We have identified from mouse the first mammalian -carotene 15,15-dioxygenase (-CD), a crucial enzyme in development and metabolism that governs the de novo entry of vitamin A from plant-derived precursors. -CD is related to the retinal pigment epithelium-expressed protein RPE65 and belongs to a diverse family that includes the plant 9-cis-epoxycarotenoid dioxygenase and bacterial lignostilbene dioxygenases. -CD expression in Escherichia coli cells engineered to produce -carotene led to the accumulation of all-trans-retinal at the expense of -carotene, confirming that -CD catalyzed the central cleavage of this vitamin A precursor. Purified recombinant -CD protein cleaves -carotene in vitro with a V max of 36 pmol of retinal/mg of enzyme/ min and a K m of 6 M. Non-provitamin A carotenoids were also cleaved, although with much lower activity. By Northern analysis, a 2.4-kilobase (kb) message was observed in liver, kidney, small intestine, and testis, tissues important in retinoid/carotenoid metabolism. This message encoded a 63-kDa cytosolic protein expressed in these tissues. A shorter transcript of 1.8 kb was found in testis and skin. Developmentally, the 2.4-kb mRNA was abundant at embryonic day 7, with lower expression at embryonic days 11, 13, and 15, suggesting a critical role for this enzyme in gastrulation. Identification of -CD in an accessible model organism will create new opportunities to study vitamin A metabolism.In vertebrates, vitamin A in its various oxidative and isomeric forms is essential for embryonic development (1), pattern formation (2, 3), and vision (4). Retinoic acid, through its interaction with the nuclear retinoic acid receptor and retinoid X receptor, profoundly affects cell differentiation and development. Because animals are unable to synthesize vitamin A de novo from endogenous isoprenoid precursors, they must instead derive it from cleavage of -carotene and certain other carotenoids with an unsubstituted -ring (e.g. ␥-and ␣-carotenes, -zeacarotene, and -cryptoxanthin). It is generally accepted that central cleavage of -carotene by a putative dioxygenase gives rise to two molecules of all-trans-retinal, whereas eccentric cleavage with subsequent processing leading to a single molecule of retinoic acid from an apocarotenal is quantitatively far less important (5). -Carotene cleavage activity is reported highest in the intestinal mucosa, but is found at high activity levels in liver, kidney, lung, and fat tissues, among other sites. However, an inability to purify the protein catalyzing this reaction has hindered thorough investigation of this crucial first step in vitamin A metabolism.Because of a loose similarity between the mammalian protein RPE65 and the neoxanthin cleavage enzymes of plants, our laboratories have considered the hypothesis that the putative -CD 1 would belong to an emerging family of carotenoidcleaving dioxygenases known mainly from examples in plants (6), but with members also in bacteria and Metazoa. The first described representative was a ba...
A detailed understanding of the mechanism of lipid transport in insects has been hampered by the inability to identify the proapolipophorin gene that encodes apolipophorins I and II, the principal protein components of lipophorin, the lipid transport vehicle. Here we provide the first molecular description of the Drosophila gene encoding a retinoid-and fatty acid-binding glycoprotein (RFABG) and present evidence that it is a member of the proapolipophorin gene family. The gene, localized to the chromosome 4 (102 F region), encodes a 3351-amino acid protein that could serve as the precursor for the ϳ70-kDa and >200-kDa polypeptides associated with RFABG. The N-terminal sequence of the ϳ70-kDa polypeptide and that predicted for the >200-kDa polypeptide showed high sequence similarity to blowfly apolipophorin II and apolipophorin I, respectively. The RFABG precursor contains a signal peptide and exhibits a multidomain mosaic protein structure, which is typical of extracellular proteins. It has structural domains similar to lipid-binding proteins, namely vitellogenins and apolipoprotein B. The protein also contains a domain similar to the D domain of von Willebrand factor and mucin. The gene is expressed in the Drosophila embryo during development in cells that make up the amnioserosa and fat bodies. Immunolocalizations using specific antibodies against RFABG reveal that the protein is initially dispersed through the embryonic amnioserosa sac and latter concentrated at skeletal muscle-epidermis apodemeal contact junctions during larval development. This novel gene may play an important role in the transport of lipids, including retinoids and fatty acids, in insects.
ARPE-19, a human retinal pigment epithelial (RPE) cell line, has been widely used in studies of RPE function as well as gene expression. Here, we report the novel finding that N-(4-hydroxyphenyl)retinamide (fenretinide), a synthetic retinoic acid derivative and a potential chemopreventive agent against cancer, induced the differentiation of ARPE-19 cells into a neuronal phenotype. The treated cells lost their epithelial phenotype and exhibited a typical neuronal shape with long processes (four to five times longer than the cell body). The onset of fenretinide-induced neuronal differentiation was dose and time dependent, started within 1-2 days, and lasted at least 4 weeks. Immunohistochemical studies indicated that the expression of neurofilament proteins (NF160 and NF200), calretinin and neural cell adhesion molecule was increased in these differentiated cells. Western blot analysis indicated that cellular retinaldehyde-binding protein, which is normally expressed in RPE cells, was decreased in treated cells. Protein analysis on a two-dimensional gel followed by matrixassisted laser desorption ionization-time of flight mass spectrometric analysis demonstrated that heat-shock protein 70 was increased after fenretinide treatment. Thus, fenretinide, a synthetic retinoid, is able to induce neuronal differentiation of human RPE cells in culture.
We have characterized NORPEG, a novel gene from human retinal pigment epithelial cells (ARPE-19), in which its expression is induced by all-trans-retinoic acid. Two transcripts (ϳ3 and ϳ5 kilobases in size) have been detected for this gene, which is localized to chromosome band 5p13.2-13.3. Placenta and testis showed the highest level of expression among various human tissues tested. Six ankyrin repeats and a long coiled-coil domain are present in the predicted sequence of the NORPEG protein, which contains 980 amino acid residues. This ϳ110-kDa protein was transiently expressed in COS-7 cells as a FLAG fusion protein and immunolocalized to the cytoplasm. Confocal microscopic analysis of the NORPEG protein in ARPE-19 cells showed threadlike projections in the cytoplasm reminiscent of the cytoskeleton. Consistent with this localization, the expressed NORPEG protein showed resistance to solubilization by Triton X-100 and KCl. An ortholog of NORPEG characterized from mouse encoded a protein that showed 91% sequence similarity to the human NOR-PEG protein. The expression of Norpeg mRNA was detected in mouse embryo at embryonic day 9.5 by in situ hybridization, and the expression appears to be developmentally regulated. In adult mouse, the highest level of expression was detected in the seminiferous tubules of testis.
Age-related macular degeneration (AMD) is a multifactorial neurodegenerative disorder. Although molecular mechanisms remain elusive, deficits in autophagy have been associated with AMD. Here we show that deficiency of calcium and integrin binding protein 2 (CIB2) in mice, leads to age-related pathologies, including sub-retinal pigment epithelium (RPE) deposits, marked accumulation of drusen markers APOE, C3, Aβ, and esterified cholesterol, and impaired visual function, which can be rescued using exogenous retinoids. Cib2 mutant mice exhibit reduced lysosomal capacity and autophagic clearance, and increased mTORC1 signaling—a negative regulator of autophagy. We observe concordant molecular deficits in dry-AMD RPE/choroid post-mortem human tissues. Mechanistically, CIB2 negatively regulates mTORC1 by preferentially binding to ‘nucleotide empty’ or inactive GDP-loaded Rheb. Upregulated mTORC1 signaling has been implicated in lymphangioleiomyomatosis (LAM) cancer. Over-expressing CIB2 in LAM patient-derived fibroblasts downregulates hyperactive mTORC1 signaling. Thus, our findings have significant implications for treatment of AMD and other mTORC1 hyperactivity-associated disorders.
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