NMR study of the binding of all-trans-retinoic acid to type II human cellular retinoic acid binding protein.

Cellular RA binding proteins are thought to play important roles in the (RA), a hormonally active metabolite of vitamin A that has profound effects on cell growth, + differentiation and morphogenesis. Binding of RA to type II human cellular RA binding proteins (CRABPII) has been investigated by NMR spectroscopy. The sequential resonance assignments of +CRABPII in the presence of RA were established by heteronuclear three-dimensional NMR at pH 7.3. The resonance assignments of the bound RA were achieved by homonucl NMR. The secondary structures of holo-CRABPII determined by NMR were ess as revealed by the crystal structure of holo-CRABPII. Most of the nuclear Overhauser effects (NOEs) between CRABPII and the bound RA were consistent with those predicted crystal structure of holo-CRABPII. The results suggested that the conformations in solution and in the crystalline state are highly similar. Compared to the ligand binding pocket, especially the ligand entrance, was stabilize Ser12-Leu18, one of the structure elements that constitute the ligand binding pocket, became more mobile upon binding of RA. Intramolecular NOEs between protons of the bo the carboxylate end of the bound RA is well fixed but the β-ionone     

Homozygous deletion of the CRABPI gene in AB1 embryonic stem cells results in increased CRABPII gene expression and decreased intracellular retinoic acid concentration

The cellular retinoic acid (RA) binding proteins I and II (CRABPI and CRABPII), intracellular proteins which bind retinoic acid with high affinity, are involved in the actions of RA, though their exact roles are not fully understood. We have generated several genetically engineered AB1 cell lines in which both alleles of the CRABPI gene have been deleted by homologous recombination. We have used these CRABPI knockout cell lines to examine the consequences of functional loss of CRABPI on RA-induced gene expression and RA metabolism in the murine embryonic stem cell line, AB1, which undergoes differentiation in response to RA. Complete lack of CRABPI results in decreased intracellular [3H]RA concentrations under conditions in which external concentrations of [3H]RA are low (1-10nM) and in an altered distribution of [3H] polar metabolites of [3H]RA in the cell and in the medium. Fewer [3H] polar metabolites are retained within the CRABPI(-/-) cells compared to the wild-type cells. These data suggest that CRABPI functions to regulate the intracellular concentrations of retinoic acid and to maintain high levels of oxidized retinoic acid metabolites such as 4-oxoretinoic acid within cells.     

Retinoic acid teratogenicity: the role of goosecoid and BMP-4.

Retinoic acid (RA) plays a pivotal role during vertebrate development, both as morphogen and as potent teratogen. While RA function in axial development has been extensively studied, little is known about the genetic control of RA teratogenicity. The knockout of the homeobox gene goosecoid in the mouse revealed similarities to RA induced embryopathy. We show that RA treatment of mouse gastrula embryos in vitro and of E10.5 embryos in utero led to a rapid but transient down-regulation of goosecoid expression. Repression was dependent on retinoid X receptors (RXR). BMP-4 was repressed by RA-treatment as well, both in embryos and in F9 teratocarcinoma cells. Our data suggest that both goosecoid and BMP-4 function as mediators of RA teratogenicity in mouse embryos.     

Retinoic acid-induced changes in differentiation-defective embryonal carcinoma RAC65 cells.

RAC65 is a mutant clone of mouse embryonal carcinoma cells, P19, which does not undergo terminal differentiation upon treatment with retinoic acid (RA). RAC65 cells express a truncated RA receptor alpha (RAR alpha) which, however, does not fully explain their defect. Here we show that RAC65 cells exhibit an additional defect in RAR alpha mRNA which may reflect a defect in RNA splicing. The parental and mutant cells also differ in their capacities to bind [3H]RA into nuclear fractions and in expression of cellular RA binding protein (CRABP) mRNA after treatment with RA. The combined data suggest that the defect in RAC65 RAR alpha results in reduced expression of the CRABP gene after RA treatment and, therefore, increased flow of RA into the nucleus.     

维生素A酸诱导小鼠F9-1胚胎性癌细胞分化过程中蛋白质的变化

本文报道了用双向凝胶电泳和彩色银染技术,分析小鼠F9-1胚胎性癌细胞在维生素A酸诱导分化过程中蛋白质的变化。发现维生素A酸处理细胞72小时后,全细胞蛋白质中有9种蛋白质消失,而新出现11种蛋白质。对细胞核蛋白质进行了双向电泳分析,观察到诱导分化后新出现8种蛋白质,其中有一些与全细胞蛋白质图谱上出现的变化完全对应。对核内低迁移率非组蛋白的分析发现,经维生素A酸处理后,一些低迁移率非组蛋白消失。蛋白质的这些变化可能与维生素A酸诱导细胞分化过程中基因活性的变化有关。