The human ortholog of the rodent testis-specific ABC transporter Abca17 is a ubiquitously expressed pseudogene (ABCA17P) and shares a common 5' end with ABCA3

Background  

During the past years, we and others discovered a series of human ATP-binding cassette (ABC) transporters, now referred to as ABC A-subfamily transporters. Recently, a novel testis-specific ABC A transporter, Abca17, has been cloned in rodent. In this study, we report the identification and characterization of the human ortholog of rodent Abca17.     

Cloning of human and rat ABCA5/Abca5 and detection of a human splice variant

We presently report the cloning of cDNA sequences encoding the novel rat ATP-binding cassette (ABC) transporter Abca5 and the orthologous human transporter, recently designated as ABCA5. Furthermore, the existence of a novel non-translated exon of the ABCA5 gene, previously assigned to an ABCA gene cluster in the chromosomal region 17q24.2-3, is demonstrated. Abca5 and ABCA5 cDNAs are predicted to give rise to proteins of 1642 amino acids which exhibit the typical domain arrangement of ABC full transporters and share 90% identity within the amino acid sequences. A cDNA representing an ABCA5 mRNA splice variant was cloned which would result in a truncated protein equivalent to an ABC half transporter. Northern blot analyses revealed expression of ABCA5 or Abca5 mRNA in several tissues, but particularly high Abca5 mRNA expression was observed in rat testis. Up-regulation of Abca5 mRNA expression during culture of primary rat hepatocytes suggests that hepatocyte cultures should provide a basis for investigation of Abca5 gene regulation and elucidation of Abca5 function.     

Inhibition of Sphingosine Kinase-2 in a Murine Model of Lupus Nephritis

Sphingosine-1-phosphate (S1P), a potent bioactive lipid, is emerging as a central mediator in inflammation and immune responses. We have previously implicated S1P and its synthetic enzyme sphingosine kinase (SK) in inflammatory and autoimmune disorders, including inflammatory bowel disease and rheumatoid arthritis. Generation of S1P requires phosphorylation of sphingosine by SK, of which there are two isoforms. Numerous studies have implicated SK1 in immune cell trafficking, inflammation and autoimmune disorders. In this study, we set out to determine the role of SK and S1P in lupus nephritis (LN). To this end, we examined S1P and dihydro-S1P (dh-S1P) levels in serum and kidney tissues from a mouse model of LN. Interestingly dh-S1P was significantly elevated in serum and kidney tissue from LN mice, which is more readily phosphorylated by SK2. Therefore, we employed the use of the specific SK2 inhibitor, ABC294640 in our murine model of LN. Treatment with ABC294640 did not improve vascular or interstitial pathology associated with LN. However, mice treated with the SK2 inhibitor did demonstrate decreases in glomerular pathology and accumulation of B and T cells in the spleen these were not statistically different from lpr mice treated with vehicle. LN mice treated with ABC294640 did not have improved urine thromboxane levels or urine proteinuria measurements. Both S1P and dh-S1P levels in circulation were significantly reduced with ABC294640 treatment; however, dh-S1P was actually elevated in kidneys from LN mice treated with ABC294640. Together these data demonstrate a role for SKs in LN; however, they suggest that inhibition of SK1 or perhaps both SK isoforms would better prevent elevations in S1P and dh-S1P and potentially better protect against LN.     

The ABC transporters of Saccharomyces cerevisiae

The ABC transporters (ATP Binding Cassette) compose one of the bigest protein family with the great medical, industrial and economical impact. They are found in all organism from bacteria to man. ABC proteins are responsible for resistance of microorganism to antibiotics and fungicides and multidrug resistance of cancer cells. Mutations in ABC transporters genes cause seriuos deseases like cystic fibrosis, adrenoleucodystrophy or ataxia. Transport catalized by ABC proteins is charged with energy from the ATP hydrolysis. The ABC superfamily contains transporters, canals, receptors. Analysis of the Saccharomyces cerevisiae genome allowed to distinguish 30 potential ABC proteins which are classified into 6 subfamilies. The structural and functional similarity of the yeast and human ABC proteins allowes to use the S. cerevisiae as a model organism for ABC transporters characterisation. In this work the present state of knowleadge on yeast S. cerevisiae ABC proteins was summarised.     

Three ATP-binding cassette transporter genes,Abca14, Abca15, and Abca16, form a cluster on mouse Chromosome 7F3

We have identified and cloned three mouse genes that belong to the ABCA subfamily of ATP-binding cassette (ABC) transporters. These three genes are arranged in a tandem head-to-tail cluster spanning about 300 kb on mouse Chromosome (Chr) 7F3. Phylogenetic analysis indicates that although the three genes are related to human and mouse ABCA3, they are not orthologs of any of the current list of 48 human ABC genes and were, therefore, named Abca14, Abca15, and Abca16. The coding region of each gene is split into 31 exons, has an open reading frame of more than 1600 amino acids, and encodes a full transporter molecule with two nucleotide-binding folds (NBF) and two transmembrane domains (TMD). All three genes are predominantly expressed in testis, which suggests that they may perform special functions in testicular development or spermatogenesis. Interestingly, the human genome contains only fragments (less than ten exons) of at least two different ABC genes in the syntenic region on Chromosome 16p12 that are scattered among other, unrelated genes and are not capable of coding functional ABC transporters.(Zhang-qun Chen and Tarmo Annilo) These authors contributed equally to this study.Sequence data from this article have been deposited with the DDBJ/EMBL/GenBank Data Libraries under accession numbers AY243470–AY243472.     

Sphingosine 1-phosphate modulates spinal nociceptive processing

Sphingosine 1-Phosphate (S1P) modulates various cellular functions such as apoptosis, cell differentiation, and migration. Although S1P is an abundant signaling molecule in the central nervous system, very little is known about its influence on neuronal functions. We found that S1P concentrations were selectively decreased in the cerebrospinal fluid of adult rats in an acute and an inflammatory pain model. Pharmacological inhibition of sphingosine kinases (SPHK) decreased basal pain thresholds and SphK2 knock-out mice, but not SphK1 knock-out mice, had a significant decrease in withdrawal latency. Intrathecal application of S1P or sphinganine 1-phosphate (dihydro-S1P) reduced the pain-related (nociceptive) behavior in the formalin assay. S1P and dihydro-S1P inhibited cyclic AMP (cAMP) synthesis, a key second messenger of spinal nociceptive processing, in spinal cord neurons. By combining fluorescence resonance energy transfer (FRET)-based cAMP measurements with Multi Epitope Ligand Cartography (MELC), we showed that S1P decreased cAMP synthesis in excitatory dorsal horn neurons. Accordingly, intrathecal application of dihydro-S1P abolished the cAMP-dependent phosphorylation of NMDA receptors in the outer laminae of the spinal cord. Taken together, the data show that S1P modulates spinal nociceptive processing through inhibition of neuronal cAMP synthesis.     

UV irradiation inhibits ABC transporters via generation of ADP-ribose by concerted action of poly(ADP-ribose) polymerase-1 and glycohydrolase

ATP-binding cassette (ABC) transporters are involved in the transport of multiple substrates across cellular membranes, including metabolites, proteins, and drugs. Employing a functional fluorochrome export assay, we found that UVB irradiation strongly inhibits the activity of ABC transporters. Specific inhibitors of poly(ADP-ribose) polymerase-1 (PARP-1) restored the function of ABC transporters in UVB-irradiated cells, and PARP-1-deficient cells did not undergo UVB-induced membrane transport inhibition. These data suggest that PARP-1 activation is necessary for ABC transporter functional downregulation. The hydrolysis of poly(ADP-ribose) by poly(ADP-ribose) glycohydrolase (PARG) was also required, since specific PARG inhibitors, which limit the production of ADP-ribose molecules, restored the function of ABC transporters. Furthermore, ADP-ribose molecules potently inhibited the activity of the ABC transporter P-glycoprotein. Hence, poly(ADP-ribose) metabolism appears to play a novel role in the regulation of ABC transporters.     

Export and functions of sphingosine-1-phosphate

The sphingolipid metabolite, sphingosine-1-phosphate (S1P), has emerged as a critical player in a number of fundamental biological processes and is important in cancer, angiogenesis, wound healing, cardiovascular function, atherosclerosis, immunity and asthma, among others. Activation of sphingosine kinases, enzymes that catalyze the phosphorylation of sphingosine to S1P, by a variety of agonists, including growth factors, cytokines, hormones, and antigen, increases intracellular S1P. Many of the biological effects of S1P are mediated by its binding to five specific G protein-coupled receptors located on the cell surface in an autocrine and/or paracrine manner. Therefore, understanding the mechanism by which intracellularly generated S1P is released out of cells is both interesting and important. In this review, we will discuss how S1P is formed and released. We will focus particularly on the current knowledge of how the S1P gradient between tissues and blood is maintained, and the role of ABC transporters in S1P release.     

草地贪夜蛾与斜纹夜蛾解毒相关基因的比较分析

草地贪夜蛾Spodoptera frugiperda近期在中国为害猖獗,由于繁殖速度快,迁飞能力强,在本土呈现爆发趋势,严重为害了我国农作物。与本地近缘物种斜纹夜蛾Spodoptera litura相比,草地贪夜蛾更偏好玉米、水稻、小麦等禾本科农作物,且对多种化学杀虫剂及转基因Bt玉米产生抗性。寄主适应性以及杀虫剂抗性与解毒代谢相关蛋白密切相关。因此,本研究对这两种夜蛾科害虫的解毒代谢相关蛋白——细胞色素P450、谷胱甘肽转移酶(GST)及ABC转运蛋白进行了全基因组水平系统的搜集和数目比较,构建系统发育树并对P450和GST部分基因扩张分支进行氨基酸差异位点分析。结果显示,在草地贪夜蛾中共鉴定出213个P450基因、58个GST基因、102个ABC基因,其中P450基因与GST基因数目远远多于斜纹夜蛾(116,37),而ABC基因数目与斜纹夜蛾(99)接近。系统发育树分析表明,草地贪夜蛾P450在CYP6、CYP9以及CYP4功能簇,GST在部分进化分支上都发生了显著基因扩张,发生显著扩张的基因中有数个氨基酸突变,其中一些突变被预测可能影响蛋白质功能。但出乎意料的是,ABC亚家族B和E在斜纹夜蛾聚集成簇并发生了显著的基因扩张现象。以上结果暗示入侵种草地贪夜蛾和本地近缘物种斜纹夜蛾在抗性方面可能发展出各自独特的应对机制。本研究为解析草地贪夜蛾解毒抗性特征机制提供基因数据,为草地贪夜蛾的生物防治及抗性研究提供参考依据。     

Quantitative measurement of sphingosine 1-phosphate by radioreceptor-binding assay

In Chinese hamster ovary cells overexpressing Edg-1, one of the sphingosine 1-phosphate (S1P) receptor subtypes, [(3)H]S1P binding was displaced by unlabeled S1P with IC(50), a half-maximal concentration to inhibit the binding, of about 20 nM. This radioreceptor binding was used for quantitative measurement of S1P. Among the various lipids employed, only sphingosylphosphorylcholine (SPC), other than S1P, practically displaced the binding; however, the potency of SPC was about 100 to 1000 times less than that of S1P. Thus, SPC bound to the S1P receptors inefficiently. Furthermore, before the application of test samples to this assay, S1P was partially purified: the lipid was extracted first into the aqueous phase and separated from other lipids under alkaline conditions, and then reextracted into the chloroform phase under acidic conditions. With this assay, we could specifically and quantitatively measure S1P from 2 to 40 pmol per assay well in biological samples including serum samples and various tissues. This assay also allowed us to measure the change in cellular S1P content in U937 cells after treatment with exogenous sphingosine.     

Critical role of ABCA1 transporter in sphingosine 1-phosphate release from astrocytes

Sphingosine 1-phosphate (S1P) is accumulated in lipoproteins, especially high-density lipoprotein (HDL), in plasma. However, it remains uncharacterized how extracellular S1P is produced in the CNS. The treatment of rat astrocytes with retinoic acid and dibutyryl cAMP, which induce apolipoprotein E (apoE) synthesis and HDL-like lipoprotein formation, stimulated extracellular S1P accumulation in the presence of its precursor sphingosine. The released S1P was present together with apoE particles in the HDL fraction. S1P release from astrocytes was inhibited by the treatment of the cells with glybenclamide or small interfering RNAs specific to ATP-binding cassette transporter A1 (ABCA1). Astrocytes from Abca1−/− mice also showed impairment of retinoic acid/dibutyryl cAMP-induced S1P release in association with the blockage of HDL-like lipoprotein formation. However, the formation of either apoE or lipoprotein itself was not sufficient, and additional up-regulation of ABCA1 was requisite to stimulate S1P release. We conclude that the S1P release from astrocytes is coupled with lipoprotein formation through ABCA1.     

ATP-binding cassette ABCC1 is involved in the release of sphingosine 1-phosphate from rat uterine leiomyoma ELT3 cells and late pregnant rat myometrium

Sphingosine 1-phosphate (S1P), a bioactive lipid generated by sphingosine kinases (SphK1/2), initiates different signalling pathways involved in physiological and pathological processes. We previously demonstrated that in rat myometrium at late (day 19) gestation, SphK1 increases the expression of COX2 via S1P generation and release. In rat uterine leiomyoma cells (ELT3), SphK1/S1P axis controls survival and proliferation. In the present study we demonstrate that PDBu activates SphK1 but not SphK2. SphK1 activation requires PKC and MAPK ERK1/2. S1P produced by PDBu is released in the medium. PDBu-induced S1P export is abolished by Ro-318220 and BIM (PKC inhibitors), by U0126 and PD98059 (MEK inhibitors), SKI-II (SphKI/2 inhibitor) and SphK1-siRNA, suggesting the involvement of PKC, ERK and SphK1 respectively. The release of S1P is insensitive to inhibitors of ATP Binding Cassette (ABC)A1 and ABCB1 transporters, but is abolished when ABCC1 transporters are inhibited by MK571 or down-regulated by ABCC1-siRNA. PDBu increases COX2 expression that is blocked by the inhibition of PKC, ERK1/2, SphK1, and when cells are treated with MK571 or transfected with ABCC1-siRNA. The induction of COX2 by the S1P release due to PDBu or by exogenous S1P involves S1P2 receptors coupled to Gi. In myometrium from rat at late gestation, the release of S1P is also strongly reduced when SphK and ABCC1 are inhibited. The data reveal that in rat leiomyoma cells and late pregnant rat myometrium, the release of S1P involves a similar signalling pathway and occurs through ABCC1.     

Multidrug transport by the ABC transporter Sav1866 from Staphylococcus aureus

Sav1866 is an ATP-binding cassette (ABC) protein from the pathogen Staphylococcus aureus and is a homologue of bacterial and human multidrug ABC transporters. Recently, the three-dimensional crystal structure of Sav1866 was determined at 3.0 A resolution [Dawson, R. J., and Locher, K. P. (2006) Nature 443, 180-185]. Although this structure is frequently used to homology model human and microbial ABC multidrug transporters by computational methods, the ability of Sav1866 to transport multiple drugs has not been described. We obtained functional expression of Sav1866 in the drug-sensitive, Gram-positive bacterium Lactococcus lactis Delta lmrA Delta lmrCD lacking major endogenous multidrug transporters. Sav1866 displayed a Hoechst 33342, verapamil, tetraphenylphosphonium, and vinblastine-stimulated ATPase activity. In growing cells, Sav1866 expression conferred resistance to Hoechst 33342. In transport assays in intact cells, Sav1866 catalyzed the translocation of amphiphilic cationic ethidium. Additionally, Sav1866 mediated the active transport of Hoechst 33342 in membrane vesicles and proteoliposomes containing purified and functionally reconstituted protein. Sav1866-mediated resistance and transport were inhibited by the human ABCB1 and ABCC1 modulator verapamil. This work represents the first demonstration of multidrug transport by Sav1866 and suggests that Sav1866 can serve as a well-defined model for studies on the molecular bases of drug-protein interactions in ABC transporters. Our methods for the overexpression, purification, and functional reconstitution of Sav1866 are described in detail.     

Demonstration of Phosphoryl Group Transfer Indicates That the ATP-binding Cassette (ABC) Transporter Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Exhibits Adenylate Kinase Activity

Cystic fibrosis transmembrane conductance regulator (CFTR) is a membrane-spanning adenosine 5′-triphosphate (ATP)-binding cassette (ABC) transporter. ABC transporters and other nuclear and cytoplasmic ABC proteins have ATPase activity that is coupled to their biological function. Recent studies with CFTR and two nonmembrane-bound ABC proteins, the DNA repair enzyme Rad50 and a structural maintenance of chromosome (SMC) protein, challenge the model that the function of all ABC proteins depends solely on their associated ATPase activity. Patch clamp studies indicated that in the presence of physiologically relevant concentrations of adenosine 5′-monophosphate (AMP), CFTR Cl⁻ channel function is coupled to adenylate kinase activity (ATP+AMP ⇆ 2 ADP). Work with Rad50 and SMC showed that these enzymes catalyze both ATPase and adenylate kinase reactions. However, despite the supportive electrophysiological results with CFTR, there are no biochemical data demonstrating intrinsic adenylate kinase activity of a membrane-bound ABC transporter. We developed a biochemical assay for adenylate kinase activity, in which the radioactive γ-phosphate of a nucleotide triphosphate could transfer to a photoactivatable AMP analog. UV irradiation could then trap the ³²P on the adenylate kinase. With this assay, we discovered phosphoryl group transfer that labeled CFTR, thereby demonstrating its adenylate kinase activity. Our results also suggested that the interaction of nucleotide triphosphate with CFTR at ATP-binding site 2 is required for adenylate kinase activity. These biochemical data complement earlier biophysical studies of CFTR and indicate that the ABC transporter CFTR can function as an adenylate kinase.     

Upregulation of the expression of endogenous Mdr1 P-glycoprotein enhances lipid translocation in MDCK cells transfected with human MRP2

Various ABC transporters can translocate lipid molecules from the cytoplasmic into the exoplasmic leaflet of the plasma membrane bilayer. Two of these, MDR1 P-glycoprotein (Pgp) and MRP1, are multidrug transporters responsible for the resistance of various cancers against chemotherapy. We wanted to study whether MRP2, an ABC transporter of the bile canalicular membrane with a substrate specificity very similar to that of MRP1, is capable of translocating lipids. The translocation of short-chain lipids across the apical membrane of MDCK cells transfected with MRP2 was significantly higher than that in untransfected controls. However, the characteristics of the lipid translocation were similar to substrate transport by MDR1 and not MRP2: transport was strongly inhibited by classic MDR1 Pgp inhibitors, was independent of cellular glutathione, and was insensitive to a drug known to inhibit MRP2 activity. When tested by immunoblot, the MRP2-transfected cells expressed high levels of MRP2 but also of endogenous Mdr1. The expression of Mdr1 was unstable during maintenance of the cell line and correlated with the rate of lipid translocation across the apical membrane. We conclude that the observed increase in lipid transport in the MDCK cells transfected with MRP2 is the consequence of the upregulation of the expression of endogenous Mdr1 and that careful characterization of endogenous Mdr1 expression is needed in studies aimed to identify substrates of plasma membrane transporters.     

Dual role for phosphoinositides in regulation of yeast and mammalian phospholipase D enzymes

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite that regulates diverse biological processes by binding to a family of G protein-coupled receptors or as an intracellular second messenger. Mammalian S1P phosphatase (SPP-1), which degrades S1P to terminate its actions, was recently cloned based on homology to a lipid phosphohydrolase that regulates the levels of phosphorylated sphingoid bases in yeast. Confocal microscopy surprisingly revealed that epitope-tagged SPP-1 is intracellular and colocalized with the ER marker calnexin. Moreover, SPP-1 activity and protein appeared to be mainly enriched in the intracellular membranes with lower expression in the plasma membrane. Treatment of SPP-1 transfectants with S1P markedly increased ceramide levels, predominantly in the intracellular membranes, diminished survival, and enhanced apoptosis. Remarkably, dihydro-S1P, although a good substrate for SPP-1 in situ, did not cause significant ceramide accumulation or increase apoptosis. Ceramide accumulation induced by S1P was completely blocked by fumonisin B1, an inhibitor of ceramide synthase, but only partially reduced by myriocin, an inhibitor of serine palmitoyltransferase, the first committed step in de novo synthesis of ceramide. Furthermore, S1P, but not dihydro-S1P, stimulated incorporation of [3H]palmitate, a substrate for both serine palmitoyltransferase and ceramide synthase, into C16-ceramide. Collectively, our results suggest that SPP-1 functions in an unprecedented manner to regulate sphingolipid biosynthesis and is poised to influence cell fate.     

ATP-binding cassette transporters modulate both coelenterazine- and D-luciferin-based bioluminescence imaging

Bioluminescence imaging (BLI) of luciferase reporters provides a cost-effective and sensitive means to image biological processes. However, transport of luciferase substrates across the cell membrane does affect BLI readout intensity from intact living cells. To investigate the effect of ATP-binding cassette (ABC) transporters on BLI readout, we generated click beetle (cLuc), firefly (fLuc), Renilla (rLuc), and Gaussia (gLuc) luciferase HEK-293 reporter cells that overexpressed different ABC transporters (ABCB1, ABCC1, and ABCG2). In vitro studies showed a significant BLI intensity decrease in intact cells compared to cell lysates, when ABCG2 was overexpressed in HEK-293/cLuc, fLuc, and rLuc cells. Selective ABC transporter inhibitors were also applied. Inhibition of ABCG2 activity increased the BLI intensity more than two-fold in HEK-293/cLuc, fLuc, and rLuc cells; inhibition of ABCB1 elevated the BLI intensity two-fold only in HEK-293/rLuc cells. BLI of xenografts derived from HEK-293/ABC transporter/luciferase reporter cells confirmed the results of inhibitor treatment in vivo. These findings demonstrate that coelenterazine-based rLuc-BLI intensity can be modulated by ABCB1 and ABCG2. ABCG2 modulates d-luciferin-based BLI in a luciferase type-independent manner. Little ABC transporter effect on gLuc-BLI intensity is observed because a large fraction of gLuc is secreted. The expression level of ABC transporters is one key factor affecting BLI intensity, and this may be particularly important in luciferase-based applications in stem cell research.