Vesicular nucleotide transporter is a molecular target of eicosapentaenoic acid for neuropathic and inflammatory pain treatment.

Eicosapentaenoic acid (EPA), an omega-3 (ω-3) polyunsaturated fatty acid, is an essential nutrient that exhibits antiinflammatory, neuroprotective, and cardiovascular-protective activities. Although EPA is used as a nutrient-based pharmaceutical agent or dietary supplement, its molecular target(s) is debatable. Here, we showed that EPA and its metabolites strongly and reversibly inhibit vesicular nucleotide transporter (VNUT), a key molecule for vesicular storage and release of adenosine triphosphate (ATP) in purinergic chemical transmission. In vitro analysis showed that EPA inhibits human VNUT-mediated ATP uptake at a half-maximal inhibitory concentration (IC50) of 67 nM, acting as an allosteric modulator through competition with Cl-. EPA impaired vesicular ATP release from neurons without affecting the vesicular release of other neurotransmitters. In vivo, VNUT-/- mice showed a delay in the onset of neuropathic pain and resistance to both neuropathic and inflammatory pain. EPA potently attenuated neuropathic and inflammatory pain in wild-type mice but not in VNUT-/- mice without affecting the basal nociception. The analgesic effect of EPA was canceled by the intrathecal injection of purinoceptor agonists and was stronger than that of existing drugs used for neuropathic pain treatment, with few side effects. Neuropathic pain impaired insulin sensitivity in previous studies, which was improved by EPA in the wild-type mice but not in the VNUT-/- mice. Our results showed that VNUT is a molecular target of EPA that attenuates neuropathic and inflammatory pain and insulin resistance. EPA may represent a unique nutrient-based treatment and prevention strategy for neurological, immunological, and metabolic diseases by targeting purinergic chemical transmission.

Vesicular nucleotide transporter mediates ATP release and migration in neutrophils.

Neutrophils migrate to sites infected by pathogenic microorganisms. This migration is regulated by neutrophil-secreted ATP, which stimulates neutrophils in an autocrine manner through purinergic receptors on the plasma membrane. Although previous studies have shown that ATP is released through channels at the plasma membrane of the neutrophil, it remains unknown whether it is also released through alternate secretory systems involving vesicular mechanisms. In this study, we investigated the possible involvement of vesicular nucleotide transporter (VNUT), a key molecule for vesicular storage and nucleotide release, in ATP secretion from neutrophils. RT-PCR and Western blotting analysis indicated that VNUT is expressed in mouse neutrophils. Immunohistochemical analysis indicated that VNUT mainly colocalized with matrix metalloproteinase-9 (MMP-9), a marker of tertiary granules, which are secretory organelles. In mouse neutrophils, ATP release was inhibited by clodronate, which is a potent VNUT inhibitor. Furthermore, neutrophils from VNUT-/- mice did not release ATP and exhibited significantly reduced migration in vitro and in vivo These findings suggest that tertiary granule-localized VNUT is responsible for vesicular ATP release and subsequent neutrophil migration. Thus, these findings suggest an additional mechanism through which ATP is released by neutrophils.

UUAT1 Is a Golgi-Localized UDP-Uronic Acid Transporter That Modulates the Polysaccharide Composition of Arabidopsis Seed Mucilage.

UDP-glucuronic acid (UDP-GlcA) is the precursor of many plant cell wall polysaccharides and is required for production of seed mucilage. Following synthesis in the cytosol, it is transported into the lumen of the Golgi apparatus, where it is converted to UDP-galacturonic acid (UDP-GalA), UDP-arabinose, and UDP-xylose. To identify the Golgi-localized UDP-GlcA transporter, we screened Arabidopsis thaliana mutants in genes coding for putative nucleotide sugar transporters for altered seed mucilage, a structure rich in the GalA-containing polysaccharide rhamnogalacturonan I. As a result, we identified UUAT1, which encodes a Golgi-localized protein that transports UDP-GlcA and UDP-GalA in vitro. The seed coat of uuat1 mutants had less GalA, rhamnose, and xylose in the soluble mucilage, and the distal cell walls had decreased arabinan content. Cell walls of other organs and cells had lower arabinose levels in roots and pollen tubes, but no differences were observed in GalA or xylose contents. Furthermore, the GlcA content of glucuronoxylan in the stem was not affected in the mutant. Interestingly, the degree of homogalacturonan methylation increased in uuat1 These results suggest that this UDP-GlcA transporter plays a key role defining the seed mucilage sugar composition and that its absence produces pleiotropic effects in this component of the plant extracellular matrix.

Deletion of the uracil permease gene confers cross-resistance to 5-fluorouracil and azoles in Candida lusitaniae and highlights antagonistic interaction between fluorinated nucleotides and fluconazole.

We characterized two additional membrane transporters (Fur4p and Dal4p) of the nucleobase cation symporter 1 (NCS1) family involved in the uptake transport of pyrimidines and related molecules in the opportunistic pathogenic yeast Candida lusitaniae. Simple and multiple null mutants were constructed by gene deletion and genetic crosses. The function of each transporter was characterized by supplementation experiments, and the kinetic parameters of the uptake transport of uracil were measured using radiolabeled substrate. Fur4p specifically transports uracil and 5-fluorouracil. Dal4p is very close to Fur4p and transports allantoin (glyoxyldiureide). Deletion of the FUR4 gene confers resistance to 5-fluorouracil as well as cross-resistance to triazoles and imidazole antifungals when they are used simultaneously with 5-fluorouracil. However, the nucleobase transporters are not involved in azole uptake. Only fluorinated pyrimidines, not pyrimidines themselves, are able to promote cross-resistance to azoles by both the salvage and the de novo pathway of pyrimidine synthesis. A reinterpretation of the data previously obtained led us to show that subinhibitory doses of 5-fluorocytosine, 5-fluorouracil, and 5-fluorouridine also were able to trigger resistance to fluconazole in susceptible wild-type strains of C. lusitaniae and of different Candida species. Our results suggest that intracellular fluorinated nucleotides play a key role in azole resistance, either by preventing azoles from targeting the lanosterol 14-alpha-demethylase or its catalytic site or by acting as a molecular switch for the triggering of efflux transport.

Inhibitors of ATP release inhibit vesicular nucleotide transporter.

Vesicular nucleotide transporter (VNUT) is responsible for vesicular ATP storage in ATP-secreting cells. In the present study, we examined the effects on VNUT-mediated transport of ATP release inhibitors such as ATP-binding cassette (ABC) proteins, hemichannels, maxi anion channels and P2X7 receptor. The ATP transport activity of proteoliposomes containing purified human VNUT was blocked by glibenclamide, carbenoxolone, 18 α-glycyrrhetinic acid, flufenamic acid, arachidonic acid and A438079 without the formation of Δψ (positive inside) as a driving force being affected. Thus, inhibitors of ATP release may inhibit VNUT and subsequent ATP release, since the previous works proved that inhibitors of ATP release blocked VNUT-mediated ATP release at the cell level.

Comparative bioenergetic assessment of transformed cells using a cell energy budget platform.

The aberrant expression and functional activity of proteins involved in ATP production pathways may cause a crisis in energy generation for cells and compromise their survival under stressful conditions such as excitation, starvation, pharmacological treatment or disease states. Under resting conditions such defects are often compensated for, and therefore masked by, alternative pathways which have significant spare capacity. Here we present a multiplexed 'cell energy budget' platform which facilitates metabolic assessment and cross-comparison of different cells and the identification of genes directly or indirectly involved in ATP production. Long-decay emitting O(2) and pH sensitive probes and time-resolved fluorometry are used to measure changes in cellular O(2) consumption, glycolytic and total extracellular acidification (ECA), along with the measurement of total ATP and protein content in multiple samples. To assess the extent of spare capacity in the main energy pathways, the cells are also analysed following double-treatment with carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone and oligomycin. The four-parametric platform operating in a high throughput format has been validated with two panels of transformed cells: mouse embryonic fibroblasts (MEFs) lacking the Krebs cycle enzyme fumarate hydratase (Fh1) and HeLa cells with reduced expression of pyrimidine nucleotide carrier 1. In both cases, a marked reduction in both respiration and spare respiratory capacity was observed, accompanied by a compensatory activation of glycolysis and consequent maintenance of total ATP levels. At the same time, in Fh1-deficient MEFs the contribution of non-glycolytic pathways to the ECA did not change.

Dual targeting to mitochondria and plastids of AtBT1 and ZmBT1, two members of the mitochondrial carrier family.

Zea mays and Arabidopsis thaliana Brittle 1 (ZmBT1 and AtBT1, respectively) are members of the mitochondrial carrier family. Although they are presumed to be exclusively localized in the envelope membranes of plastids, confocal fluorescence microscopy analyses of potato, Arabidopsis and maize plants stably expressing green fluorescent protein (GFP) fusions of ZmBT1 and AtBT1 revealed that the two proteins have dual localization to plastids and mitochondria. The patterns of GFP fluorescence distribution observed in plants stably expressing GFP fusions of ZmBT1 and AtBT1 N-terminal extensions were fully congruent with that of plants expressing a plastidial marker fused to GFP. Furthermore, the patterns of GFP fluorescence distribution and motility observed in plants expressing the mature proteins fused to GFP were identical to those observed in plants expressing a mitochondrial marker fused to GFP. Electron microscopic immunocytochemical analyses of maize endosperms using anti-ZmBT1 antibodies further confirmed that ZmBT1 occurs in both plastids and mitochondria. The overall data showed that (i) ZmBT1 and AtBT1 are dually targeted to mitochondria and plastids; (ii) AtBT1 and ZmBT1 N-terminal extensions comprise targeting sequences exclusively recognized by the plastidial compartment; and (iii) targeting sequences to mitochondria are localized within the mature part of the BT1 proteins.

The nucleotide sugar transporters AtUTr1 and AtUTr3 are required for the incorporation of UDP-glucose into the endoplasmic reticulum, are essential for pollen development and are needed for embryo sac progress in Arabidopsis thaliana.

Uridine 5'-diphosphate (UDP)-glucose is transported into the lumen of the endoplasmic reticulum (ER), and the Arabidopsis nucleotide sugar transporter AtUTr1 has been proposed to play a role in this process; however, different lines of evidence suggest that another transporter(s) may also be involved. Here we show that AtUTr3 is involved in the transport of UDP-glucose and is located at the ER but also at the Golgi. Insertional mutants in AtUTr3 showed no obvious phenotype. Biochemical analysis in both AtUTr1 and AtUTr3 mutants indicates that uptake of UDP-glucose into the ER is mostly driven by these two transporters. Interestingly, the expression of AtUTr3 is induced by stimuli that trigger the unfolded protein response (UPR), a phenomenon also observed for AtUTr1, suggesting that both AtUTr1 and AtUTr3 are involved in supplying UDP-glucose into the ER lumen when misfolded proteins are accumulated. Disruption of both AtUTr1 and AtUTr3 causes lethality. Genetic analysis showed that the atutr1 atutr3 combination was not transmitted by pollen and was poorly transmitted by the ovules. Cell biology analysis indicates that knocking out both genes leads to abnormalities in both male and female germ line development. These results show that the nucleotide sugar transporters AtUTr1 and AtUTr3 are required for the incorporation of UDP-glucose into the ER, are essential for pollen development and are needed for embryo sac progress in Arabidopsis thaliana.

Convergent evolution and orphan genes in the Fur4p-like family and characterization of a general nucleoside transporter in Aspergillus nidulans.

The function of seven paralogues phylogenetically related to the Saccharomyces cerevisiae Fur4p together with a number of functionally related transporters present in Aspergillus nidulans has been investigated. After deletion of the cognate genes we checked the incorporation of radiolabelled substrates, utilization of nitrogen sources, resistance to toxic analogues and supplementation of auxotrophies. FurA and FurD encode allantoin and uracil transporters respectively. No function was found for FurB, FurC, FurE, FurF and FurG. As we failed to identify Fur-related transporters for uridine, pyridoxine or thiamine, we deleted other possible candidates for these functions. A FCY2-like gene carrying in its 5' UTR a putative thiamine pyrophosphate riboswitch, and which encodes a protein similar to the pyridoxine transporter of yeast (Tpn1p), does not encode either a major thiamine or a pyridoxine transporter. CntA, a member of the concentrative nucleoside transporter family, is a general nucleoside permease, while no function was found for PnpA, a member of the equilibrative transporter family. Phylogenetic analysis shows that within the ascomycetes, the same transport activity could be catalysed by totally unrelated proteins and that within the Fur subfamily convergent evolution towards uracil and allantoin transport activity has occurred at least three and two independent times respectively.

Identification and characterization of a novel plastidic adenine nucleotide uniporter from Solanum tuberosum.

Homologs of BT1 (the Brittle1 protein) are found to be phylogenetically related to the mitochondrial carrier family and appear to occur in both mono- and dicotyledonous plants. Whereas BT1 from cereals is probably involved in the transport of ADP-glucose, which is essential for starch metabolism in endosperm plastids, BT1 from a noncereal plant, Solanum tuberosum (StBT1), catalyzes an adenine nucleotide uniport when functionally integrated into the bacterial cytoplasmic membrane. Import studies into intact Escherichia coli cells harboring StBT1 revealed a narrow substrate spectrum with similar affinities for AMP, ADP, and ATP of about 300-400 mum. Transiently expressed StBT1-green fluorescent protein fusion protein in tobacco leaf protoplasts showed a plastidic localization of the StBT1. In vitro synthesized radioactively labeled StBT1 was targeted to the envelope membranes of isolated spinach chloroplasts. Furthermore, we showed by real time reverse transcription-PCR a ubiquitous expression pattern of the StBT1 in autotrophic and heterotrophic potato tissues. We therefore propose that StBT1 is a plastidic adenine nucleotide uniporter used to provide the cytosol and other compartments with adenine nucleotides exclusively synthesized inside plastids.

Enhanced resistance to Phytophthora infestans and Alternaria solani in leaves and tubers, respectively, of potato plants with decreased activity of the plastidic ATP/ADP transporter.

Recently, it has been reported that tubers of transgenic potato ( Solanum tuberosum L.) plants with decreased activity of the plastidic ATP/ADP transporter (AATP1) contain less starch, despite having an increased glucose level [P. Geigenberger et al. (2001) Plant Physiol 125:1667-1678]. The metabolic alterations correlated with enhanced resistance to the bacterium Erwinia carotovora. Here it is shown that transgenic potato tubers, possessing less starch yet increased glucose levels due to the expression of a cytoplasm-localized yeast invertase, exhibit drastic susceptibility to E. carotovora. In addition, it is demonstrated that AATP1 anti-sense tubers show an increased capacity to ward off the pathogenic fungus Alternaria solani. In contrast to AATP1 anti-sense tubers, the corresponding leaf tissue does not show changes in carbohydrate accumulation. However, upon elicitor treatment, AATP1 anti-sense leaves possess an increased capacity to release H(2)O(2) and activate various defence-related genes, reactions that are associated with substantially delayed appearance of disease symptoms caused by Phytophthora infestans. Grafting experiments between AATP1 anti-sense plants and wild-type plants indicate the presence of a signal that is generated in AATP1 rootstocks and primes wild-type scions for potentiated activation of cellular defence responses in leaves. Together, the results suggest that (i) the enhanced pathogen tolerance of AATP1 anti-sense tubers is not due to "high sugar resistance", (ii) the increased disease resistance of AATP1 anti-sense tubers is effective against different types of pathogen and (iii) a systemic signal induced by antisensing the plastidic ATP/ADP transporter in potato tubers confers increased resistance to pathogens.