Molecular and phylogenetic characterization of a novel putative membrane transporter (SLC10A7), conserved in vertebrates and bacteria

The 'Solute Carrier Family SLC10' consists of six annotated members in humans, comprising two bile acid carriers (SLC10A1 and SLC10A2), one steroid sulfate transporter (SLC10A6), and three orphan carriers (SLC10A3 to SLC10A5). In this study we report molecular characterization and expression analysis of a novel member of the SLC10 family, SLC10A7, previously known as C4orf13. SLC10A7 proteins consist of 340-343 amino acids in humans, mice, rats, and frogs and show an overall amino acid sequence identity of >85%. SLC10A7 genes comprise 12 coding exons and show broad tissue expression pattern. When expressed in Xenopus laevis oocytes and HEK293 cells, SLC10A7 was detected in the plasma membrane but revealed no transport activity for bile acids and steroid sulfates. By immunofluorescence analysis of dual hemagglutinin (HA)- and FLAG-labeled SLC10A7 proteins in HEK293 cells, we established a topology of 10 transmembrane domains with an intracellular cis orientation of the N-terminal and C-terminal ends. This topology pattern is clearly different from the seven-transmembrane domain topology of the other SLC10 members but similar to hitherto uncharacterized non-vertebrate SLC10A7-related proteins. In contrast to the established SLC10 members, which are restricted to the taxonomic branch of vertebrates, SLC10A7-related proteins exist also in yeasts, plants, and bacteria, making SLC10A7 taxonomically the most widespread member of this carrier family. Vertebrate and bacterial SLC10A7 proteins exhibit >20% sequence identity, which is higher than the sequence identity of SLC10A7 to any other member of the SLC10 carrier family.     

Phosphodiesterase 10A Is Tethered to a Synaptic Signaling Complex in Striatum

Phosphodiesterase 10A (PDE10A) is a dual substrate PDE that can hydrolyze both cGMP and cAMP. In brain, PDE10A is almost exclusively expressed in the striatum. In several studies, PDE10A has been implicated in regulation of striatal output using either specific inhibitors or PDE10A knock-out mice and has been suggested as a promising target for novel antipsychotic drugs. In striatal medium spiny neurons, PDE10A is localized at the plasma membrane and in dendritic spines close to postsynaptic densities. In the present study, we identify PDE10A as the major cAMP PDE in mouse striatum and monitor PKA-dependent PDE10A phosphorylation. With recombinantly expressed PDE10A we demonstrate that phosphorylation does not alter PDE10A activity. In striatum, PDE10A was found to be associated with the A kinase anchoring protein AKAP150 suggesting the existence of a multiprotein signaling complex localizing PDE10A to a specific functional context at synaptic membranes. Furthermore, the cAMP effector PKA, the NMDA receptor subunits NR2A and -B, as well as PSD95, were tethered to the complex. In agreement, PDE10A was almost exclusively found in multiprotein complexes as indicated by migration in high molecular weight fractions in size exclusion chromatography. Finally, affinity of PDE10A to the signaling complexes formed around AKAP150 was reduced by PDE10A phosphorylation. The data indicate that phosphorylation of PDE10 has an impact on the interaction with other signaling proteins and adds an additional line of complexity to the role of PDE10 in regulation of synaptic transmission.     

Helix stabilization of amphipathic peptides by hydrocarbon stapling increases cholesterol efflux by the ABCA1 transporter

Apolipoprotein mimetic peptides are short amphipathic peptides that efflux cholesterol from cells by the ABCA1 transporter and are being investigated as therapeutic agents for cardiovascular disease. We examined the role of helix stabilization of these peptides in cholesterol efflux. A 23-amino acid long peptide (Ac-VLEDSFKVSFLSALEEYTKKLNTQ-NH2) based on the last helix of apoA-I (A10) was synthesized, as well as two variants, S1A10 and S2A10, in which the third and fourth and third and fifth turn of each peptide, respectively, were covalently joined by hydrocarbon staples. By CD spectroscopy, the stapled variants at 24 °C were more helical in aqueous buffer than A10 (A10 17%, S1A10 62%, S2A10 97%). S1A10 and S2A10 unlike A10 were resistant to proteolysis by pepsin and chymotrypsin. S1A10 and S2A10 showed more than a 10-fold increase in cholesterol efflux by the ABCA1 transporter compared to A10. In summary, hydrocarbon stapling of amphipathic peptides increases their helicity, makes them resistant to proteolysis and enhances their ability to promote cholesterol efflux by the ABCA1 transporter, indicating that this peptide modification may be useful in the development of apolipoprotein mimetic peptides.     

Effects of H-2 and vitamin A on eye defects in congenic mice.

Pregnant mice congenic with C57BL/10 (B10.A, B10.BR, B10.D2, B10.A(2R), B10.A(5R), B10.A(15R), B10.A(1R), B10.A(18R), and B10.OL) were fed Purina Mouse Chow or the same diet plus 200 IU of vitamin A daily. The pregnant dams were sacrificed on the 18th day of gestation and the fetuses were sexed and examined for defects in eye development. It was found that the frequency of microphthalmia and anophthalmia in the female progeny of mice fed Mouse Chow was 7.4-9.2% in B10.A and B10.BR, 4.0-5.5% in B10.A(18R), B10, B10.A(5R), B10.A(1R), B10.A(15R), and B10.A(2R), and 0.8% and 1.4% in B10.D2 and B10.OL mice, respectively. On average, the frequency of these defects in the female progeny was 6.2 times greater than that in males (P less than 0.001). The right eye was 5.8 times more often affected than the left (P less than 0.001). The addition of vitamin A to the diet increased the frequency of these eye abnormalities in all strains, suggesting that this effect is not mediated by loci associated with H-2, as is the case with vitamin A-enhanced cleft palate. The addition of vitamin A to the diet did not affect the ratios of affected males to females, affected right to left eye, or microphthalmia to anophthalmia. The results suggest that there are two loci on chromosome 17, one centromeric to E beta and one telemeric to C4, that interact to determine to some degree the frequency of microphthalmia and anophthalmia.     

A new DNA marker (D10S94) very tightly linked to the multiple endocrine neoplasia type 2A (MEN2A) locus.

Combined somatic cell hybrid and linkage studies between D10S94 and five pericentromeric loci (FNRB, D10Z1, MEN2A, RBP3, and D10S15) have localized the new DNA sequence pcl1/A1S-6-c23 at D10S94 to 10q11.2. No recombinants were observed between D10S94 and D10Z1 or MEN2A. D10S94 maps in proximal 10q11.2 very near to MEN2A. There are three possible orders for the six loci that we investigated from the centromeric region of chromosome 10. At present the genetic data do not allow us to order MEN2A with respect to D10Z1 and D10S94. The three possible orders are FNRB-D10Z1-D10S94-MEN2A-RBP3-D10S15, FNRB-D10Z1-MEN2A-D10S94-RBP3-D10S15, and FNRB-MEN2A-D10Z1-D10S94-RBP3-D10S15. In view of the fact that no recombinants between D10S94 and MEN2A or between D10S94 and D10Z1 were observed, the combined haplotypes formed from RFLPs and D10Z1 and D10S94 will increase the informativeness and accuracy of genotype prediction for at-risk members of the families having the MEN 2A syndrome, particularly when the affected parent is female. The localization of D10S94 with respect to MEN2A will prove valuable in experiments directed toward cloning the MEN2A locus.     

The porcine 2A10 antigen is homologous to human CD163 and related to macrophage differentiation

The mAb 2A10 recognizes a 120-kDa protein with sequence homology to the human CD163 and whose expression is restricted to the cells of the porcine monocyte/macrophage lineage. While most of tissue macrophages express high levels of 2A10 Ag, bone marrow cells and a subset of blood monocytes are negative for this marker. The percentage of 2A10+ blood monocytes ranges between 5-50% depending on the donor. The phenotypic analysis indicates that these cells are more similar to mature macrophages than 2A10- monocytes. 2A10+ monocytes express higher levels of swine histocompatibility leukocyte Ag II, CD16, and the adhesion molecules very late Ag-4 (CD49d) and LFA-1 (CD11a) than 2A10- monocytes, while CD14 and SWC1 expression is lower. Both monocyte subsets also differ in their functional capabilities. 2A10+ monocytes induce a greater allogeneic response on T lymphocytes than 2A10- cells. LPS-stimulated 2A10+ and 2A10- monocytes both produce proinflammatory cytokines (TNF-alpha and IL-1alpha), but antiinflammatory IL-10 is only detected on the latter population. When 2A10- monocytes were cultured in medium containing pig serum, they acquired some phenotypic features of 2A10+ cells, expressing the 2A10 Ag. In contrast, when they were cultured in the presence of L929 supernatant as a source of GM-CSF, the 2A10 Ag expression remained low, scarcely increasing over basal levels. 2A10+ cells cultured with pig serum developed features that resemble monocyte-derived dendritic cells. These results indicate that 2A10+ monocytes could constitute a cell population in a more advanced maturation stage than 2A10- circulating monocytes.     

Physical and functional interactions between vaccinia virus F10 protein kinase and virion assembly proteins A30 and G7

An early step in vaccinia virus morphogenesis, the association of crescent membranes with electron-dense granular material, is perturbed when expression of the viral protein encoded by the A30L or G7L open reading frame is repressed. Under these conditions, we found that phosphorylation of the A17 membrane protein, which is mediated by the F10 kinase, was severely reduced. Furthermore, A30 and G7 stimulated F10-dependent phosphorylation of A17 in the absence of other viral late proteins. Evidence for physical interactions between A30, G7, and F10 was obtained by their coimmunoprecipitation with antibody against A30 or F10. In addition, phosphorylation of A30 was dependent on the F10 kinase and autophosphorylation of F10 was stimulated by A30 and G7. Nevertheless, the association of A30, G7, and F10 occurred even with mutated, catalytically inactive forms of F10. Just as A30 and G7 are mutually dependent on each other for stability, F10 was nearly undetectable in the absence of A30 and G7. The reverse is not true, however, as repression of F10 did not diminish A30 or G7. Interaction of F10 with A30 and G7 presumably occurred within the virus factory areas of the cytoplasm, where each was concentrated. F10 localized predominantly in the cortical region of immature virions, beneath the membrane where A17 is located. F10 remained associated with the particulate core fraction of mature virions after treatment with a nonionic detergent and reducing agent. The formation of protein complexes such as the one involving A30, G7, and F10 may be a mechanism for the regulated packaging and processing of virion components.     

Increased social interaction in mice deficient of the striatal medium spiny neuron-specific phosphodiesterase 10A2

Cyclic nucleotide phosphodiesterase 10A (PDE10A) is a member of phosphodiesterase families that degrade cAMP and/or cGMP in distinct intracellular sites. PDE10A has a dual activity on hydrolysis of both cAMP and cGMP, and is prominently expressed in the striatum and the testis. Previous studies suggested that PDE10A is involved in regulation of locomotor activity and potentially related to psychosis, but concrete physiological roles of PDE10A remains elusive yet. In this study, we genetically inactivated PDE10A2, a prominent isoform of PDE10A in the brain, in mice, and demonstrate that PDE10A2 deficiency results in increased social interaction without any major influence on different other behaviors, along with increased levels of striatal cAMP. We also demonstrate that PDE10A2 is selectively distributed in medium spiny neurons, but not interneurons, of the striatal complex. Thus, our results establish a physiological role for PDE10A2 in regulating cAMP pathway and social interaction, and suggest that cAMP signaling cascade in striatal medium spiny neurons might be involved in regulating social interaction behavior in mice.     

Correlation between HLA antigens with clinical features of mixed infection of hepatitis A and HBV-carriers

It is showed that HAV+HBV mixed infection is a genetically determined pathology. Following HLA-antigens were immunogenetic markers of the disease: HLA-A10, B21, Cw2, Cw5, A10-A19, 88-813, B21-B35, A3-821, A9-B21. Lower risk of disease development was associated with HLA-B5, A2-Cw3, A3-Cw4, B35-Cw4, A3-B35-Cw4. Atypical forms of the hepatitis A were often met in carriers of HLA-Cw5, 827-835, A3-814, A3-B21, A9-B21, whereas typical forms - in carriers of HLA-A10, Cw2, A10-A19, B8-813, 821-835. Mild forms of hepatitis A were associated with the presence of HLA-A10,B22, A10-A19, B8-B13, A3-B21, A9-B8, A10-814, A10-822, A10-Cw3 in the patients' phenotype, whereas intermediate and severe forms - with the presence of HLA-B17, 817-818, 821 - 835, A28-B21, B18-Cw2. The findings about distribution of HLA-antigens and their combinations in mixed hepatitis A+B can be used in attempt of their prediction and prevention.     

Effects of H-2 complex and non-H-2 background on urethane-induced chromosomal aberrations in mice

The incidence of in vivo urethane-induced chromosomal aberrations was examined in H-2 congenic strains of mice with B10 and A backgrounds. Chromosome analysis of bone-marrow cells could divide 7 lines of A.H-2 congenic strains into 2 groups: one with a higher frequency of chromosomal aberrations such as in A/Wy (haplotype H-2a), A/J (H-2a), A.AL (H-2al) and A.TL (H-2tl), and the other consisting of A.TH (H-2t2), A.CA (H-2f), A.BY (H-2b) and A.SW (H-2s). The same tendency was also observed in the spleen cells. Among B10.H-2 congenic mice, B10.A (H-2a), B10.BR (H-2k), B10.A(3R) (H-2i3), B10.A(5R) (H-2i5) and B10.S(9R) (H-2t4) exhibited significantly higher rates of induced chromosomal aberrations than those in B10 (H-2b), B10.S (H-2s), B10.A(2R) (H-2h2), B10.A(4R) (H-2h4) and B10.S(7R) (H-2t2). To determine the effect on non-H-2 genetic backgrounds on urethane-induced chromosomal aberrations, 4 pairs of strains which have the same H-2 haplotypes, such as in B10 vs. A.BY (H-2b), B10.A vs. A/Wy (H-2a), B10.S vs. A.SW (H-2s), and B10.S(7R) vs. A.TH (H-2t2), were compared. The strains with a B10 background exhibited significantly higher frequencies of deletions and lower frequencies of exchanges than the strains with an A background. These data suggested that at least two genes are involved in the regulation of urethane-induced chromosomal aberrations in mice, one of which is mapped between the S and D regions in the H-2 complex, and another not belonging to H-2.     

Phospholipid Flippase ATP10A Translocates Phosphatidylcholine and Is Involved in Plasma Membrane Dynamics

We showed previously that ATP11A and ATP11C have flippase activity toward aminophospholipids (phosphatidylserine (PS) and phosphatidylethanolamine (PE)) and ATP8B1 and that ATP8B2 have flippase activity toward phosphatidylcholine (PC) (Takatsu, H., Tanaka, G., Segawa, K., Suzuki, J., Nagata, S., Nakayama, K., and Shin, H. W. (2014) J. Biol. Chem. 289, 33543–33556). Here, we show that the localization of class 5 P4-ATPases to the plasma membrane (ATP10A and ATP10D) and late endosomes (ATP10B) requires an interaction with CDC50A. Moreover, exogenous expression of ATP10A, but not its ATPase-deficient mutant ATP10A(E203Q), dramatically increased PC flipping but not flipping of PS or PE. Depletion of CDC50A caused ATP10A to be retained at the endoplasmic reticulum instead of being delivered to the plasma membrane and abrogated the increased PC flipping activity observed by expression of ATP10A. These results demonstrate that ATP10A is delivered to the plasma membrane via its interaction with CDC50A and, specifically, flips PC at the plasma membrane. Importantly, expression of ATP10A, but not ATP10A(E203Q), dramatically altered the cell shape and decreased cell size. In addition, expression of ATP10A, but not ATP10A(E203Q), delayed cell adhesion and cell spreading onto the extracellular matrix. These results suggest that enhanced PC flipping activity due to exogenous ATP10A expression alters the lipid composition at the plasma membrane, which may in turn cause a delay in cell spreading and a change in cell morphology.     

3-phenylacetylamino-2,6-piperidinedione inhibition of rat Nb2 lymphoma cell mitogenesis

3-Phenylacetylamino-2,6-piperidinedione (A10), an amino acid analog, has been reported to possess antineoplastic activity against certain neoplastic tissues. The antimitogenic properties of A10 were studied by determining its effect on prolactin (PRL)- and interleukin 2 (IL-2)-stimulated mitogenic responses in the rat Nb2 lymphoma cell line. The addition of A10 (1-12 mM) to PRL (0.4 ng/ml)-stimulated cells inhibited growth in a dose-dependent manner. DNA synthesis patterns studied by thymidine incorporation demonstrated that A10 was significantly inhibitory (25% at 20 hr; 50% at 40 hr, P less than 0.01). IL-2 stimulation of mitogenesis was also sensitive to A10 inhibition. The inhibition of PRL stimulated mitogenesis was reversible when A10 was removed after 24 hr of culture and A10 showed no toxicity in a chromium release assay. These data suggest that A10 effects may be cytostatic, rather than cytotoxic.     

Effects of H-2 and vitamin A on micrognathia in congenic mice.

Pregnant mice congenic with C57BL/10 (B10.A, B10.BR, B10.D2, B10.A(2R), B10.A(5R), B10.A(15R), B10.A(1R), B10.A(18R), and B10.OL) were fed Purina Mouse Chow or the same diet plus 200 IU of vitamin A daily. The pregnant dams were sacrificed on the eighteenth day of gestation, and the fetuses were sexed and examined for defects in mandibular development. On average, micrognathia occurred five times more frequently in female (1.5%) than male (0.3%) fetuses. The addition of vitamin A to the diet affected only females, reducing the frequency of this defect to that observed in males from dams fed the control diet. Micrognathia was strongly associated with micro- or anophthalmia, but not with defects of the palate. C57BL/10 fetuses had the highest frequency of micrognathia (3.2%) and B10.D2 and B10.A(5R) fetuses had the lowest (0.1%). The results suggest that a locus distal to C4 and perhaps proximal to Qa-1 may exert a moderate influence on mandibular development and a second locus proximal to E beta may have a weak effect.     

Selection of an attenuated Coxsackievirus B3 variant, using a monoclonal antibody reactive to myocyte antigen.

Previously, we described a heart-reactive monoclonal antibody (MAb), 10A1, derived from a coxsackievirus B3 (CVB3)-infected mouse. This MAb selectively inhibits infection of HeLa cells and myocytes with the myocarditic virus variant (CVB3W). A plaque-purified variant (H3) of CVB3W was isolated from the heart of an infected animal, and a second virus (H3-10A1) was obtained by growing H3 in HeLa cells in the presence of MAb 10A1. As with the parental CVB3W virus, H3 infection of HeLa cells can be inhibited by MAb 10A1, but the antibody-selected H3-10A1 variant is resistant to MAb inhibition (presumably an escape mutant). BALB/c mice infected with 10(6) PFU of CVB3W, H3, or H3-10A1 resulted in approximately 90% animal mortality with CVB3W or H3 and less than 10% mortality with H3-10A1, suggesting that the escape mutant is less pathogenic. Additionally, hearts from animals infected with H3-10A1 demonstrated only half the amount of myocarditis observed in either CVB3W- or H3-infected mice. Cardiac virus titers were also reduced approximately 200-fold in H3-10A1-infected animals compared with those in mice given the pathogenic variants. In vitro studies indicate that H3-10A1 is less effective in inhibiting cellular RNA and protein synthesis and show reduced virus replication compared with that of pathogenic viruses in cultured myocytes.     

Trp22, Trp24, and Tyr8 play a pivotal role in the binding of the family 10 cellulose-binding module from Pseudomonas xylanase A to insoluble ligands

Aromatic amino acids are believed to play a pivotal role in carbohydrate-binding proteins, by forming hydrophobic stacking interactions with the sugar rings of their target ligands. Family 10 cellulose-binding modules (CBM10s), present in a number of cellulases and xylanases expressed by Pseudomonas fluorescens subsp. cellulosa, contain two tyrosine and three tryptophan residues which are highly conserved. To investigate whether these amino acids play an important role in the interaction of CBM10 from P. fluorescens subsp. cellulosa xylanase A (Pf Xyn10A) with cellulose, each of these residues was changed to alanine in CBM10 expressed as a discrete module or fused to the catalytic domain of Pf Xyn10A (CBM10-CD), and the capacity of the mutant proteins of CBM10-CD to bind the polysaccharide was evaluated. The data showed that W22A, W24A, and Y8A bound very weakly to cellulose compared to the wild-type protein, while Y12A retained its capacity to interact with the glucose polymer. When the W7A mutation was introduced into CBM10 the protein domain did not accumulate in Escherichia coli. In contrast, the W7A mutant of CBM10-CD was efficiently expressed in E. coli, although the protein bound very weakly to cellulose. NMR spectra of wild-type CBM10, W22A, and W24A were very similar, suggesting that the mutations did not significantly affect the protein fold. Titration of wild-type CBM10, W22A, and W24A with N-bromosuccinimide indicated that Trp22 and Trp24 were on the surface of the protein, while Trp7 was buried. Collectively, these data indicate that Trp22, Trp24, and Tyr8 play a direct role in the binding of Pf Xyn10A CBM10 to cellulose. The results are discussed in the light of the three-dimensional structure of CBM10 [Raghothama, S., Simpson, P. J., Szabó, L., Nagy, T., Gilbert, H. J., and Williamson, M. P. (2000) Biochemistry 39, 978-984].     

Isolation and characterization of PDE10A, a novel human 3', 5'-cyclic nucleotide phosphodiesterase.

A gene encoding a novel human 3', 5'-cyclic nucleotide phosphodiesterase (PDE) was identified and characterized. PDE10A1 encodes a protein that is 779 amino acids in length. An incomplete cDNA for a second 5'-splice variant, PDE10A2, was isolated. The proteins encoded by the two variants share 766 amino acids in common. This common region includes an amino-terminal domain with partial homology to the cGMP-binding domains of PDE2, PDE5 and PDE6 as well as a carboxy-terminal region with homology to the catalytic regions of mammalian PDEs. Northern analysis revealed that PDE10A is widely expressed. The PDE10A gene was mapped to three yeast artificial chromosomes (YACs) that contain human DNA from chromosome 6q26-27. A recombinant protein corresponding to the 766 amino acid region common to PDE10A1 and PDE10A2 was expressed in yeast. It hydrolyzed both cAMP and cGMP. Inhibitors that are selective for other PDE families are poor inhibitors of PDE10A; however, PDE10A is inhibited by the non-specific PDE inhibitor, IBMX.