and E417D on isomerohydrolase activity. Although both mutations decreased the stability of RPE65 and altered its sub-cellular localization, E417Q abolished isomerohydrolase activity whereas the E417D mutant retained partial enzymatic activity suggesting that the negative charge of E417 is important for RPE65 catalytic activity. Loss of charge at this position may represent a mechanism by which the E417Q mutation causes blindness in LCA patients.     

Binding to lipid membrane induces conformational changes in RPE65: implications for its isomerohydrolase activity

The visual cycle is a multi-step pathway to recycle 11-cis retinal, the chromophore for both rod and cone visual pigments. The isomerohydrolase RPE65, a membrane-associated enzyme, converts atRE (all-trans-retinyl ester) to 11-cis-retinol, a key step in the visual cycle. Previously, it has been shown that membrane association of RPE65 is essential for its catalytic activity. Using purified recombinant chicken RPE65 and an in vitro liposome-based floatation assay, we present evidence that the RPE65 membrane-binding affinity was significantly facilitated by incorporation of atRE, the substrate of RPE65, into liposomal membrane. Using tryptophan emission fluorescence quenching and CD spectroscopy, we showed that, upon membrane binding, RPE65 undergoes conformational changes at both the tertiary and secondary structural levels. Specifically, tryptophan fluorescence quenching showed that the tertiary RPE65 structure became more open towards the hydrophilic environment upon its association with the membrane. Simultaneously, a decrease in the α-helix content of RPE65 was revealed upon binding with the lipid membrane containing atRE. These results demonstrated that RPE65's functional activity depends on its conformational changes caused by its association with the membrane.     

Alpha-phenyl-N-tert-butylnitrone (PBN) prevents light-induced degeneration of the retina by inhibiting RPE65 protein isomerohydrolase activity

α-Phenyl-N-tert-butylnitrone (PBN), a free radical spin trap, has been shown previously to protect retinas against light-induced neurodegeneration, but the mechanism of protection is not known. Here we report that PBN-mediated retinal protection probably occurs by slowing down the rate of rhodopsin regeneration by inhibiting RPE65 activity. PBN (50 mg/kg) protected albino Sprague-Dawley rat retinas when injected 0.5-12 h before exposure to damaging light at 2,700 lux intensity for 6 h but had no effect when administered after the exposure. PBN injection significantly inhibited in vivo recovery of rod photoresponses and the rate of recovery of functional rhodopsin photopigment. Assays for visual cycle enzyme activities indicated that PBN inhibited one of the key enzymes of the visual cycle, RPE65, with an IC(50) = 0.1 mm. The inhibition type for RPE65 was found to be uncompetitive with K(i) = 53 μm. PBN had no effect on the activity of other visual cycle enzymes, lecithin retinol acyltransferase and retinol dehydrogenases. Interestingly, a more soluble form of PBN, N-tert-butyl-α-(2-sulfophenyl) nitrone, which has similar free radical trapping activity, did not protect the retina or inhibit RPE65 activity, providing some insight into the mechanism of PBN specificity and action. Slowing down the visual cycle is considered a treatment strategy for retinal diseases, such as Stargardt disease and dry age-related macular degeneration, in which toxic byproducts of the visual cycle accumulate in retinal cells. Thus, PBN inhibition of RPE65 catalytic action may provide therapeutic benefit for such retinal diseases.     

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.     

RPE65 from cone-dominant chicken is a more efficient isomerohydrolase compared with that from rod-dominant species

Cones recover their photosensitivity faster than rods after bleaching. It has been suggested that a higher rate regeneration of 11-cis-retinal, the chromophore for visual pigments, is required for cones to continuously function under bright light conditions. RPE65 is the isomerohydrolase catalyzing a key step in regeneration of 11-cis-retinal. The present study investigated whether RPE65 in a cone-dominant species is more efficient in its enzymatic activity than that from roddominant species. In vitro isomerohydrolase activity assay showed that isomerohydrolase activity in the chicken retinal pigment epithelium (RPE) was 11.7-fold higher than in the bovine RPE, after normalization by RPE65 protein levels. Similar to that of human and bovine, the isomerohydrolase activity in chicken RPE was blocked by two specific inhibitors of lecithin retinal acyltransferase, indicating that chicken RPE65 also uses all-trans-retinyl ester as the direct substrate. To exclude the possibility that the higher isomerohydrolase activity in the chicken RPE could arise from another unknown isomerohydrolase, we expressed chicken and human RPE65 using the adenovirus system in a stable cell line expressing lecithin retinal acyltransferase. Under the same conditions, isomerohydrolase activity of recombinant chicken RPE65 was 7.7-fold higher than that of recombinant human RPE65, after normalization by RPE65 levels. This study demonstrates that RPE65 from the cone-dominant chicken RPE possesses significantly higher specific retinol isomerohydrolase activity, when compared with RPE65 from rod-dominant species, consistent with the faster regeneration rates of visual pigments in cone-dominant retinas.     

Determining radical penetration of lipid bilayers with new lipophilic spin traps

Predicting the susceptibility of lipid moieties to radical attack requires a determination of the depth of radical penetration into a lipid membrane. We thus synthesized three homologous series of lipophilic spin traps--DMPO analogs 2-alkanoyl-2-methyl-1-pyrroline N-oxides (11) and PBN derivatives 4-alkoxyphenyl N-tert-butylnitrones (18) and 4-alkoxyphenyl N-admantylnitrones (20). The intercalation depth of these spin traps within the liposomal bilayer was determined via the previously reported NMR technique, which correlates the chemical shift and the micropolarity (measured in ET(30) units) experienced by the pivotal nitronyl carbon. Hydroxyl and alpha-hydroxyalkyl radicals were generated in the extraliposomal aqueous phase and the lowest depth at which a radical could be spin trapped was determined. The ESR data indicate that these radicals can exit the aqueous phase, penetrate the lipid bilayer past the head groups (ET(30)=63 kcal/mol) and the glycerol ester (ET(30)=52 kcal/mol), and pass down to an ET(30) polarity of at least 44 kcal/mol. The latter depth presumably corresponds to the upper portion of the lipid slab. It is likely, if not probable, that having come this far they can abstract the allylic/diallylic hydrogens resident in the midslab at ET(30) values of >31 kcal/mol.     

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.     

Aromatic phosphonates inhibit the lysophospholipase D activity of autotaxin

Autotaxin (ATX) is an attractive target for the anticancer therapeutics that inhibits angiogenesis, invasion and migration. ATX is an extracellular lysophospholipase D that hydrolyzes lysophosphatidylcholine to form the bioactive lipid lysophosphatidic acid. The aromatic phosphonate S32826 was the first described nanomolar inhibitor of ATX. However, the tridecylamide substituent on aromatic ring contributed to its poor solubility and bioavailability, severely limiting its utility in vivo. c Log P calculations revealed that the lipophilicity of S32826 could be lowered by shortening its hydrophobic chain and by introducing substituents alpha to the phosphonate. Herein, we describe the synthesis of a small set of α-substituted phosphonate analogs of S32826, and we show that shortening the chain and adding α-halo or α-hydroxy substituents increased solubility; however, ATX inhibition was reduced by most substitutions. An optimal compound was identified for examination of biological effects of ATX inhibition in vivo.     

Aromatic Residues in the Substrate Cleft of RPE65 Protein Govern Retinol Isomerization and Modulate Its Progression

Previously, we showed that mutating RPE65 residue Phe-103 preferentially produces 13-cis-retinol instead of 11-cis-retinol, supporting a carbocation/radical cation mechanism of retinol isomerization. We asked whether this modulation of specificity can occur with residues other than Phe-103 and what role it plays in substrate binding and isomerization. We modeled the substrate-binding cleft of RPE65 to identify residues lining its surface. Many are phenylalanines and tyrosines, including three Phe residues (Phe-61, Phe-312, and Phe-526) forming an arch-like arrangement astride the cleft and Tyr-338. Also, Phe-418 sits at the neck of the cleft, lending a bend to the volume enclosed by the cleft. All mutations of Phe-61, Phe-312, and Phe-418 result in severely impaired or inactive enzyme. However, mutation of Phe-526 and Tyr-338, like Phe-103, decreases 11-cis-retinol formation, whereas increasing the 13-cis isomer. Significantly, 2 of these 3 residues, Phe-103 and Tyr-338, are located on putatively mobile interstrand loops. We propose that residual densities located in the binding cleft of the RPE65 structure represents a post-cleavage snapshot consistent not only with a fatty acid product, as originally modeled, but also an 11-cis-retinol product. Substrate docking simulations permit 11-cis- or 13-cis-retinyl ester binding in this relatively closed cleft, with the latter favored in F103L, F526A, and Y338A mutant structures, but prohibit binding of all-trans-retinyl ester, suggesting that isomerization occurs early in the temporal sequence, with O-alkyl ester cleavage occurring later. These findings provide insight into the mechanism of isomerization central to the visual cycle.     

Potential therapeutic antioxidants that combine the radical scavenging ability of myricetin and the lipophilic chain of vitamin E to effectively inhibit microsomal lipid peroxidation

The flavonol myricetin, reacts with oxygen-centred galvinoxyl radicals 28 times faster than d-alpha-tocopherol (vitamin E), the main lipid-soluble antioxidant in biological membranes. Moreover, each myricetin molecule reduces twice as many such radicals as vitamin E. However, myricetin fails to protect vitamin E-deficient microsomes from lipid peroxidation as assessed by the formation of thiobarbituric acid reactive substances (TBARS). Novel and potentially therapeutic antioxidants have been prepared that combine the radical-scavenging ability of a myricetin-like head group with a lipophilic chain similar to that of vitamin E. C(6)-C(12) alkyl chains are attached to the A-ring of either a 3,3',4',5'-tetrahydroxyflavone or a 3,2',4',5'-tetrahydroxyflavone head group to give lipophilic flavonoids (C log P = 4 to 10) that markedly inhibit iron-ADP catalysed oxidation of microsomal preparations. Orientation of the head group as well as total lipophilicity are important determinants of antioxidant efficacy. MM2 models indicate that our best straight chain 7-alkylflavonoids embed to the same depth in the membrane as vitamin E. The flavonoid head groups are prepared by aldol condensation followed by Algar-Flynn-Oyamada (AFO) oxidation or by Baker-Venkataraman rearrangement. The alkyl tails are introduced by Suzuki or Negishi palladium-catalysed cross-coupling or by cross-metathesis catalysed by first generation Grubbs catalyst, which tolerate phenolic hydroxyl and ketone groups.     

Spin traps inhibit formation of hydrogen peroxide via the dismutation of superoxide: implications for spin trapping the hydroxyl free radical.

To enhance the sensitivity of EPR spin trapping for radicals of limited reactivity, high concentrations (10-100 mM) of spin traps are routinely used. We noted that in contrast to results with other hydroxyl radical detection systems, superoxide dismutase (SOD) often increased the amount of hydroxyl radical-derived spin adducts of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) produced by the reaction of hypoxanthine, xanthine oxidase and iron. One possible explanation for these results is that high DMPO concentrations (approximately 100 mM) inhibit dismutation of superoxide (O2.-) to hydrogen peroxide (H2O2). Therefore, we examined the effect of DMPO on O2.- dismutation to H2O2. Lumazine +/- 100 mM DMPO was placed in a Clark oxygen electrode following which xanthine oxidase was added. The amount of H2O2 formed in this reaction was determined by introducing catalase and measuring the amount of generated via O2.- dismutation as compared to direct divalent O2 reduction. In the presence of 100 mM DMPO, H2O2 generation decreased 43%. DMPO did not scavenge H2O2 nor alter the rate of O2.- production. The effect of DMPO was concentration-dependent with inhibition of H2O2 production observed at [DMPO] greater than 10 mM. Inhibition of H2O2 production by DMPO was not observed if SOD was present or if the rate of O2.- formation increased. The spin trap 2-methyl-2-nitroso-propane (MNP, 10 mM) also inhibited H2O2 formation (81%). However, alpha-phenyl-N-tert-butylnitrone (PBN, 10 mM), 3,3,5,5 tetramethyl-1-pyrroline N-oxide (M4PO, 100 mM), alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN, 100 mM) had no effect. These data suggest that in experimental systems in which the rate of O2.- generation is low, formation of H2O2 and thus other H2O2-derived species (e.g., OH) may be inhibited by commonly used concentrations of some spin traps. Thus, under some experimental conditions spin traps may potentially prevent production of the very free radical species they are being used to detect.     

Mercuric chloride-induced spin or ligation state changes in ferric or ferrous human cystathionine beta-synthase inhibit enzyme activity.

Cystathionine beta-synthase is a key heme and pyridoxal phosphate-dependent enzyme involved in homocysteine metabolism in humans. The role of the recently discovered heme in this protein remains an important open question. The axial ligands to the heme in both the ferrous and ferric states have been assigned as cysteine and histidine residues, respectively. In this study, we have examined the effect of ligation and spin state changes in the heme on the activity of the enzyme. Treatment of the ferric enzyme with HgCl2 results in the conversion of six-coordinate low-spin heme to five-coordinate high-spin heme and is paralleled by a loss of activity. In contrast, treatment of the ferrous enzyme with HgCl2 results in replacement of the cysteine ligand by an unidentified sixth ligand and retention of the six-coordinate state, and is also accompanied by loss of enzyme activity. Treatment of the five-coordinate HgCl2-treated enzyme with thiols, such as homocysteine, results in reversion to a six-coordinate state. Resonance Raman spectroscopy with 34S-labeled enzyme reveals the return of the endogenous thiol ligand under these conditions and rules out direct coordination by the thiolate of homocysteine to the heme.