Comparative study on the chromophore binding sites of rod and red-sensitive cone visual pigments by use of synthetic retinal isomers and analogues

A comparative study on the chromophore (retinal) binding sites of the opsin (R-photopsin) from chicken red-sensitive cone visual pigment (iodopsin) and that scotopsin) from bovine rod pigment (rhodopsin) was made by the aid of geometric isomers of retinal (all-trans, 13-cis, 11-cis, 9-cis, and 7-cis) and retinal analogues including fluorinated (14-F, 12-F, 10-F, and 8-F) and methylated (12-methyl) 11-cis-retinals. The stereoselectivity of R-photopsin for the retinal isomers and analogues was almost identical with that of scotopsin, indicating that the shapes of the chromophore binding sites of both opsins are similar, although the former appears to be somewhat more restricted than the latter. The rates of pigment formation from R-photopsin were considerably greater than those from scotopsin. In addition, all the iodopsin isomers and analogues were more susceptible to hydroxylamine than were the rhodopsin ones. These observations suggest that the retinal binding site of iodopsin is located near the protein surface. On the basis of the spectral properties of fluorinated analogues, a polar group in the chromophore binding site of iodopsin as well as rhodopsin was estimated to be located near the hydrogen atom at the C10 position of the retinylidene chromophore. A large difference in wavelength between the absorption maxima of iodopsin and rhodopsin was significantly reduced in the 9-cis and 7-cis pigments. On the assumption that the retinylidene chromophore is anchored rigidly at the alpha-carbon of the lysine residue and loosely at the cyclohexenyl ring, each of the two isomers would have the Schiff-base nitrogen at a position altered from that of the 11-cis pigments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spectroscopic study of the batho-to-lumi transition during the photobleaching of rhodopsin using ring-modified retinal analogues

Photochemical and subsequent thermal reactions of rhodopsin containing 9-cis-retinal [Rh(9)] or one of four analogues with 9-cis geometries formed from ring-modified retinals, alpha-retinal [alpha Rh(9)], acyclic retinal [AcRh(9)], acyclic alpha-retinal [Ac alpha Rh(9)], and 5-isopropyl-alpha-retinal [P alpha Rh(9)] were investigated by low-temperature spectrophotometry and nanosecond laser photolysis. Irradiation of each pigment at -180 degrees C produced a photosteady-state mixture containing the original 9-cis pigment, its 11-cis pigment, and a photoproduct, indicating that the primary process of each pigment is a photoisomerization of its chromophore. The photoproduct produced by the irradiation of AcRh(9) had an absorption spectrum red shifted from the original AcRh(9) and was identified as the batho intermediate of AcRh(9). It was converted to the lumi intermediate through a metastable species, the BL intermediate, which has never been detected in Rh(9) at low temperature and whose absorption maximum was at shorter wavelengths than that of the batho intermediate. In contrast, the absorption maxima of the photoproducts produced from the other analogue pigments were at shorter wavelengths than those of the original pigments. They were identified as BL intermediates on the basis of their absorption maxima and thermal stabilities. The formation time constant of the lumi intermediate at room temperature was found to be dependent on the extent of modification of the ring portion of the chromophore, decreasing with the complete truncation of the cyclohexenyl ring [Ac alpha Rh(9)] and increasing with the attachment of the isopropyl group to the ring [P alpha Rh(9)].(ABSTRACT TRUNCATED AT 250 WORDS)     

Substrate specificity and kinetics for VP14, a carotenoid cleavage dioxygenase in the ABA biosynthetic pathway

The plant growth regulator, abscisic acid (ABA), is synthesized via the oxidative cleavage of an epoxy-carotenoid. Specifically, a double bond is cleaved by molecular oxygen and an aldehyde is formed at the site of cleavage in both products. The Vp14 gene from maize encodes an oxidative cleavage enzyme for ABA biosynthesis and the recombinant VP14 protein catalyzes the cleavage reaction in vitro. The enzyme has a strict requirement for a 9-cis double bond adjacent to the site of cleavage (the 11-12 bond), but shows some plasticity in other features of carotenoids that are cleaved. A kinetic analysis with the 9-cis isomer of five carotenoids displays several substrate activity relationships. One of the carotenoids was not readily cleaved, but inhibited the cleavage of another substrate in mixed assays. Of the remaining four carotenoids used in this study, three of the substrates have similar V(max) values. The V(max) for the cleavage of one carotenoid substrate was significantly higher. Molecular modeling and several three-dimensional quantitative substrate-activity relationship programs were used to analyze these results. In addition to a 9-cis double bond, the presence and orientation of the ring hydroxyl affects substrate binding or the subsequent cleavage. Additional variations that affect substrate cleavage are proposed.     

Retinoid signalling and gene expression in neuroblastoma cells: RXR agonist and antagonist effects on CRABP-II and RARbeta expression

9-cis Retinoic acid (RA) induces gene expression in neuroblastoma cells more effectively and with different kinetics than other RA isomers, and could be acting in part through Retinoid X Receptors (RXRs). The aim of this study was to characterise the effects of an RXR agonist and RXR homodimer antagonist on the induction of cellular RA binding protein II (CRABP-II) and RA receptor-beta (RARbeta) in neuroblastoma cells in response to different retinoids. The RXR agonist, LDG1069, was as effective as all-trans RA in inducing gene expression, but less effective than 9-cis RA. The RXR-homodimer antagonist, LG100754, inhibited the induction of CRABP-II mRNA in SH-SY5Y neuroblastoma cells by 9-cis RA or the RXR-specific agonist LGD1069, but had no effect when used with all-trans RA. Conversely, LG100754 did not inhibit induction of RARbeta mRNA by 9-cis or all-trans RA, or by LGD1069. RAR- and RXR-specific ligands used together induced CRABP-II and RARbeta as effectively as 9-cis RA. These results demonstrate the value of combining RXR- and RAR-specific ligands to regulate RA-inducible gene expression. The possibility that RXR-homodimers mediate, in part, the induction of CRABP-II by 9-cis RA and RXR-specific ligands is discussed.     

Solid-state NMR analysis of ligand--receptor interactions reveals an induced misfit in the binding site of isorhodopsin

Rhodopsin is the photosensitive protein of the rod photoreceptor in the vertebrate retina and is a paradigm for the superfamily of G-protein-coupled receptors (GPCRs). Natural rhodopsin contains an 11-cis-retinylidene chromophore. We have prepared the 9-cis analogue isorhodopsin in a natural membrane environment using uniformly (13)C-enriched 9-cis retinal. Subsequently, we have determined the complete (1)H and (13)C assignments with ultra-high field solid-state magic angle spinning NMR. The 9-cis substrate conforms to the opsin binding pocket in isorhodopsin in a manner very similar to that of the 11-cis form in rhodopsin, but the NMR data reveal an improper fit of the 9-cis chromophore in this binding site. We introduce the term "induced misfit" to describe this event. Downfield proton NMR ligation shifts (Deltasigma(lig)(H) > 1 ppm) are observed for the 16,17,19-H and nearby protons of the ionone ring and for the 9-methyl protons. They provide converging evidence for global, nonspecific steric interactions between the chromophore and protein, and contrast with the specific interactions over the entire ionone ring and its substituents detected for rhodopsin. The Deltasigma(lig)(C) pattern of the polyene chain confirms the positive charge delocalization in the polyene associated with the protonation of the Schiff base nitrogen. In line with the misalignment of the ionone ring, an additional and anomalous perturbation of the (13)C response is detected in the region of the 9-cis bond. This provides evidence for strain in the isomerization region of the polyene and supports the hypothesis that perturbation of the conjugation around the cis bond induced by the protein environment assists the selective photoisomerization.     

Spectral tuning in salamander visual pigments studied with dihydroretinal chromophores.

In visual pigments, opsin proteins regulate the spectral absorption of a retinal chromophore by mechanisms that change the energy level of the excited electronic state relative to the ground state. We have studied these mechanisms by using photocurrent recording to measure the spectral sensitivities of individual red rods and red (long-wavelength-sensitive) and blue (short-wavelength-sensitive) cones of salamander before and after replacing the native 3-dehydro 11-cis retinal chromophore with retinal analogs: 11-cis retinal, 3-dehydro 9-cis retinal, 9-cis retinal, and 5,6-dihydro 9-cis retinal. The protonated Schiff's bases of analogs with unsaturated bonds in the ring had broader spectra than the same chromophores bound to opsins. Saturation of the bonds in the ring reduced the spectral bandwidths of the protonated Schiff's bases and the opsin-bound chromophores and made them similar to each other. This indicates that torsion of the ring produces spectral broadening and that torsion is limited by opsin. Saturating the 5,6 double bond in retinal reduced the perturbation of the chromophore by opsin in red and in blue cones but not in red rods. Thus an interaction between opsin and the chromophoric ring shifts the spectral maxima of the red and blue cone pigments, but not that of the red rod pigment.     

A new and efficient strategy for the synthesis of shimofuridin analogs: 2'-O-(4-O-stearoyl-alpha-L-fucopyranosyl)thymidine and -uridine

Two shimofuridin analogs: 2'-O-(4-O-stearoyl-alpha-L-fucopyranosyl)thymidine (2) and -uridine (3) have been synthesized using D-arabinose, L-fucose, thymine, uracil, and stearoyl chloride as the starting materials. The synthetic procedures involve the facile preparation of 1-(3,5-di-O-benzyl-beta-D-ribofuranosyl)thymine (9) and -uracil (10) by coupling of 1,2-anhydro-3,5-di-O-benzyl-alpha-D-ribofuranose (8) with silylated thymine and uracil, and then stereoselective formation of the 1,2-cis (alpha) interglycoside bonds through condensation of the nucleoside derivatives 9 and 10 with 2-(2,3-di-O-benzyl-4-O-stearoyl-beta-L-fucopyranosylsulfonyl) pyrimidine (18). The 1,2-anhydro-3,5-di-O-benzyl-alpha-D-ribofuranose (8) was prepared by an improved procedure from D-arabinose.     

Photosensitivity of 10-substituted visual pigment analogues: detection of a specific secondary opsin-retinal interaction

The photosensitivities of the bovine rhodopsin and gecko pigment 521 analogues regenerated from C-10-substituted analogues of 11-cis- and 9-cis-retinals were determined by two different methods. A similar reactivity trend was noted for both pigment systems as revealed in the photosensitivity of the gecko pigments and relative quantum yields of the bovine analogues. The 10-fluoro-11-cis photopigments had a photosensitivity less than, but approaching, that of the native (11-cis) visual pigment while the 10-fluoro-9-cis photopigments had a much lower photosensitivity than the parent 9-cis regenerated pigment. The results are interpreted in terms of recently described models of rhodopsin architecture and of the primary molecular reaction of visual pigments to light. The unusually low photoreactivity of the 10-fluoro-9-cis pigment molecule is viewed as the result of a regiospecific hydrogen-bonding interaction of the electronegative fluorine atom to the opsin.     

The barbamide biosynthetic gene cluster: a novel marine cyanobacterial system of mixed polyketide synthase (PKS)-non-ribosomal peptide synthetase (NRPS) origin involving an unusual trichloroleucyl starter unit

Barbamide was extracted from the marine cyanobacterium Lyngbya majuscula strain 19L as a chlorinated lipopeptide for its potent molluscicidal activity. Precursor incorporation studies indicated that it is derived from acetate, L-phenylalanine, L-leucine and L-cysteine. The gene cluster responsible for biosynthesis of barbamide (bar) was cloned and characterized in this study. DNA sequence analysis of cosmid pLM49 revealed a cluster of 12 open reading frames (barA-barK) extending 26 kb including the expected polyketide synthase and non-ribosomal peptide synthetase modules and tailoring genes. The genetic architecture and domain organization of the bar cluster supports the assignment based on the apparent co-linearity of the systems. The activity assay of adenylation domains of barD (A(D)), barE (A(E)) and barG (A(G2) for module 2) in an amino acid-dependent ATP-pyrophosphate exchange experiment supports the conclusion that barbamide is synthesized from acetate, L-phenylalanine, L-cysteine and L-leucine with trichloroleucine as a direct precursor by a mixed polyketide synthase/non-ribosomal polypeptide synthetase. Assembly of barbamide includes unique biochemical mechanisms for chlorination, one-carbon truncation during chain elongation, E-double bond formation and thiazole ring formation.     

9-cis and all-trans retinoic acid induce a similar phenotype in human teratocarcinoma cells

Abstract. Prior work has shown that all-trans retinoic acid (t-RA) treatment of the human teratocarcinoma (TC) cell line NTERA-2 clone D1 (abbreviated NT2/D1) induces a neuronal phenotype and other cell lineages. This study sought to explore the potential of 9-cis retinoic acid (9-cis RA) as a differentiation-inducing agent of this multipotent cell. Findings reported here show that 9-cis RA induced a phenotype similar to t-RA treatment of NT2/D1 cells. This similarity extended to their effects on the nuclear receptors retinoic acid receptor-β (RAR-β) and retinoid X receptor-α (RXR-α). Both retinoids prominently augmented RAR-β expression and transactivated a reporter plasmid containing putative RAR response elements (RAREs) with direct repeats separated by five nucleotides (DR5). Both retinoids had no appreciable effect on RXR-α expression and both minimally transactivated a reporter plasmid containing putative RXR response elements (RXREs) with direct repeats separated by one nucleotide (DR1). These studies suggest that 9-cis RA and t-RA activate common events during retinoid-mediated NT2/D1 differentiation. This hypothesis was supported by the finding that NT2/D1 cells rendered refractory to t-RA (NTZ/D1-R1) were also resistant to 9-cis RA. To discover alterations that could confer retinoid-refractoriness, retinoid receptor expression was examined in NT2/D1-R1 cells. In contrast to NT2/D1, the NT2/D1-R1 cell was found to have reduced RXR-α expression at the level of total cellular RNA. These studies establish the effectiveness of 9-cis RA as a differentiation agent of human TC cells and demonstrate that retinoids with different nuclear receptor affinities can induce similar phenotypes in NT2/D1 cells. In addition, the findings in the retinoid resistant NT2/Dl-R1 cell implicate a role for specific retinoid receptors in this human TC differentiation program.     

9-cis-retinoic acid decreases the level of its cognate receptor, retinoid X receptor, through acceleration of the turnover.

In this study, we investigated the ligand-mediated regulation of retinoid X receptor (RXR) in two human cell lines (HepG2 and JEG-3 cells), which have been reported to express RXRalpha predominantly. Western blot analysis revealed that a treatment with 1 microM 9-cis-retinoic acid (9-cis RA) for 24 h decreased RXRalpha protein level to 72 +/- 9 and 75 +/- 7% in HepG2 and JEG-3 cells, respectively, when the levels in the non-treated cells were expressed as 100%. The decrease was not due to the changes in steady-state level of RXRalpha mRNA or its stability as revealed by Northern blot analysis. However, the 9-cis RA treatment decreased the half-life of RXRalpha protein as determined by pulse-chase analysis. It was thus demonstrated that 9-cis RA downregulates RXRalpha by increasing the turnover of the protein in the two cell lines. The ligand-dependent downregulation of RXRalpha protein may be important for several hormonal signalings, in which the receptors heterodimerize with RXR.     

Kinetic analysis of mouse retinal dehydrogenase type-2 (RALDH2) for retinal substrates

Retinal dehydrogenase (RALDH) isozymes catalyze the terminal oxidation of retinol into retinoic acid (RA) that is essential for embryogenesis and tissue differentiation. To understand the role of mouse type 2 RALDH in synthesizing the ligands (all-trans and 9-cis RA) needed to bind and activate nuclear RA receptors, we determined the detailed kinetic properties of RALDH2 for various retinal substrates. Purified recombinant RALDH2 showed a pH optimum of 9.0 for all-trans retinal oxidation. The activity of the enzyme was lower at 37 degrees C compared to 25 degrees C. The efficiency of conversion of all-trans retinal to RA was 2- and 5-fold higher than 13-cis and 9-cis retinal, respectively. The K(m) for all-trans and 13-cis retinal were similar (0.66 and 0.62 microM, respectively). However, the K(m) of RALDH2 for 9-cis retinal substrate (2.25 microM) was 3-fold higher compared to all-trans and 13-cis retinal substrates. Among several reagents tested for their ability to either inhibit or activate RALDH2, citral and para-hydroxymercuribenzoic acid (p-HMB) inhibited and MgCl(2) activated the reaction. Comparison of the kinetic properties of RALDH2 for retinal substrates and its activity towards various reagents with those of previously reported rat kidney RALDH1 and human liver aldehyde dehydrogenase-1 showed distinct differences. Since RALDH2 has low K(m) and high catalytic efficiency for all-trans retinal, it may likely be involved in the production of all-trans RA in vivo.     

9-顺式维甲酸在原代培养猪前体脂肪细胞分化中的调控作用

采用RT-PCR、油红O染色法、油红O染色提取法和分光光度计法,探讨不同浓度9-顺式维甲酸(9-cisRA)(0-10$mol/L)对体外原代培养猪前体脂肪细胞分化的影响及其可能机制。结果表明,9-cisRA在脂肪细胞分化中因浓度不同而发挥不同作用。低浓度9-cisRA(0-10nmol/L)促进前体脂肪细胞分化,并上调RXRα、PPARγmRNA表达,增加前体脂肪细胞分化标志酶3-磷酸甘油脱氢酶(glyc-erol-3-phosphate dehydrogenase,GPDH)的活性;高浓度9-cisRA(100nmol/L-10$mol/L)则抑制前体脂肪细胞分化,并下调RXRα、PPARγmRNA表达,减少GPDH的活性。结果提示9-cisRA在猪前体脂肪细胞分化中,可能通过调控RXRα和PPARγ基因表达变化来发挥其促进或抑制作用。     

A novel human cytochrome P450, CYP26C1, involved in metabolism of 9-cis and all-trans isomers of retinoic acid

Retinoids are potent regulators of cell proliferation, cell differentiation, and morphogenesis and are important therapeutic agents in oncology and dermatology. The gene regulatory activity of endogenous retinoids is effected primarily by retinoic acid isomers (all-trans and 9-cis) that are synthesized from retinaldehyde precursors in a broad range of tissues and act as ligands for nuclear retinoic acid receptors. The catabolism of all-trans-retinoic acid (atRA) is an important mechanism of controlling RA levels in cell and tissues. We have previously identified two cytochrome P450s, P450RAI-1 and P450RAI-2 (herein named CYP26A1 and CYP26B1), which were shown to be responsible for catabolism of atRA both in the embryo and the adult. In this report, we describe the identification, molecular cloning, and substrate characterization of a third member of the CYP26 family, named CYP26C1. Transiently transfected cells expressing CYP26C1 convert atRA to polar water-soluble metabolites similar to those generated by CYP26A1 and -B1. Competition studies with all-trans, 13-cis, and 9-cis isomers of retinoic acid demonstrated that atRA was the preferred substrate for CYP26C1. Although CYP26C1 shares extensive sequence similarity with CYP26A1 and CYP26B1, its catalytic activity appears distinct from those of other CYP26 family members. Specifically, CYP26C1 can also recognize and metabolize 9-cis-RA and is much less sensitive than the other CYP26 family members to the inhibitory effects of ketoconazole. CYP26C1 is not widely expressed in the adult but is inducible by RA in HPK1a, transformed human keratinocyte cell lines. This third CYP26 member may play a specific role in catabolizing both all-trans and 9-cis isomers of RA.     

9-cis and all-trans retinoic acid induce a similar phenotype in human teratocarcinoma cells

Abstract. Prior work has shown that all-trans retinoic acid (t-RA) treatment of the human teratocarcinoma (TC) cell line NTERA-2 clone D1 (abbreviated NT2/D1) induces a neuronal phenotype and other cell lineages. This study sought to explore the potential of 9-cis retinoic acid (9-cis RA) as a differentiation-inducing agent of this multipotent cell. Findings reported here show that 9-cis RA induced a phenotype similar to t-RA treatment of NT2/D1 cells. This similarity extended to their effects on the nuclear receptors retinoic acid receptor-β (RAR-β) and retinoid X receptor-α (RXR-α). Both retinoids prominently augmented RAR-β expression and transactivated a reporter plasmid containing putative RAR response elements (RAREs) with direct repeats separated by five nucleotides (DR5). Both retinoids had no appreciable effect on RXR-α expression and both minimally transactivated a reporter plasmid containing putative RXR response elements (RXREs) with direct repeats separated by one nucleotide (DR1). These studies suggest that 9-cis RA and t-RA activate common events during retinoid-mediated NT2/D1 differentiation. This hypothesis was supported by the finding that NT2/D1 cells rendered refractory to t-RA (NT2/D1-R1) were also resistant to 9-cis RA. To discover alterations that could confer retinoid-refractoriness, retinoid receptor expression was examined in NT2/D1-R1 cells. In contrast to NT2/D1, the NT2/D1-R1 cell was found to have reduced RXR-α expression at the level of total cellular RNA. These studies establish the effectiveness of 9-cis RA as a differentiation agent of human TC cells and demonstrate that retinoids with different nuclear receptor affinities can induce similar phenotypes in NT2/D1 cells. In addition, the findings in the retinoid resistant NT2/D1-R1 cell implicate a role for specific retinoid receptors in this human TC differentiation program.     

Regiospecific effect of 1-octanol on cis-trans isomerization of unsaturated fatty acids in the solvent-tolerant strain Pseudomonas putida S12

The solvent-tolerant bacterium Pseudomonas putida S12, which adapts its membrane lipids to the presence of toxic solvents by a cis to trans isomerization of unsaturated fatty acids, was used to study possible in vivo regiospecificity of the isomerase. Cells were supplemented with linoleic acid (C18:2delta9-cis,delta12-cis), a fatty acid that cannot be synthesized by this bacterium, but which was incorporated into membrane lipids up to an amount of 15% of total fatty acids. After addition of 1-octanol, which was used as an activator of the cis-trans isomerase, the linoleic acid was converted into the delta9-trans,delta12-cis isomer, while the delta9-cis,delta12-trans and delta9-trans,epsilon12-trans isomers were not synthesized. Thus, for the first time, regiospecific in vivo formation of novel, mixed cis/trans isomers of dienoic fatty acid chains was observed. The maximal conversion (27-36% of the chains) was obtained at 0.03-0.04% (v/v) octanol, after 2 h. The observed regiospecificity of the enzyme, which is located in the periplasmic space, could be due to penetration of the enzyme to a specific depth in the membrane as well as to specific molecular recognition of the substrate molecules.     

Retinoic acid isomers produce malformations in postembryonic development of the Japanese flounder,Paralichthys olivaceus

We previously reported that characteristic deformities were induced by retinoic acid (RA) treatment of the Japanese flounder, Paralichthys olivaceus, at 6-9 days post-hatching (dph). To evaluate the toxic potency of nuclear retinoid receptors in induction of deformities by RA, we here investigated the effects of retinoic acid isomers on postembryonic development of this species. Larvae were exposed to either 25 nM of all-trans RA (atRA), 9-cis RA (9cRA) or 13-cis RA (13cRA) at 6-9 dph. All RA isomers induced deformities in the lower jaw, caudal fin and vertebrae. In the lower jaw, growth retardation of the dentary was evident. In the vertebrae, the major abnormalities were hypertrophy of the centrum, central fusion, and an increase in the number of abdominal vertebrae. Caudal fin deformities included deformity of caudal bone complex and absence of the entire caudal fin. The absence of the hypural primordium at 12 dph was the first sign of abnormality in caudal fin development, and resulted in complete blocking of the caudal fin development. Among the RA isomers, atRA induced the most severe deformity in all skeletons examined. Retinoic acid receptor (RAR) expression was activated by atRA and 9cRA, and pitx2 expression was inhibited in the lower jaw by atRA and 9cRA. Vitamin D receptor (VDR) expression was specifically inhibited by atRA treatment, suggesting that RA inhibits the lower jaw growth by suppressing the expression of these genes. These results suggest that RA exerted toxic effects on the skeletal systems, mainly through the RAR pathway.