Localization of a fatty acid binding protein and its transcript in the developing chick retina

Godbout, Roseline; Marusyk, Halyna; Bisgrove, Dwayne A.; Dabbagh, Laith; Poppema, Sibrand

doi:10.1016/s0014-4835(05)80006-5

Cited by 20 publications

(20 citation statements)

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“…We obtained 8 mg of protein in the S4 fraction, 1 mg in P2, 0.5 mg in P3, and 2 mg in P4 fraction. The procedures related to the immunoelectron microscopy have been previously described (44).…”

Section: Methodsmentioning

confidence: 99%

Overexpression of a DEAD Box Protein (DDX1) in Neuroblastoma and Retinoblastoma Cell Lines

Godbout

Packer

Bie

1998

Journal of Biological Chemistry

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The DEAD box gene, DDX1, is a putative RNA helicase that is co-amplified with MYCN in a subset of retinoblastoma (RB) and neuroblastoma (NB) tumors and cell lines. Although gene amplification usually involves hundreds to thousands of kilobase pairs of DNA, a number of studies suggest that co-amplified genes are only overexpressed if they provide a selective advantage to the cells in which they are amplified. Here, we further characterize DDX1 by identifying its putative transcription and translation initiation sites. We analyze DDX1 protein levels in MYCN/DDX1-amplified NB and RB cell lines using polyclonal antibodies specific to DDX1 and show that there is a good correlation with DDX1 gene copy number, DDX1 transcript levels, and DDX1 protein levels in all cell lines studied. DDX1 protein is found in both the nucleus and cytoplasm of DDX1-amplified lines but is localized primarily to the nucleus of nonamplified cells. Our results indicate that DDX1 may be involved in either the formation or progression of a subset of NB and RB tumors and suggest that DDX1 normally plays a role in the metabolism of RNAs located in the nucleus of the cell.DEAD box proteins are a family of putative RNA helicases that are characterized by eight conserved amino acid motifs, one of which is the ATP hydrolysis motif containing the core amino acid sequence DEAD (Asp-Glu-Ala-Asp) (1-3). Over 40 members of the DEAD box family have been isolated from a variety of organisms including bacteria, yeast, insects, amphibians, mammals, and plants. The prototypic DEAD box protein is the translation initiation factor, eukaryotic initiation factor 4A, which, when combined with eukaryotic initiation factor 4B, unwinds double-stranded RNA (4). Other DEAD box proteins, such as p68, Vasa, and An3, can effectively and independently destabilize/unwind short RNA duplexes in vitro (5-7). Although some DEAD box proteins play general roles in cellular processes such as translation initiation (eukaryotic initiation factor 4A (4)), RNA splicing (PRP5, PRP28, and SPP81 in yeast (8 -10)), and ribosomal assembly (SrmB in Escherichia coli (11)), the function of most DEAD box proteins remains unknown. Many of the DEAD box proteins found in higher eukaryotes are tissue-or stage-specific. For example, PL10 mRNA is expressed only in the male germ line, and its product has been proposed to have a specific role in translational regulation during spermatogenesis (12). Vasa and ME31B are maternal proteins that may be involved in embryogenesis (13,14). p68, found in dividing cells (15), is believed to be required for the formation of nucleoli and may also have a function in the regulation of cell growth and division (16,17). Other DEAD box proteins are implicated in RNA degradation, mRNA stability, and RNA editing (18 -20).The human DEAD box protein gene DDX1 1 was identified by differential screening of a cDNA library enriched in transcripts present in the two RB cell lines Y79 and RB522A (21). The longest DDX1 cDNA insert isolated from this library was 2.4 kb with an ope...

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Section: Methodsmentioning

confidence: 99%

Overexpression of a DEAD Box Protein (DDX1) in Neuroblastoma and Retinoblastoma Cell Lines

Godbout

Packer

Bie

1998

Journal of Biological Chemistry

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“…In addition, a family of proteins known as the fatty acid-binding proteins are capable of binding to fatty acid moieties. The chick retina fatty acid protein displays little expression in post-embryonic chick retinas (57)(58)(59). Neither of these proteins share any sequence identity to the rod soluble ␦ subunit cDNA reported here.…”

Section: Figmentioning

confidence: 99%

Solubilization of Membrane-bound Rod Phosphodiesterase by the Rod Phosphodiesterase Recombinant δ Subunit

Florio

Prusti²,

Beavo

1996

Journal of Biological Chemistry

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Retinal rod and cone phosphodiesterases are oligomeric enzymes that consist of a dimeric catalytic core (␣ 2 in cones and ␣␤ in rods) with inhibitory subunits (␥) that regulate their activity. In addition, a 17-kDa protein referred to as the ␦ subunit co-purifies with the rod soluble phosphodiesterase and the cone phosphodiesterase. We report here partial protein sequencing of the rod ␦ subunit and isolation of a cDNA clone encoding it. The predicted amino acid sequence is unrelated to any other known protein. Of eight bovine tissue mRNA preparations examined by Northern analysis, the strongest ␦ subunit-specific signal was present in the retina. A less intense signal was seen in the brain and adrenal mRNA. In bovine retinal sections, rod ␦ subunit anti-peptide antibodies label rod but not cone outer segments. ␦ subunit, added back to washed outer segment membranes, solubilizes a large fraction of the membrane-bound phosphodiesterase, indicating that this subunit binds to the classical membrane associated phosphodiesterase. The subunit forms a tight complex with native, but not trypsin-released phosphodiesterase, suggesting that the isoprenylated carboxyl termini of the catalytic subunits may be involved in binding of the ␦ subunit to the phosphodiesterase holoenzyme.Phototransduction in the retina involves a cascade of regulated biochemical processes (for recent reviews, see Refs. 1 and 2). Key components in this light-activated biochemical cascade are the retinal specific phosphodiesterases (PDE6s).1 These phosphodiesterases catalyze the conversion of cGMP to 5Ј-GMP when activated by the G protein, transducin. The retinal phosphodiesterases are multimeric proteins composed of catalytic and inhibitory subunits. The large subunits of the rod and cone phosphodiesterases (the ␣ and ␤ subunits in rods and the ␣Ј subunit in cones) dimerize to form the catalytic core of the isozymes. The smaller 11-and 13-kDa subunits (rod ␥ and cone ␥) serve as inhibitors of the rod and cone phosphodiesterases, respectively (3-5). Hurwitz et al. (6) identified a 17-kDa protein that immunoprecipitated with bovine retinal phosphodiesterases. Gillespie and Beavo (7) and Gillespie et al. (8) later demonstrated that a protein of this size co-purified with the isotonically soluble rod and cone phosphodiesterases, but not with the membrane-bound rod phosphodiesterase. A function for this 17-kDa protein subunit (also referred to as the ␦ or 15-kDa subunit) has not been described.The rod membrane-bound phosphodiesterase is loosely associated with the membrane at least in part due to C-terminal isoprenyl and carboxymethyl post-translational modifications. Several investigators (9, 10) have demonstrated that the ␣ subunit is modified by a farnesyl (C-15) group and the ␤ subunit is modified by a geranylgeranyl (C-20) group on their carboxyl termini. In addition both COOH termini are methylated as reported by Swanson and Applebury (11) and others (12). Mutation of the conserved cysteine residue to serine in the CAAX isoprenylation motif in the ...

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“…At d7, 60% of retinal cells are proliferating (16) and R-FABP transcript levels are high (24,26). The relatively large size of the eyes at this late proliferative stage facilitates the accumulation of the large quantities of retinal tissue re- ϩ RNA was extracted from d5, d7, d10, and d16 chick retinas.…”

Section: Discussionmentioning

confidence: 99%

“…Interestingly, B-FABP was not detected in the retina of the developing mouse embryo (37). We have found R-FABP to be highly expressed from day 3 (d3) (the earliest stage tested) to d7 in the chick retina as well as from d5 to d19 in the chick brain (24,26), suggesting a role in chick retinal developmentmaturation in addition to the roles proposed for B-FABP in the brain.…”

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confidence: 85%

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Involvement of AP-2 in Regulation of theR-FABPGene in the Developing Chick Retina

Bisgrove

Monckton

Godbout

1997

Molecular and Cellular Biology

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Little is known regarding the molecular pathways that underlie the retinal maturation process. We are studying the regulation of the retinal fatty-acid-binding protein (R-FABP) gene, highly expressed in retinal precursor cells, to identify DNA regulatory elements and transcriptional factors involved in retinal development. Although the upstream sequence of the R-FABP gene is extremely GC rich, CpG methylation in this region is not implicated in the regulation of this gene because the 5 flanking DNA remains unmethylated with tissue differentiation when there is a dramatic decrease in R-FABP transcript levels. Using a combination of DNase I hypersensitivity experiments, gel shift assays, and DNase I footprinting, we have found three sites of DNA-protein interaction within 205 bp of 5 flanking DNA in the undifferentiated retina and four sites in the differentiated retina. DNA transfection analysis indicates that the first two footprints located within 150 bp of 5 flanking DNA are required for high levels of transcription in primary undifferentiated retinal cultures. The first footprint includes a putative TATA box and Sp1 binding sites while the second footprint contains a consensus AP-2 DNA binding site. Supershift experiments using antibodies to AP-2 and methylation interference experiments indicate that an AP-2-like transcription factor present in both late-proliferative-stage retina and differentiated retina binds to the upstream region of the R-FABP gene. A combination of data including the expression profile of AP-2 during retinal development and DNA transfection analysis using constructs mutated at critical residues within the AP-2 binding site suggests that AP-2 is a repressor of R-FABP transcription.The fatty-acid-binding protein (FABP) family consists of a number of structurally related proteins with characteristic cellular, tissue, and developmental distribution patterns. Members of this family include heart FABP, intestinal FABP, adipocyte lipid-binding protein, myelin P 2 , cellular retinoicacid-binding proteins, and cellular retinol-binding proteins (1, 51). Some FABPs, such as adipocyte FABP and myelin P 2 , are restricted to one tissue or organ type while others are more broadly expressed. For example, intestinal FABP is found in both intestine and stomach while liver FABP is present in liver, intestine, kidney, and stomach (reviewed in reference 51). Roles proposed for these proteins include the uptake, intracellular solubilization, storage, and/or delivery of fatty acids and retinoids (1). A role in signal transduction has also been proposed for heart FABP and adipocyte lipid-binding protein via phosphorylation of a tyrosine residue at position 19 by the insulin receptor (8, 41). Furthermore, mammary-derived growth inhibitor (also called heart FABP [46]) and liver FABP appear to be involved in the differentiation of mammary epithelial cells and in the control of hepatocyte cell proliferation, respectively (35,36,60). Some FABPs are located in both the nucleus and the cytoplasm, suggesting that the...

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Localization of a fatty acid binding protein and its transcript in the developing chick retina

Cited by 20 publications

References 20 publications

Overexpression of a DEAD Box Protein (DDX1) in Neuroblastoma and Retinoblastoma Cell Lines

Overexpression of a DEAD Box Protein (DDX1) in Neuroblastoma and Retinoblastoma Cell Lines

Solubilization of Membrane-bound Rod Phosphodiesterase by the Rod Phosphodiesterase Recombinant δ Subunit

Involvement of AP-2 in Regulation of theR-FABPGene in the Developing Chick Retina

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