Distinct transcriptional signatures in purified circulating immune cells drive heterogeneity in disease location in IBD.

OBJECTIVE: To infer potential mechanisms driving disease subtypes among patients with inflammatory bowel disease (IBD), we profiled the transcriptome of purified circulating monocytes and CD4 T-cells. DESIGN: RNA extracted from purified monocytes and CD4 T-cells derived from the peripheral blood of 125 endoscopically active patients with IBD was sequenced using Illumina HiSeq 4000NGS. We used complementary supervised and unsupervised analytical methods to infer gene expression signatures associated with demographic/clinical features. Expression differences and specificity were validated by comparison with publicly available single cell datasets, tissue-specific expression and meta-analyses. Drug target information, druggability and adverse reaction records were used to prioritise disease subtype-specific therapeutic targets. RESULTS: Unsupervised/supervised methods identified significant differences in the expression profiles of CD4 T-cells between patients with ileal Crohn's disease (CD) and ulcerative colitis (UC). Following a pathway-based classification (Area Under Receiver Operating Characteristic - AUROC=86%) between ileal-CD and UC patients, we identified MAPK and FOXO pathways to be downregulated in UC. Coexpression module/regulatory network analysis using systems-biology approaches revealed mediatory core transcription factors. We independently confirmed that a subset of the disease location-associated signature is characterised by T-cell-specific and location-specific expression. Integration of drug-target information resulted in the discovery of several new (BCL6, GPR183, TNFAIP3) and repurposable drug targets (TUBB2A, PRKCQ) for ileal CD as well as novel targets (NAPEPLD, SLC35A1) for UC. CONCLUSIONS: Transcriptomic profiling of circulating CD4 T-cells in patients with IBD demonstrated marked molecular differences between the IBD-spectrum extremities (UC and predominantly ileal CD, sandwiching colonic CD), which could help in prioritising particular drug targets for IBD subtypes.

In vivo evidence for GDP-fucose transport in the absence of transporter SLC35C1 and putative transporter SLC35C2.

Slc35c1 encodes an antiporter that transports GDP-fucose into the Golgi and returns GMP to the cytoplasm. The closely related gene Slc35c2 encodes a putative GDP-fucose transporter and promotes Notch fucosylation and Notch signaling in cultured cells. Here, we show that HEK293T cells lacking SLC35C1 transferred reduced amounts of O-fucose to secreted epidermal growth factor-like repeats from NOTCH1 or secreted thrombospondin type I repeats from thrombospondin 1. However, cells lacking SLC35C2 did not exhibit reduced fucosylation of these epidermal growth factor-like repeats or thrombospondin type I repeats. To investigate SLC35C2 functions in vivo, WW6 embryonic stem cells were targeted for Slc35c2. Slc35c2[-/-] mice were viable and fertile and exhibited no evidence of defective Notch signaling during skeletal or T cell development. By contrast, mice with inactivated Slc35c1 exhibited perinatal lethality and marked skeletal defects in late embryogenesis, typical of defective Notch signaling. Compound Slc35c1[-/-]Slc35c2[-/-] mutants were indistinguishable in skeletal phenotype from Slc35c1[-/-] embryos and neonates. Double mutants did not exhibit the exacerbated skeletal defects predicted if SLC35C2 was functionally important for Notch signaling in vivo. In addition, NOTCH1 immunoprecipitated from Slc35c1[-/-]Slc35c2[-/-] neonatal lung carried fucose detected by binding of Aleuria aurantia lectin. Given that the absence of both SLC35C1, a known GDP-fucose transporter, and SLC35C2, a putative GDP-fucose transporter, did not lead to afucosylated NOTCH1 nor to the severe Notch signaling defects and embryonic lethality expected if all GDP-fucose transport were abrogated, at least one more mechanism of GDP-fucose transport into the secretory pathway must exist in mammals.

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.

Single-nucleotide polymorphisms of the SLC17A9 and P2RY12 genes are significantly associated with phantom tooth pain.

Phantom tooth pain (PTP) is a rare and specific neuropathic pain that occurs after pulpectomy and tooth extraction, but its cause is not understood. We hypothesized that there is a genetic contribution to PTP. We focused on solute carrier family 17 member 9 (SLC17A9)/vesicular nucleotide transporter (VNUT) and purinergic receptor P2Y12 (P2RY12), both of which have been associated with neuropathic pain and pain transduction signaling in the trigeminal ganglion in rodents. We sought to corroborate these associations in humans. We investigated gene polymorphisms that contribute to PTP. We statistically examined the association between genetic polymorphisms and PTP vulnerability in 150 patients with orofacial pain, including PTP, and 500 healthy subjects. We found that the rs735055 polymorphism of the SLC17A9 gene and rs3732759 polymorphism of the P2RY12 gene were associated with the development of PTP. Carriers of the minor allele of rs735055 and individuals who were homozygous for the major allele of rs3732759 had a higher rate of PTP. Carriers of the minor allele of rs735055 reportedly had high SLC17A9 mRNA expression in the spinal cord, which may increase the storage and release of adenosine triphosphate. Individuals who were homozygous for the major allele of rs3732759 may have higher P2RY12 expression that is more active in microglia. Therefore, these carriers may be more susceptible to PTP. These results suggest that specific genetic polymorphisms of the SLC17A9 and P2RY12 genes are involved in PTP. This is the first report on genes that are associated with PTP in humans.

Lysosomal ATP Transporter SLC17A9 Controls Cell Viability via Regulating Cathepsin D.

SLC17A9 (solute carrier family 17 member 9) functions as an ATP transporter in lysosomes as well as other secretory vesicles. SLC17A9 inhibition or silence leads to cell death. However, the molecular mechanisms causing cell death are unclear. In this study, we report that cell death induced by SLC17A9 deficiency is rescued by the transcription factor EB (TFEB), a master gene for lysosomal protein expression, suggesting that SLC17A9 deficiency may be the main cause of lysosome dysfunction, subsequently leading to cell death. Interestingly, Cathepsin D, a lysosomal aspartic protease, is inhibited by SLC17A9 deficiency. Heterologous expression of Cathepsin D successfully rescues lysosomal dysfunction and cell death induced by SLC17A9 deficiency. On the other hand, the activity of Cathepsin B, a lysosomal cysteine protease, is not altered by SLC17A9 deficiency, and Cathepsin B overexpression does not rescue lysosomal dysfunction and cell death induced by SLC17A9 deficiency. Our data suggest that lysosomal ATP and SLC17A9 play critical roles in lysosomal function and cell viability by regulating Cathepsin D activity.

Arabidopsis guard cell chloroplasts import cytosolic ATP for starch turnover and stomatal opening.

Stomatal opening requires the provision of energy in the form of ATP for proton pumping across the guard cell (GC) plasma membrane and for associated metabolic rearrangements. The source of ATP for GCs is a matter of ongoing debate that is mainly fuelled by controversies around the ability of GC chloroplasts (GCCs) to perform photosynthesis. By imaging compartment-specific fluorescent ATP and NADPH sensor proteins in Arabidopsis, we show that GC photosynthesis is limited and mitochondria are the main source of ATP. Unlike mature mesophyll cell (MC) chloroplasts, which are impermeable to cytosolic ATP, GCCs import cytosolic ATP through NUCLEOTIDE TRANSPORTER (NTT) proteins. GCs from ntt mutants exhibit impaired abilities for starch biosynthesis and stomatal opening. Our work shows that GCs obtain ATP and carbohydrates via different routes from MCs, likely to compensate for the lower chlorophyll contents and limited photosynthesis of GCCs.

Docetaxel-triggered SIDT2/NOX4/JNK/HuR signaling axis is associated with TNF-α-mediated apoptosis of cancer cells.

Previous studies have confirmed that docetaxel (DTX) treatment increases TNF-α production in cancer cells, but its mechanism of action remains unclear. Therefore, this study aimed to determine the signaling axis by which DTX induced the expression of TNF-α in U937 leukemia and MCF-7 breast carcinoma cells. DTX treatment promoted Ca2+-controlled autophagy and SIDT2 expression, resulting in lysosomal degradation of miR-25 in U937 cells. Downregulation of miR-25 increased NOX4 mRNA stability and protein expression. NOX4-stimulated ROS generation led to JNK-mediated phosphorylation of cytosolic HuR at Ser221, thereby increasing TNF-α protein expression by stabilizing TNF-α mRNA. Consequently, DTX induced TNF-α-dependent death in U937 cells. Depletion of HuR using siRNA or abolishment of JNK activation reduced TNF-α expression and eliminated DTX-mediated cytotoxicity. Knockdown of SIDT2 or pretreatment with chloroquine (a lysosome inhibitor) reduced DTX-induced NOX4 and TNF-α expression and mitigated JNK-mediated HuR phosphorylation. Altogether, our data indicate that DTX triggers HuR-mediated TNF-α mRNA stabilization through the Ca2+/SIDT2/NOX4/ROS/JNK axis, thereby inducing TNF-α-dependent apoptosis in U937 cells. In addition, DTX induces apoptosis in MCF-7 cells through SIDT2/NOX4/JNK/HuR axis-mediated TNF-α expression.

Long non-coding RNA LIFR-AS1 suppressed the proliferation, angiogenesis, migration and invasion of papillary thyroid cancer cells via the miR-31-5p/SIDT2 axis.

Long non-coding RNA LIFR-AS1 is low-expressed in many cancers, but its functions in papillary thyroid carcinoma (PTC) were not defined and require further study. The relationship between LIFR-AS1 expression and clinicopathological characteristics of patients with PTC was statistically analyzed. The downregulation of LIFR-AS1 in PTC tissues and cell lines was predicted by bioinformatics analysis and verified by qRT-PCR. After overexpressing or silencing LIFR-AS1, the regulatory role of LIFR-AS1 in PTC was examined by performing MTT, colony formation, wound healing, Transwell, ELISA, tube formation and xenograft tumor experiment. MiR-31-5p and SID1 transmembrane family member 2 (SIDT2) expressions in PTC tissues or cell lines were detected by qRT-PCR, Western blot, or in situ hybridization. The relationship between miR-31-5p and LIFR-AS1/SIDT2 was predicted by LncBase, TargetScan or Pearson correlation test and then verified by Dual-Luciferase Reporter assay, RNA pull-down assay and qRT-PCR. The regulatory effect of LIFR-AS1/miR-31-5p/SIDT2 axis on the biological behaviors of PTC cells was confirmed by functional experiments and rescue experiments mentioned above. The tumor size and lymphatic metastasis were correlated with LIFR-AS1 overexpression. Overexpressed LIFR-AS1 suppressed tumorigenesis in vivo. LIFR-AS1 and SIDT2 expressions were suppressed in PTC tissues, while that of miR-31-5p was elevated in PTC tissues. LIFR-AS1 was negatively correlated with miR-31-5p. LIFR-AS1 sponged miR-31-5p to upregulate SIDT2, thereby inhibiting the viability, proliferation, migration, invasion, and the secretion of vascular endothelial growth factor (VEGF) of PTC cells and angiogenesis of human umbilical vein endothelial cells (HUVECs). This paper demonstrates that LIFR-AS1/miR-31-5p/SIDT2 axis modulated the development of PTC.

Identification of a Signature Comprising 5 Soluble Carrier Family Genes to Predict the Recurrence of Papillary Thyroid Carcinoma.

RNA-sequencing data and relevant clinical data in The Cancer Genome Atlas for 502 samples of papillary thyroid cancer (PTC) were analyzed to determine the prognostic value of soluble carrier family genes in PTC. We analyzed soluble carrier family gene expression and function in the samples. Clustering identified 2 clusters in the data. Risk characteristics were identified using LASSO and Univariate Cox regression analysis, which divided the patients into low and high-risk groups. The expression levels of 88 soluble carrier genes were significantly different between tumors and normal tissue. The 2 PTC clusters had different clinical outcomes and distributions of gene expression. The expression levels of SFXN1, SLC12A4, SLC35A1, SLC35E1, and SLCO1C1 were markedly different between the 2 groups. The high risk and low risk groups had significant different prognoses (P < 0.05). Significant differences were identified for disease free survival (DFS), sex and T stage between the 2 subgroups. The risk score was identified as an independent prognostic variable (P < 0.05) and as a predictor of clinicopathological variables. In patients with PTC, solute carrier gene expression showed differential associations with clinicopathological variables. The 5 genes could be used as prognostic factors for PTC, particularly to predict PTC recurrence.

Genome-Wide Association Study Identifies a Functional SIDT2 Variant Associated With HDL-C (High-Density Lipoprotein Cholesterol) Levels and Premature Coronary Artery Disease.

Objective: Low HDL-C (high-density lipoprotein cholesterol) is the most frequent dyslipidemia in Mexicans, but few studies have examined the underlying genetic basis. Our purpose was to identify genetic variants associated with HDL-C levels and cardiovascular risk in the Mexican population. Approach and Results: A genome-wide association studies for HDL-C levels in 2335 Mexicans, identified four loci associated with genome-wide significance: CETP, ABCA1, LIPC, and SIDT2. The SIDT2 missense Val636Ile variant was associated with HDL-C levels and was replicated in 3 independent cohorts (P=5.9×10−18 in the conjoint analysis). The SIDT2/Val636Ile variant is more frequent in Native American and derived populations than in other ethnic groups. This variant was also associated with increased ApoA1 and glycerophospholipid serum levels, decreased LDL-C (low-density lipoprotein cholesterol) and ApoB levels, and a lower risk of premature CAD. Because SIDT2 was previously identified as a protein involved in sterol transport, we tested whether the SIDT2/Ile636 protein affected this function using an in vitro site-directed mutagenesis approach. The SIDT2/Ile636 protein showed increased uptake of the cholesterol analog dehydroergosterol, suggesting this variant affects function. Finally, liver transcriptome data from humans and the Hybrid Mouse Diversity Panel are consistent with the involvement of SIDT2 in lipid and lipoprotein metabolism. Conclusions: This is the first genome-wide association study for HDL-C levels seeking associations with coronary artery disease in the Mexican population. Our findings provide new insight into the genetic architecture of HDL-C and highlight SIDT2 as a new player in cholesterol and lipoprotein metabolism in humans.

Knockout of the CMP-Sialic Acid Transporter SLC35A1 in Human Cell Lines Increases Transduction Efficiency of Adeno-Associated Virus 9: Implications for Gene Therapy Potency Assays.

Recombinant adeno-associated viruses (AAV) have emerged as an important tool for gene therapy for human diseases. A prerequisite for clinical approval is an in vitro potency assay that can measure the transduction efficiency of each virus lot produced. The AAV serotypes are typical for gene therapy bind to different cell surface structures. The binding of AAV9 on the surface is mediated by terminal galactose residues present in the asparagine-linked carbohydrates in glycoproteins. However, such terminal galactose residues are rare in cultured cells. They are masked by sialic acid residues, which is an obstacle for the infection of many cell lines with AAV9 and the respective potency assays. The sialic acid residues can be removed by enzymatic digestion or chemical treatment. Still, such treatments are not practical for AAV9 potency assays since they may be difficult to standardize. In this study, we generated human cell lines (HEK293T and HeLa) that become permissive for AAV9 transduction after a knockout of the CMP-sialic acid transporter SLC35A1. Using the human aspartylglucosaminidase (AGA) gene, we show that these cell lines can be used as a model system for establishing potency assays for AAV9-based gene therapy approaches for human diseases.

Clodronate, an inhibitor of the vesicular nucleotide transporter, ameliorates steatohepatitis and acute liver injury.

The vesicular nucleotide transporter (VNUT) is responsible for the vesicular storage and release of ATP from various ATP-secreting cells, and it plays an essential role in purinergic signaling. Although extracellular ATP and its degradation products are known to mediate various inflammatory responses via purinoceptors, whether vesicular ATP release affects steatohepatitis and acute liver injury is far less understood. In the present study, we investigated the effects of clodronate, a potent and selective VNUT inhibitor, on acute and chronic liver inflammation in mice. In a model of methionine/choline-deficient diet-induced non-alcoholic steatohepatitis (NASH), the administration of clodronate reduced hepatic inflammation, fibrosis, and triglyceride accumulation. Clodronate also protected mice against high-fat/high-cholesterol diet-induced steatohepatitis. Moreover, prophylactic administration of clodronate prevented D-galactosamine and lipopolysaccharide-induced acute liver injury by reducing inflammatory cytokines and hepatocellular apoptosis. In vitro, clodronate inhibited glucose-induced vesicular ATP release mediated by VNUT and reduced the intracellular level and secretion of triglycerides in isolated hepatocytes. These results suggest that VNUT-dependent vesicular ATP release plays a crucial role in the recruitment of immune cells, cytokine production, and the aggravation of steatosis in the liver. Pharmacological inhibition of VNUT may provide therapeutic benefits in liver inflammatory disorders, including NASH and acute toxin-induced injury.

Cytidine Monophosphate N-Acetylneuraminic Acid Synthetase and Solute Carrier Family 35 Member A1 Are Required for Reovirus Binding and Infection.

Engagement of cell surface receptors by viruses is a critical determinant of viral tropism and disease. The reovirus attachment protein σ1 binds sialylated glycans and proteinaceous receptors to mediate infection, but the specific requirements for different cell types are not entirely known. To identify host factors required for reovirus-induced cell death, we conducted a CRISPR-knockout screen targeting over 20,000 genes in murine microglial BV2 cells. Candidate genes required for reovirus to cause cell death were highly enriched for sialic acid synthesis and transport. Two of the top candidates identified, CMP N-acetylneuraminic acid synthetase (Cmas) and solute carrier family 35 member A1 (Slc35a1), promote sialic acid expression on the cell surface. Two reovirus strains that differ in the capacity to bind sialic acid, T3SA+ and T3SA-, were used to evaluate Cmas and Slc35a1 as potential host genes required for reovirus infection. Following CRISPR-Cas9 disruption of either gene, cell surface expression of sialic acid was diminished. These results correlated with decreased binding of strain T3SA+, which is capable of engaging sialic acid. Disruption of either gene did not alter the low-level binding of T3SA-, which does not engage sialic acid. Furthermore, infectivity of T3SA+ was diminished to levels similar to those of T3SA- in cells lacking Cmas and Slc35a1 by CRISPR ablation. However, exogenous expression of Cmas and Slc35a1 into the respective null cells restored sialic acid expression and T3SA+ binding and infectivity. These results demonstrate that Cmas and Slc35a1, which mediate cell surface expression of sialic acid, are required in murine microglial cells for efficient reovirus binding and infection.IMPORTANCE Attachment factors and receptors are important determinants of dissemination and tropism during reovirus-induced disease. In a CRISPR cell survival screen, we discovered two genes, Cmas and Slc35a1, which encode proteins required for sialic acid expression on the cell surface and mediate reovirus infection of microglial cells. This work elucidates host genes that render microglial cells susceptible to reovirus infection and expands current understanding of the receptors on microglial cells that are engaged by reovirus. Such knowledge may lead to new strategies to selectively target microglial cells for oncolytic applications.

Biosynthesis of GlcNAc-rich N- and O-glycans in the Golgi apparatus does not require the nucleotide sugar transporter SLC35A3.

Nucleotide sugar transporters, encoded by the SLC35 gene family, deliver nucleotide sugars throughout the cell for various glycosyltransferase-catalyzed glycosylation reactions. GlcNAc, in the form of UDP-GlcNAc, and galactose, as UDP-Gal, are delivered into the Golgi apparatus by SLC35A3 and SLC35A2 transporters, respectively. However, although the UDP-Gal transporting activity of SLC35A2 has been clearly demonstrated, UDP-GlcNAc delivery by SLC35A3 is not fully understood. Therefore, we analyzed a panel of CHO, HEK293T, and HepG2 cell lines including WT cells, SLC35A2 knockouts, SLC35A3 knockouts, and double-knockout cells. Cells lacking SLC35A2 displayed significant changes in N- and O-glycan synthesis. However, in SLC35A3-knockout CHO cells, only limited changes were observed; GlcNAc was still incorporated into N-glycans, but complex type N-glycan branching was impaired, although UDP-GlcNAc transport into Golgi vesicles was not decreased. In SLC35A3-knockout HEK293T cells, UDP-GlcNAc transport was significantly decreased but not completely abolished. However, N-glycan branching was not impaired in these cells. In CHO and HEK293T cells, the effect of SLC35A3 deficiency on N-glycan branching was potentiated in the absence of SLC35A2. Moreover, in SLC35A3-knockout HEK293T and HepG2 cells, GlcNAc was still incorporated into O-glycans. However, in the case of HepG2 cells, no qualitative changes in N-glycans between WT and SLC35A3 knockout cells nor between SLC35A2 knockout and double-knockout cells were observed. These findings suggest that SLC35A3 may not be the primary UDP-GlcNAc transporter and/or different mechanisms of UDP-GlcNAc transport into the Golgi apparatus may exist.

Effect of sidt2 Gene on Cell Insulin Resistance and Its Molecular Mechanism.

BACKGROUND: Sidt2 (SID1 transmembrane family, member 2) is a multiple transmembrane lysosomal membrane protein newly discovered in our previous study. In the previous study, we used gene targeting technique to make a mouse model of sidt2 gene knockout (sidt2-/-). It was found that sidt2-/- mice showed elevated fasting blood glucose and impaired glucose tolerance, showing a disorder of glucose metabolism, suggesting that sidt2 may be closely related to insulin resistance. We used 3T3-L1 adipocytes, C2-C12 myoblasts, and HEPA1-6 hepatoma cells as subjects to observe the effects of sidt2 on insulin-stimulated glucose uptake and the abovementioned insulin signal transduction pathways, and then to explore the effect of sidt2 on peripheral tissue insulin resistance and its possible molecular mechanism. METHODS: (1) Lentiviruses with sidt2 gene knockout and puromycin resistance were constructed by Crispr/cas9 vector and transfected into 3T3-L1 adipocytes, C2-C12 myoblasts, and HEPA1-6 hepatoma cells to construct sidt2 knockout cell line model. (2) Glucose uptake of 3T3-L1 adipocytes, C2-C12 myoblasts, and HEPA1-6 hepatoma cells stimulated by insulin was detected by glucose detection kit, and the results were analyzed. (3) Sidt2 knockout group and control group of 3T3-L1 adipocytes, C2-C12 myoblast, and HEPA1-6 hepatoma cells were cultured according to the routine method. The total proteins of the above cells were extracted, and the expression of PAKT (thr308), PI3-K, and PIRS-1 (ser307) in the IRS-1 signaling pathway of the three groups was detected by western blot technique. RESULTS: (1) The sidt2 elimination models of 3T3-L1 adipocytes, C2-C12 myoblasts, and HEPA1-6 hepatoma cells were successfully constructed. (2) It was found that the glucose uptake of cells in the sidt2 knockout group was lower than that in normal group under insulin stimulation through the detection of glucose concentration in the cell culture medium. (3) It was found that the expression of PAKT (thr308) and PI3-K protein decreased and the expression of PIRS-1 (ser307) protein increased in sidt2-/- group compared to the control group. CONCLUSIONS: sidt2 knockout can reduce glucose uptake in peripheral tissue under insulin stimulation, which may lead to peripheral tissue insulin resistance by affecting the IRS-1 signal pathway.

Physiopathological roles of vesicular nucleotide transporter (VNUT), an essential component for vesicular ATP release.

Vesicular nucleotide transporter (VNUT) is the last identified member of the SLC17 organic anion transporter family, which plays a central role in vesicular storage in ATP-secreting cells. The discovery of VNUT demonstrated that, despite having been neglected for a long time, vesicular ATP release represents a major pathway for purinergic chemical transmission, which had been mainly attributed to ATP permeation channels. This article summarizes recent advances in our understanding of the mechanism of VNUT and its physiopathological roles as well as the development of inhibitors. Regulating the activity and/or the expression of VNUT represents a new and promising therapeutic strategy for the treatment of multiple diseases.

VNUT/SLC17A9, a vesicular nucleotide transporter, regulates osteoblast differentiation.

Osteoblasts release adenosine triphosphate (ATP) out of the cell following mechanical stress. Although it is well established that extracellular ATP affects bone metabolism via P2 receptors [such as purinergic receptor P2X7 (P2X7R) and purinergic receptor P2Y2 (P2Y2R)], the mechanism of ATP release from osteoblasts remains unknown. Recently, a vesicular nucleotide transporter [VNUT, solute carrier family 17 member 9 (SLC17A9)] that preserves ATP in vesicles has been identified. The purpose of this study was to elucidate the role of VNUT in osteoblast bone metabolism. mRNA and protein expression of VNUT were confirmed in mouse bone and in osteoblasts by quantitative real-time PCR (qPCR) and immunohistochemistry. Next, when compressive force was applied to MC3T3-E1 cells by centrifugation, the expression of Slc17a9, P2x7r, and P2y2r was increased concomitant with an increase in extracellular ATP levels. Furthermore, compressive force decreased the osteoblast differentiation capacity of MC3T3-E1 cells. shRNA knockdown of Slc17a9 in MC3T3-E1 cells reduced levels of extracellular ATP and also led to increased osteoblast differentiation after the application of compressive force as assessed by qPCR analysis of osteoblast markers such as Runx2, Osterix, and alkaline phosphatase (ALP) as well as ALP activity. Consistent with these observations, knockdown of P2x7r or P2y2r by siRNA partially rescued the downregulation of osteoblast differentiation markers, caused by mechanical loading. In conclusion, our results demonstrate that VNUT is expressed in osteoblasts and that VNUT inhibits osteoblast differentiation in response to compressive force by mechanisms related to ATP release and P2X7R and/or P2Y2R activity.

Novel Insights into Selected Disease-Causing Mutations within the SLC35A1 Gene Encoding the CMP-Sialic Acid Transporter.

Congenital disorders of glycosylation (CDG) are a group of rare genetic and metabolic diseases caused by alterations in glycosylation pathways. Five patients bearing CDG-causing mutations in the SLC35A1 gene encoding the CMP-sialic acid transporter (CST) have been reported to date. In this study we examined how specific mutations in the SLC35A1 gene affect the protein's properties in two previously described SLC35A1-CDG cases: one caused by a substitution (Q101H) and another involving a compound heterozygous mutation (T156R/E196K). The effects of single mutations and the combination of T156R and E196K mutations on the CST's functionality was examined separately in CST-deficient HEK293T cells. As shown by microscopic studies, none of the CDG-causing mutations affected the protein's proper localization in the Golgi apparatus. Cellular glycophenotypes were characterized using lectins, structural assignment of N- and O-glycans and analysis of glycolipids. Single Q101H, T156R and E196K mutants were able to partially restore sialylation in CST-deficient cells, and the deleterious effect of a single T156R or E196K mutation on the CST functionality was strongly enhanced upon their combination. We also revealed differences in the ability of CST variants to form dimers. The results of this study improve our understanding of the molecular background of SLC35A1-CDG cases.

High SLC17A9 expression correlates with poor survival in gastric carcinoma.

Aim: To elucidate the clinicopathological significance and prognostic value of SLC17A9 expression in gastric carcinoma (GC). Methods: SLC17A9 mRNA level and its relationship with TP53 mutation was analyzed. SLC17A9 protein expression was examined by immunohistochemistry in 161 patients. Results: SLC17A9 mRNA and protein expression were higher in GC tissues than in adjacent normal tissues (p < 0.01). SLC17A9 mRNA expression was higher in GC tissues having mutated TP53 than in tissues with wild-type TP53 (p < 0.001). High SLC17A9 expression was also significantly associated with poor overall survival and recurrence-free survival and was also found to be an independent prognostic factor for long-term survival in GC patients.Conclusion: Our results show that SLC17A9 may serve as a potential prognostic biomarker in GC patients.

The transporters SLC35A1 and SLC30A1 play opposite roles in cell survival upon VSV virus infection.

Host factor requirements for different classes of viruses have not been fully unraveled. Replication of the viral genome and synthesis of viral proteins within the human host cell are associated with an increased demand for nutrients and specific metabolites. With more than 400 acknowledged members to date in humans, solute carriers (SLCs) represent the largest family of transmembrane proteins dedicated to the transport of ions and small molecules such as amino acids, sugars and nucleotides. Consistent with their impact on cellular metabolism, several SLCs have been implicated as host factors affecting the viral life cycle and the cellular response to infection. In this study, we aimed at characterizing the role of host SLCs in cell survival upon viral infection by performing unbiased genetic screens using a focused CRISPR knockout library. Genetic screens with the cytolytic vesicular stomatitis virus (VSV) showed that the loss of two SLCs genes, encoding the sialic acid transporter SLC35A1/CST and the zinc transporter SLC30A1/ZnT1, affected cell survival upon infection. Further characterization of these genes suggests a role for both of these transporters in the apoptotic response induced by VSV, offering new insights into the cellular response to oncolytic virus infections.