Neuronal differentiation defects in induced pluripotent stem cells derived from a Prader-Willi syndrome patient.

Prader-Willi syndrome (PWS) is a neurodevelopmental disorder caused by a lack of expression of paternally inherited genes located in the15q11.2-q13 chromosome region. An obstacle in the study of human neurological diseases is the inaccessibility of brain material. Generation of induced pluripotent stem cells (iPSC cells) from patients can partially overcome this problem. We characterized the cellular differentiation potential of iPS cells derived from a PWS patient with a paternal 15q11-q13 deletion. A gene tip transcriptome array revealed very low expression of genes in the 15q11.2-q13 chromosome region, including SNRPN, SNORD64, SNORD108, SNORD109, and SNORD116, in iPS cells of this patient compared to that in control iPS cells. Methylation-specific PCR analysis of the SNRPN gene locus indicated that the PWS region of the paternal chromosome was deleted or methylated in iPS cells from the patient. Both the control and patient-derived iPS cells were positive for Oct3/4, a key marker of pluripotent cells. After 11 days of differentiation into neural stem cells (NSCs), Oct3/4 expression in both types of iPS cells was decreased. The NSC markers Pax6, Sox1, and Nestin were induced in NSCs derived from control iPS cells, whereas induction of these NSC markers was not apparent in NSCs derived from iPS cells from the patient. After 7 days of differentiation into neurons, neuronal cells derived from control iPS cells were positive for ßIII-tubulin and MAP2. However, neuronal cells derived from patient iPS cells only included a few immunopositive neurons. The mRNA expression levels of the neuronal marker ßIII-tubulin were increased in neuronal cells derived from control iPS cells, while the expression levels of ßIII-tubulin in neuronal cells derived from patient iPS cells were similar to those of NSCs. These results indicate that iPS cells derived from a PWS patient exhibited neuronal differentiation defects.

Protective and therapeutic effects of fucoxanthin against sunburn caused by UV irradiation.

Mild exposure to ultraviolet (UV) radiation is also harmful and hazardous to the skin and often causes a photosensitivity disorder accompanied by sunburn. To understand the action of UV on the skin we performed a microarray analysis to isolate UV-sensitive genes. We show here that UV irradiation promoted sunburn and downregulated filaggrin (Flg); fucoxanthin (FX) exerted a protective effect. In vitro analysis showed that UV irradiation of human dermal fibroblasts caused production of intracellular reactive oxygen species (ROS) without cellular toxicity. ROS production was diminished by N-acetylcysteine (NAC) or FX, but not by retinoic acid (RA). In vivo analysis showed that UV irradiation caused sunburn and Flg downregulation, and that FX, but not NAC, RA or clobetasol, exerted a protective effect. FX stimulated Flg promoter activity in a concentration-dependent manner. Flg promoter deletion and chromatin immunoprecipitation analysis showed that caudal type homeo box transcription factor 1 (Cdx1) was a key factor for Flg induction. Cdx1 was also downregulated in UV-exposed skin. Therefore, our data suggested that the protective effects of FX against UV-induced sunburn might be exerted by promotion of skin barrier formation through induction of Flg, unrelated to quenching of ROS or an RA-like action.

GPR87 mediates lysophosphatidic acid-induced colony dispersal in A431 cells.

We have previously reported that an orphan G protein-coupled receptor GPR87 was activated by lysophosphatidic acid (LPA) and that it induced an increase in the intracellular Ca(2+) levels in the CHO cells genetically engineered to express GPR87-Gα16 fusion protein. Because the Ca(2+) response was blocked by the LPA receptor antagonist Ki16425, GPR87 was suggested to be a putative LPA receptor. However, further studies are required to confirm whether GPR87 is an LPA receptor. A previous study showed that colonies of A431 cells treated with LPA showed rapid and synchronized dissociation. Because A431 cells have been shown to express GPR87, we used these cells to examine whether GPR87 acted as an LPA receptor. When A431 cells were treated with gpr87-specific siRNA, the expression of GPR87 was decreased and LPA-induced colony dispersal was significantly reduced. Treatment of the cells with lpa1 siRNA had an additive effect in decrease in the colony dispersal. Studies on the LPA-mediated signaling pathway in A431 cells indicated that transactivation of the epidermal growth factor receptor (EGFR) by LPA led to cell scattering. PD153035, an inhibitor of tyrosine-kinase of EGFR, and BB94, an inhibitor of metalloprotease which produces a ligand for EGFR, significantly prevented the LPA-induced scattering of A431 cells pretreated with lpa1 or gpr87-siRNA. These results strongly suggested that GPR87 acts as an LPA receptor and induces colony dispersal via the transactivation of EGFR in A431 cells.

Expression of orphan G-protein coupled receptor GPR174 in CHO cells induced morphological changes and proliferation delay via increasing intracellular cAMP.

We established cell lines that stably express orphan GPCR GPR174 using CHO cells, and studied physiological and pharmacological features of the receptor. GPR174-expressing cells showed cell-cell adhesion with localization of actin filaments to cell membrane, and revealed significant delay of cell proliferation. Since the morphological changes of GPR174-cells were very similar to mock CHO cells treated with cholera toxin, we measured the concentration of intracellular cAMP. The results showed the concentration was significantly elevated in GPR174-cells. By measuring intracellular cAMP concentration in GPR174-cells, we screened lipids and nucleotides to identify ligands for GPR174. We found that lysophosphatidylserine (LysoPS) stimulated increase in intracellular cAMP in a dose-dependent manner. Moreover, phosphorylation of Erk was elevated by LysoPS in GPR174 cells. These LysoPS responses were inhibited by NF449, an inhibitor of Gα(s) protein. These results suggested that GPR174 was a putative LysoPS receptor conjugating with Gα(s), and its expression induced morphological changes in CHO cells by constitutively activating adenylyl cycles accompanied with cell conjunctions and delay of proliferation.

In silico study on the substrate binding manner in human myo-inositol monophosphatase 2.

The human IMPA2 gene encoding myo-inositol monophosphatase 2 is highly implicated with bipolar disorder but the substrates and the reaction mechanism of myo-inositol monophosphatase 2 have not been well elucidated.9 In the present study, we constructed 3D models of three- and two-Mg(2+)-ion bound myo-inositol monophosphatase 2, and studied substrate-binding manners using the docking program AutoDock3. The subsequent study showed that the three-metal-ion model could interact with myo-inositol monophosphates, as follows: The phosphate moiety coordinated three Mg(2+) ions, and the inositol ring formed hydrogen bonds with the amino acids conserved in the family. Furthermore, the OH group vicinal to the phosphate group formed a hydrogen bond with a non-bridging oxygen atom of the phosphate. These interactions have been proposed as crucial for forming the transitional state, bipyramidal structure in the bovine myo-inositol monophosphatase. We therefore propose that the human myo-inositol monophosphatase 2 interacts with myo-inositol monophosphates in the three-metal-ion bound form, and proceeds the dephosphorylation through the three-metal-ion theory.

Biochemical studies on sphingolipids of Artemia franciscana: novel neutral glycosphingolipids.

Neutral glycosphingolipids containing one to six sugars in their oligosaccharide chains have been isolated from cysts of the brine shrimp Artemia franciscana. The structures of these glycolipids were identified by methylation analysis, partial acid hydrolysis, gas-liquid chromatography, combined gas-liquid chromatography-mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and proton nuclear magnetic resonance spectroscopy to be Glcß1-Cer, Manß1-4Glcß1-Cer, Fucα1-3Manß1-4Glcß1-Cer, GlcNAcß1-3Manß1-4Glcß1-Cer, GlcNAcα1-2Fucα1-3Manß1-4Glcß1-Cer, GalNAcß1-4GlcNAcß1-3Manß1-4Glcß1-Cer, GalNAcß1-4(Fucα1-3)GlcNAcß1-3Manß1-4Glcß1-Cer (CPS), and GalNAcß1-4(GlcNAcα1-2Fucα1-3)GlcNAcß1-3Manß1-4Glcß1-Cer (CHS). Two glycosphingolipids, CPS and CHS, were characterized as novel structures. Because Artemia contains a certain series of glycosphingolipids (-Fucα3Manß4GlcßCer), which differ from the core sugar sequences reported thus far, we tentatively designated the glycosphingolipids characterized as nonarthro-series ones. Furthermore, CHS exhibited a hybrid structure of arthro-series and nonarthro-series sugar chain. Two novel glycosphingolipids were characterized from the brine shrimp Artemia franciscana; one was composed of arthrotetraose and a branching fucose attached to N-acetylglucosamine residue, and the other was composed of CPS with an additional N-acetylglucosamine residue attached to the branching fucose.

Identification of the orphan GPCR, P2Y(10) receptor as the sphingosine-1-phosphate and lysophosphatidic acid receptor.

Phylogenetic analysis of transmembrane regions of GPCRs using PHYLIP indicated that the orphan receptor P2Y(10) receptor was classified into the cluster consisting nucleotide and lipid receptors. Based on the results, we studied the abilities of nucleotides and lipids to activate the P2Y(10) receptors. As a result, sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) evoked intracellular Ca(2+) increases in the CHO cells stably expressing the P2Y(10) fused with a G(16alpha) protein. These Ca(2+) responses were inhibited by S1P receptor and LPA receptor antagonists. The introduction of siRNA designed for P2Y(10) receptor into the P2Y(10)-CHO cells effectively blocked both S1P- and LPA-induced Ca(2+) increases. RT-PCR analysis showed that the mouse P2Y(10) was expressed in reproductive organs, brain, lung and skeletal muscle, suggesting the receptor plays physiological roles throughout the whole body. In conclusion, the P2Y(10) receptor is the first receptor identified as a dual lysophospholipid receptor.

The orphan GPCR GPR87 was deorphanized and shown to be a lysophosphatidic acid receptor.

In CHO cells stably expressing the GPR87 fused with a G16alpha protein, lysophosphatidic acid (LPA) evoked an intracellular Ca(2+) increase in a high affinity manner. The Ca(2+) increase was reversibly blocked by the LPA receptor antagonists and inhibited by pretreatment of the cells with GPR87-specific siRNAs. GPR87 was shown to be closer to the P2Y and P2Y-related receptors than LPA receptors by ClustalW analyses. However, none of nucleotides and their derivatives activated GPR87. The human gpr87 is located on the chromosome 3q25 in a cluster containing p2y12,13,14. RT-PCR analysis showed that the mouse GPR87 was expressed in placenta, ovary, testis, prostate, brain, and skeletal muscle. The 3D model of GPR87-LPA complex indicated that the ligand interacted with R115 and K296 of GPR87, which are well conserved in the P2Y receptors. These results suggest that the GPR87 is a LPA receptor which evolved from a common ancestor of P2Y receptors.

Effect of ATP on preadipocyte migration and adipocyte differentiation by activating P2Y receptors in 3T3-L1 cells.

The effect of extracellular ATP on adipogenesis was investigated using the mouse 3T3-L1 cell line. Incubation of cells with ATP (1-100 microM) for 5 min induced actin filament reorganization and membrane ruffling mediated through P2Y receptors. Enhancement of preadipocyte migration into fat cell clusters is one of the essential processes of adipose tissue development in vivo and cell migration assays revealed that stimulation of P2Y receptors enhanced chemokinesis (migration) in a concentration dependent manner. In this cell line, growth arrest is required before initiation of differentiation and growth-arrested post-confluent cells can be converted into adipocytes by the presence of the adipogenic hormones dexamethasone, 3-isobutyl-1-methylxanthine and insulin. On the other hand, those hormones alone do not trigger differentiation in proliferating cells. ATP did not induce differentiation when applied alone to either proliferating or postconfluent cells. By contrast, proliferating cells (density <50%) preincubated with ATP for 5 min and subsequently given the adipogenic hormones in the continued presence of ATP, underwent adipocyte differentiation mediated through phospholipase C-coupled P2Y receptors. These adipocytes were found to show very similar characteristics, including morphology and intracellular triacylglycerol accumulation compared with adipocytes differentiated from post-confluent preadipocytes with those adipogenic hormones. When proliferating cells were preincubated with ATP before the addition of the adipogenic hormones, gene expression of aP2 (adipose protein 2) was markedly increased within 6 days, whereas without ATP pretreatment the expression level stayed very low. These results suggest that extracellular ATP renders preadipocytes responsive to adipogenic hormones during the growth phase.

Identification of endogenous surrogate ligands for human P2Y12 receptors by in silico and in vitro methods.

Endogenous ligands acting on a human P2Y12 receptor, one of the G-protein coupled receptors, were searched by in silico screening against our own database, which contains more than 500 animal metabolites. The in silico screening using the docking software AutoDock resulted in selection of cysteinylleukotrienes (CysLTs) and 5-phosphoribosyl 1-pyrophosphate (PRPP), with high free energy changes, in addition to the known P2Y12 ligands such as 2MeSADP and ADP. These candidates were subjected to an in vitro Ca2+ assay using the CHO cells stably expressing P2Y12-G16alpha fusion proteins. We found that CysLTE4 and PRPP acted on the P2Y12 receptor as agonists with the EC50 values of 1.3 and 7.8 nM, respectively. Furthermore, we analyzed the phylogenetic relationship of the P2Y, P2Y-like, and CysLT receptors based on sequence alignment followed by evolutionary analyses. The analyses showed that the P2Y12, P2Y13, P2Y14, GPR87, CysLT-1, and CysLT-2 receptors formed a P2Y-related receptor subfamily with common sequence motifs in the transmembrane regions.

Detection of pairwise residue proximity by covariation analysis for 3D-structure prediction of G-protein-coupled receptors.

G-protein-coupled receptor (GPCR) is one of the most important targets for medicines. Homology modeling based on the crystal structure of bovine rhodopsin is currently the most frequently used method for GPCR targeted drug design. Information about residue-residue contacts and the structural specificity in the subfamily is essential for constructing more precise 3D structures, to distinguish the structural differences between the template and targets. In this study, we adopted the covariation analysis to extract information about residue-residue interactions from the amino acid sequence. In the opsin family, a large number of adjacent covarying residue pairs were detected. The detected residue pairs have a strong tendency to gather in some regions important for the structure and function. These results suggest that the covariation analysis is practically utilized to detect adjacent residue pairs and also to apply for predicting functional sites. Analyses of other GPCR subfamilies, olfactory receptor and chemokine receptor families, demonstrated that some adjacent covarying residue pairs were common. Thus, the covariation analysis has possibilities in the substantial improvement of the 3D-structure modeling of GPCRs and in the detection of functional sites such as the ligand-binding sites.

Statistical sequence analyses of G-protein-coupled receptors: structural and functional characteristics viewed with periodicities of entropy, hydrophobicity, and volume.

G-protein-coupled receptors (GPCRs) play a crucial role in signal transduction and receive a wide variety of ligands. GPCRs are a major target in drug design, as nearly 50% of all contemporary medicines act on GPCRs. GPCRs are membrane proteins possessing a common structural feature, seven transmembrane helices. In order to design an effective drug to act on a GPCR, knowledge of the three-dimensional (3D) structure of the target GPCR is indispensable. However, as GPCRs are membrane bound, their 3D structures are difficult to obtain. Thus we conducted statistical sequence analyses to find information about 3D structure and ligand binding using the receptors' primary sequences. We present statistical sequence analyses of 270 human GPCRs with regard to entropy (Shannon entropy in sequence alignment), hydrophobicity and volume, which are associated with the alpha-helical periodicity of the accessibility to the surrounding lipid. We found periodicity such that the phase changes once in the middle of each transmembrane region, both in the entropy plot and in the hydrophobicity plot. The phase shift in the entropy plot reflects the variety of ligands and the generality of the mechanism of signal transduction. The two periodic regions in the hydrophobicity plot indicate the regions facing the hydrophobic lipid chain and the polar phospholipid headgroup. We also found a simple periodicity in the plot of volume deviation, which suggests conservation of the stable structural packing among the transmembrane helices.

Analysis of the set of GABA(A) receptor genes in the human genome.

The genes of the ionotropic gamma-aminobutyric acid receptor (GABR) subunits have shown an unusual chromosomal clustering, but only now can this be fully specified by analyses of the human genome. We have characterized the genes encoding the 18 known human GABR subunits, plus one now located here, for their precise locations, sizes, and exon/intron structures. Clusters of 17 of the 19, distributed between five chromosomes, are specified in detail, and their possible significance is considered. By applying search algorithms designed to recognize sequences of all known GABR-type subunits in species from man down to nematodes, we found no new GABR subunit is detectable in the human genome. However, the sequence of the human orthologue of the rat GABR rho3 receptor subunit was uncovered by these algorithms, and its gene could be analyzed. Consistent with those search results, orthologues of the beta4 and gamma4 subunits from the chicken, not cloned from mammals, were not detectable in the human genome by specific searches for them. The relationships are consistent with the mammalian subunit being derived from the beta line and epsilon from the gamma line, with mammalian loss of beta4 and gamma4. In their structures the human GABR genes show a basic pattern of nine coding exons, with six different genomic mechanisms for the alternative splicing found in various subunits. Additional noncoding exons occur for certain subunits, which can be regulatory. A dicysteine loop and its exon show remarkable constancy between all GABR subunits and species, of deduced functional significance.

Identification of endogenous surrogate ligands for human P2Y receptors through an in silico search.

G protein-coupled receptors (GPCRs) are distributed widely throughout the human body, and nearly 50% of current medicines act on a GPCR. GPCRs are considered to consist of seven transmembrane alpha-helices that form an alpha-helical bundle in which agonists and antagonists bind. A 3D structure of the target GPCR is indispensable for designing novel medicines acting on a GPCR. We have previously constructed the 3D structure of human P2Y(1) (hP2Y(1)) receptor, a GPCR, by homology modeling with the 3D structure of bovine rhodopsin as a template. In the present study, we have employed an in silico screening for compounds that could bind to the hP2Y(1)-receptor model using AutoDock 3.0. We selected 21 of the 30 top-ranked compounds, and by measuring intracellular Ca(2+) concentration, we identified 12 compounds that activated or blocked the hP2Y(1) receptor stably expressed in recombinant CHO cells. 5-Phosphoribosyl-1-pyrophosphate (PRPP) was found to activate the hP2Y(1) receptor with a low ED(50) value of 15 nM. The Ca(2+) assays showed it had no significant effect on P2Y(2), P2Y(6), or P2X(2) receptors, but acted as a weak agonist on the P2Y(12) receptor. This is the first study to rationally identify surrogate ligands for the P2Y-receptor family.

Construction of hypothetical three-dimensional structure of P2Y1 receptor based on Fourier transform analysis.

G protein-coupled receptors constitute a large family of homologous transmembrane proteins that represents one of the most important classes of confirmed drug targets. For novel drug discovery, the 3D structure of target protein is indispensable. To construct hypothetical 3D structures of G protein-coupled receptors, several prediction methods have been proposed. But none of the them has confirmed a correct ligand binding site. In this study we constructed the 3D structure of bovine rhodopsin using the prediction method proposed by Donnelly et al., with some modification. We found that our 3D model showed a good agreement with the reported retinal binding site. Using the similar method, we constructed the 3D structure of the P2Y1 receptor; one of the G protein-coupled receptors, and showed a binding site of an endogenous ligand, ADP, on the basis of the 3D model and in vitro experimental data. These results should be valuable for design of a specific antagonist for P2Y1 receptor.