Isomerization of 11-cis-retinoids to all-trans-retinoids in vitro and in vivo.

The regeneration of 11-cis-retinal, the universal chromophore of the vertebrate retina, is a complex process involving photoreceptors and adjacent retinal pigment epithelial cells (RPE). 11-cis-Retinal is coupled to opsins in both rod and cone photoreceptor cells and is photoisomerized to all-trans-retinal by light. Here, we show that RPE microsomes can catalyze the reverse isomerization of 11-cis-retinol to all-trans-retinol (and 13-cis-retinol), and membrane exposure to UV light further enhances the rate of this reaction. This conversion is inhibited when 11-cis-retinol is in a complex with cellular retinaldehyde-binding protein (CRALBP), providing a clear demonstration of the protective effect of retinoid-binding proteins in retinoid processes in the eye, a function that has been long suspected but never proven. The reverse isomerization is nonenzymatic and specific to alcohol forms of retinoids, and it displays stereospecific preference for 11-cis-retinol and 13-cis-retinol but is much less efficient for 9-cis-retinol. The mechanism of reverse isomerization was investigated using stable isotope-labeled retinoids and radioactive tracers to show that this reaction occurs with the retention of configuration of the C-15 carbon of retinol through a mechanism that does not eliminate the hydroxyl group, in contrast to the enzymatic all-trans-retinol to 11-cis-retinol reaction. The activation energy for the conversion of 11-cis-retinol to all-trans-retinol is 19.5 kcal/mol, and 20.1 kcal/mol for isomerization of 13-cis-retinol to all-trans-retinol. We also demonstrate that the reverse isomerization occurs in vivo using exogenous 11-cis-retinol injected into the intravitreal space of wild type and Rpe65-/- mice, which have defective forward isomerization. This study demonstrates an uncharacterized activity of RPE microsomes that could be important in the normal flow of retinoids in the eye in vivo during dark adaptation.

Cloning and characterization of a human beta,beta-carotene-15,15'-dioxygenase that is highly expressed in the retinal pigment epithelium.

Retinoids play a critical role in vision, as well as in development and cellular differentiation. beta,beta-Carotene-15,15'-dioxygenase (Bcdo), the enzyme that catalyzes the oxidative cleavage of beta,beta-carotene into two retinal molecules, plays an important role in retinoid synthesis. We report here the first cloning of a mammalian Bcdo. Human BCDO encodes a protein of 547 amino acid residues that demonstrates 68% identity with chicken Bcdo. It is expressed highly in the retinal pigment epithelium (RPE) and also in kidney, intestine, liver, brain, stomach, and testis. The gene spans approximately 20 kb, is composed of 11 exons and 10 introns, and maps to chromosome 16q21-q23. A mouse orthologue was also identified, and its predicted amino acid sequence is 83% identical with human BCDO. Biochemical analysis of baculovirus expressed human BCDO demonstrates the predicted beta,beta-carotene-15,15'-dioxygenase activity. The expression pattern of BCDO suggests that it may provide a local supplement to the retinoids available to photoreceptors, as well as a supplement to the retinoid pools utilized elsewhere in the body. In addition, the finding that many of the enzymes involved in retinoid metabolism are mutated in retinal degenerations suggests that BCDO may also be a candidate gene for retinal degenerative disease.

Characterization of a dehydrogenase activity responsible for oxidation of 11-cis-retinol in the retinal pigment epithelium of mice with a disrupted RDH5 gene. A model for the human hereditary disease fundus albipunctatus.

In the vertebrate retina, the final step of visual chromophore production is the oxidation of 11-cis-retinol to 11-cis-retinal. This reaction is catalyzed by 11-cis-retinol dehydrogenases (11-cis-RDHs), prior to the chromophore rejoining with the visual pigment apo-proteins. The RDH5 gene encodes a dehydrogenase that is responsible for the majority of RDH activity. In humans, mutations in this gene are associated with fundus albipunctatus, a disease expressed by delayed dark adaptation of both cones and rods. In this report, an animal model for this disease, 11-cis-rdh-/- mice, was used to investigate the flow of retinoids after a bleach, and microsomal membranes from the retinal pigment epithelium of these mice were employed to characterize remaining enzymatic activities oxidizing 11-cis-retinol. Lack of 11-cis-RDH leads to an accumulation of cis-retinoids, particularly 13-cis-isomers. The analysis of 11-cis-rdh-/- mice showed that the RDH(s) responsible for the production of 11-cis-retinal displays NADP-dependent specificity toward 9-cis- and 11-cis-retinal but not 13-cis-retinal. The lack of 13-cis-RDH activity could be a reason why 13-cis-isomers accumulate in the retinal pigment epithelium of 11-cis-rdh-/- mice. Furthermore, our results provide detailed characterization of a mouse model for the human disease fundus albipunctatus and emphasize the importance of 11-cis-RDH in keeping the balance between different components of the retinoid cycle.

Mechanism of rhodopsin activation as examined with ring-constrained retinal analogs and the crystal structure of the ground state protein.

The guanine nucleotide-binding protein (G-protein)-coupled receptor superfamily (GPCR) is comprised of a large group of membrane proteins involved in a wide range of physiological signaling processes. The functional switch from a quiescent to an active conformation is at the heart of GPCR action. The GPCR rhodopsin has been studied extensively because of its key role in scotopic vision. The ground state chromophore, 11-cis-retinal, holds the transmembrane region of the protein in the inactive conformation. Light induces cis-trans isomerization and rhodopsin activation. Here we show that rhodopsin regenerated with a ring-constrained 11-cis-retinal analog undergoes photoisomerization; however, it remains marginally active because isomerization occurs without the chromophore-induced conformational change of the opsin moiety. Modeling the locked chromophore analogs in the active site of rhodopsin suggests that the beta-ionone ring rotates but is largely confined within the binding site of the natural 11-cis-retinal chromophore. This constraint is a result of the geometry of the stable 11-cis-locked configuration of the chromophore analogs. These results suggest that the native chromophore cis-trans isomerization is merely a mechanism for repositioning of the beta-ionone ring which ultimately leads to helix movements and determines receptor activation.

Phosphorylation of RGS9-1 by an endogenous protein kinase in rod outer segments.

Inactivation of the visual G protein transducin, during recovery from photoexcitation, is regulated by RGS9-1, a GTPase-accelerating protein of the ubiquitous RGS protein family. Incubation of dark-adapted bovine rod outer segments with [gamma-(32)P]ATP led to RGS9-1 phosphorylation by an endogenous kinase in rod outer segment membranes, with an average stoichiometry of 0.2-0.45 mol of phosphates/mol of RGS9-1. Mass spectrometry revealed a single major site of phosphorylation, Ser(475). The kinase responsible catalyzed robust phosphorylation of recombinant RGS9-1 and not of an S475A mutant. A synthetic peptide corresponding to the region surrounding Ser(475) was also phosphorylated, and a similar peptide with the S475A substitution inhibited RGS9-1 phosphorylation. The RGS9-1 kinase is a peripheral membrane protein that co-purifies with rhodopsin in sucrose gradients and can be extracted in buffers of high ionic strength. It is not inhibited or activated significantly by a panel of inhibitors or activators of protein kinase A, protein kinase G, rhodopsin kinase, CaM kinase II, casein kinase II, or cyclin-dependent kinase 5, at concentrations 50 or more times higher than their reported IC(50) or K(i) values. It was inhibited by the protein kinase C inhibitor bisindolylmaleimide I and by lowering Ca(2+) to nanomolar levels with EGTA; however, it was not stimulated by the addition of phorbol ester, under conditions that significantly enhanced rhodopsin phosphorylation. A monoclonal antibody specific for the Ser(475)-phosphorylated form of RGS9-1 recognized RGS9-1 in immunoblots of dark-adapted mouse retina. Retinas from light-adapted mice had much lower levels of RGS9-1 phosphorylation. Thus, RGS9-1 is phosphorylated on Ser(475) in vivo, and the phosphorylation level is regulated by light and by [Ca(2+)], suggesting the importance of the modification in light adaptation.

Stereoisomeric specificity of the retinoid cycle in the vertebrate retina.

Understanding of the stereospecificity of enzymatic reactions that regenerate the universal chromophore required to sustain vision in vertebrates, 11-cis-retinal, is needed for an accurate molecular model of retinoid transformations. In rod outer segments (ROS), the redox reaction involves all-trans-retinal and pro-S-NADPH that results in the production of pro-R-all-trans-retinol. A recently identified all-trans-retinol dehydrogenase (photoreceptor retinol dehydrogenase) displays identical stereospecificity to that of the ROS enzyme(s). This result is unusual, because photoreceptor retinol dehydrogenase is a member of a short chain alcohol dehydrogenase family, which is often pro-S-specific toward their hydrophobic alcohol substrates. The second redox reaction occurring in retinal pigment epithelium, oxidation of 11-cis-retinol, which is largely catalyzed by abundantly expressed 11-cis-retinol dehydrogenase, is pro-S-specific to both 11-cis-retinol and NADH. However, there is notable presence of pro-R-specific activities. Therefore, multiple retinol dehydrogenases are involved in regeneration of 11-cis-retinal. Finally, the cellular retinaldehyde-binding protein-induced isomerization of all-trans-retinol to 11-cis-retinol proceeds with inversion of configuration at the C(15) carbon of retinol. Together, these results provide important additions to our understanding of retinoid transformations in the eye and a prelude for in vivo studies that ultimately may result in efficient pharmacological intervention to restore and prevent deterioration of vision in several inherited eye diseases.

Substrate specificity of mammalian prenyl protein-specific endoprotease activity.

We have previously identified proteolytic activity in rat liver microsomes that cleaves an intact tripeptide, VIS, from S-farnesylated-CVIS tetrapeptide. This enzymatic activity, termed prenyl protein-specific endoprotease (PPEP) activity, has been solubilized in CHAPS and purified 5-fold. To probe the peptide recognition features of PPEP activity, 64 tripeptides [N-acetyl-C(S-farnesyl)a1a2] were prepared and tested as competitive inhibitors of PPEP activity-catalyzed hydrolysis of N-acetyl-C(S-farnesyl)VI[3H]S. It was found that PPEP activity prefers large hydrophobic residues in the a1 and a2 positions. A subset of N-acetyl-C(S-farnesyl)a1a2 peptides were prepared in radiolabeled form, and it was found that PPEP activity preferences for these substrates correlated well in most cases with the inhibition data. The exception is that R in the a1 position does not prevent binding of peptide to PPEP activity, but such peptides are poor substrates. The anionic residue D in the a2 position is not tolerated by PPEP activity. Five farnesylated radiolabeled tetrapeptides, Ac-C(F)FM[3H]L, Ac-C(F)LI[3H]L, Ac-C(F)LL[3H]L, Ac-C(F)LM[3H]L, and Ac-C(F)VI[3H]L were prepared, and PPEP activity kinetic studies revealed that they are good substrates and show comparable KM values (2.2-13.5 microM). Ac-C(F)RL[3H]S is a poor substrate. The reported peptide binding preferences of PPEP activity should be useful in designing compounds that block the C-terminal proteolysis of prenylated proteins. Nonprenylated peptides do not bind to PPEP activity, and replacement of the farnesyl group with ann-pentadecyl group modestly reduces binding. Peptide-membrane partitioning studies were used together with theoretical arguments to fully understand the substrate specificity of PPEP activity toward these compounds.

A prenylated protein-specific endoprotease in rat liver microsomes that produces a carboxyl-terminal tripeptide.

The maturation of proteins that contain the C-terminal sequence Cys-Ali-Ali-Xaa (where Ali is usually an aliphatic amino acid and Xaa is a number of different amino acids) involves the attachment of a farnesyl or geranylgeranyl group to the cysteine residue, proteolytic removal of the C-terminal three amino acids, and methylation of the prenylated cysteine alpha-carboxyl group. Two prenylated and radiolabeled peptides were prepared in order to detect the proteolysis step(s) in a cell-free system and to determine the reaction products. These peptides are ECB-NPFRQRRFFC(S-geranylgeranyl)AI[3H]L and ECB-C(S-farnesyl)VI[3H]S (ECB is an extended-chain biotin group) which are patterned after the C-termini of geranylgeranylated and farnesylated G protein gamma-subunits. Incubation of these peptides with rat liver microsomes, but not cytosol, results in the production of radiolabeled dipeptides (I[3H]L and I[3H]S) and tripeptides (AI[3H]L and VI[3H]S) as the major products and smaller amounts of amino acids ([3H]L and [3H]S). A multitude of independent experiments shows that the dipeptides are produced from the tripeptides by secondary proteolysis. Although a portion of the [3H]S produced comes directly from ECB-C(S-farnesyl)VI[3H]S, the KM of > 30 microM for this reaction is significantly higher than the KM of 1.1 microM for the production of VI[3H]S from the farnesylated peptide. This suggests that the carboxypeptidase is not part of the pathway for the maturation of prenylated proteins. Nonprenylated peptides at concentrations of 10-100-fold higher than those of the prenylated substrates did not reduce the amount of tripeptide produced.(ABSTRACT TRUNCATED AT 250 WORDS)