Plasma Membrane MCC/Eisosome Domains Promote Stress Resistance in Fungi.

There is growing appreciation that the plasma membrane orchestrates a diverse array of functions by segregating different activities into specialized domains that vary in size, stability, and composition. Studies with the budding yeast Saccharomyces cerevisiae have identified a novel type of plasma membrane domain known as the MCC (membrane compartment of Can1)/eisosomes that correspond to stable furrows in the plasma membrane. MCC/eisosomes maintain proteins at the cell surface, such as nutrient transporters like the Can1 arginine symporter, by protecting them from endocytosis and degradation. Recent studies from several fungal species are now revealing new functional roles for MCC/eisosomes that enable cells to respond to a wide range of stressors, including changes in membrane tension, nutrition, cell wall integrity, oxidation, and copper toxicity. The different MCC/eisosome functions are often intertwined through the roles of these domains in lipid homeostasis, which is important for proper plasma membrane architecture and cell signaling. Therefore, this review will emphasize the emerging models that explain how MCC/eisosomes act as hubs to coordinate cellular responses to stress. The importance of MCC/eisosomes is underscored by their roles in virulence for fungal pathogens of plants, animals, and humans, which also highlights the potential of these domains to act as novel therapeutic targets.

Oryza sativa Lysine-Histidine-type Transporter 1 functions in root uptake and root-to-shoot allocation of amino acids in rice.

In agricultural soils, amino acids can represent vital nitrogen (N) sources for crop growth and yield. However, the molecular mechanisms underlying amino acid uptake and allocation are poorly understood in crop plants. This study shows that rice (Oryza sativa L.) roots can acquire aspartate at soil concentration, and that japonica subspecies take up this acidic amino acid 1.5-fold more efficiently than indica subspecies. Genetic association analyses with 68 representative japonica or indica germplasms identified rice Lysine-Histidine-type Transporter 1 (OsLHT1) as a candidate gene associated with the aspartate uptake trait. When expressed in yeast, OsLHT1 supported cell growth on a broad spectrum of amino acids, and effectively transported aspartate, asparagine and glutamate. OsLHT1 is localized throughout the rice root, including root hairs, epidermis, cortex and stele, and to the leaf vasculature. Knockout of OsLHT1 in japonica resulted in reduced root uptake of amino acids. Furthermore, in 15 N-amino acid-fed mutants versus wild-type, a higher percentage of 15 N remained in roots instead of being allocated to the shoot. 15 N-ammonium uptake and subsequently the delivery of root-synthesized amino acids to Oslht1 shoots were also significantly decreased, which was accompanied by reduced shoot growth. These results together provide evidence that OsLHT1 functions in both root uptake and root to shoot allocation of a broad spectrum of amino acids in rice.

Characterisation of genes differentially expressed in macrophages by virulent and attenuated Mycobacterium tuberculosis through RNA-Seq analysis.

Tuberculosis (TB) remains a global healthcare issue. Understanding the host-pathogen interactions in TB is vital to develop strategies and therapeutic tools for the control of Mycobacterium tuberculosis (Mtb). In this study, transcriptome analyses of macrophages infected with either the virulent Mtb strain H37Rv (Rv) or the avirulent Mtb strain H37Ra (Ra) were carried out and 750 differentially expressed genes (DEGs) were identified. As expected, the DEGs were mainly involved in the induction of innate immune responses against mycobacterial infections. Among the DEGs, solute carrier family 7 member 2 (Slc7a2) was more strongly expressed in Ra-infected macrophages. Induction of SLC7A2 was important for macrophages to control the intracellular survival of Mtb. Our results imply that SLC7A2 plays an important role in macrophages during Mtb infection. Our findings could prove useful for the development of new therapeutic strategies to control TB infection.

Genetic ablation of solute carrier family 7a3a leads to hepatic steatosis in zebrafish during fasting.

UNLABELLED: Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disorder caused by abnormal lipid metabolisms, such as reduced hepatic fatty acid oxidation (FAO), but intracellular control of FAO under physio- and pathological conditions remains largely undefined. Here, we demonstrate that deprivation of Slc7a3a leads to hepatic steatosis in fasted zebrafish as a result of defects in arginine-dependent nitric oxide (NO) synthesis. Fast-induced hepatic steatosis in slc7a3a-null mutants can be rescued by treatments with NO donor, cyclic guanosine monophosphate analog, adenosine-monophosphate-activated protein kinase (AMPK) activator, or peroxisome proliferator-activated receptor alpha (PPAR-α) agonist. In contrast, inhibitors of NO synthases, AMPK, or soluble guanylate cyclase and liver-specifically expressed dominant negatives of peroxisome proliferator-activated receptor-gamma coactivator 1 alpha and PPAR-α are sufficient to induce hepatic steatosis in fasted wild-type larvae. Moreover, knockdown of Slc7a3 in mice or SLC7A3 in human liver cells impaired AMPK-PPAR-α signaling and resulted in lipid accumulation under fasting or glucose starvation, respectively. CONCLUSION: These findings have revealed a NO-AMPK-PPAR-α-signaling pathway that is crucial for the control of hepatic FAO in vertebrates.

The SLC3 and SLC7 families of amino acid transporters.

Amino acids are necessary for all living cells and organisms. Specialized transporters mediate the transfer of amino acids across plasma membranes. Malfunction of these proteins can affect whole-body homoeostasis giving raise to diverse human diseases. Here, we review the main features of the SLC3 and SLC7 families of amino acid transporters. The SLC7 family is divided into two subfamilies, the cationic amino acid transporters (CATs), and the L-type amino acid transporters (LATs). The latter are the light or catalytic subunits of the heteromeric amino acid transporters (HATs), which are associated by a disulfide bridge with the heavy subunits 4F2hc or rBAT. These two subunits are glycoproteins and form the SLC3 family. Most CAT subfamily members were functionally characterized and shown to function as facilitated diffusers mediating the entry and efflux of cationic amino acids. In certain cells, CATs play an important role in the delivery of L-arginine for the synthesis of nitric oxide. HATs are mostly exchangers with a broad spectrum of substrates and are crucial in renal and intestinal re-absorption and cell redox balance. Furthermore, the role of the HAT 4F2hc/LAT1 in tumor growth and the application of LAT1 inhibitors and PET tracers for reduction of tumor progression and imaging of tumors are discussed. Finally, we describe the link between specific mutations in HATs and the primary inherited aminoacidurias, cystinuria and lysinuric protein intolerance.

Solute carrier protein family may involve in radiation-induced radioresistance of non-small cell lung cancer.

PURPOSE: To find new signaling pathways that may be involved in the cellular response to ionizing radiation. METHODS: Two radioresistant subclones (A549/R and SPCA1/R) derived from lung adenocarcinoma cell lines A549 and SPCA1 were established after eight rounds of sublethal irradiation. The new subclones were tested for radioresistant features using clonogenic assay and apoptosis analysis. The genes expressed differentially were screened with cDNA microarray analysis consisting of 48,000 transcript probes and confirmed by quantitative real-time PCR. RESULTS: Stable and significant radioresistance was observed in the screened subclones. The microarray analysis showed 65 genes were up-regulated and 141 genes down-regulated in SPCA1/R cells. The up-regulated and down-regulated genes were 708 and 230 in A549/R cells, respectively. Twenty-seven altered genes were consistent in both subclones. Interestingly, members the of human solute carrier (SLC) gene superfamily were among in 27 genes. CONCLUSIONS: The differentially expressed genes in both cell lines may contribute to their radioresistant phenotype. This extensive list of genes identified in the experiment provides a large body of potentially valuable information for studying the molecular mechanism(s) of radiosensitivity and identification of candidate molecular markers of radiation sensitivity. Thus, to our knowledge, the SLC gene superfamily is the first being reported to involve in acquired radioresistance.

Effect of the cation-chloride cotransporter inhibitor furosemide in a rat model of postoperative pain.

OBJECTIVE: Recently, evidence has accumulated to show that cation-chloride cotransporters (CCCs) participate in the modulation of pain transmission at the spinal cord level. To investigate whether CCC inhibitors might affect surgical pain, we examined the effect of furosemide in a rat incisional pain model. DESIGN: We examined pain thresholds using von Frey filaments in intact and incision model Sprague-Dawley rats before and after intrathecal furosemide administration (100 µg/20 µL). Twenty-four rats were divided into four groups (N=6), groups A and B were intact rats, which received furosemide and its solvent, respectively. Groups C and D were incision rats, which received furosemide and its solvent, respectively. Non-parametric tests were used to calculate pain thresholds, and P<0.05 was considered significant. RESULTS: Furosemide decreased the pain threshold (vs solvent) in intact rats for 2 hours after administration and caused excitatory behavior. However, furosemide increased the pain threshold in incision model rats ([1] at the incision point: at 20 minutes, 2 hours, 3 hours, 4 hours, and on the second to fifth days after incision; [2] at the remote point: at 20 minutes, 3-5 hours, and from the 3rd to the 7th day after incision) and improved wound recovery. CONCLUSIONS: Intrathecal administration of the CCC inhibitor furosemide had antinociceptive effects in rats with incisional pain. Furosemide may be a novel treatment for postoperative pain.

Facilitated transporters mediate net efflux of amino acids to the fetus across the basal membrane of the placental syncytiotrophoblast.

Fetal growth depends on placental transfer of amino acids from maternal to fetal blood. The mechanisms of net amino acid efflux across the basal membrane (BM) of the placental syncytiotrophoblast to the fetus, although vital for amino acid transport, are poorly understood. We examined the hypothesis that facilitated diffusion by the amino acid transporters TAT1, LAT3 and LAT4 plays an important role in this process, with possible effects on fetal growth. Amino acid transfer was measured in isolated perfused human placental cotyledons (n = 5 per experiment) using techniques which distinguish between different transport processes. Placental TAT1, LAT3 and LAT4 proteins were measured, and mRNA expression levels (measured using real-time quantitative-PCR) were related to fetal and neonatal anthropometry and dual-energy X-ray absorptiometry measurements of neonatal lean mass in 102 Southampton Women's Survey (SWS) infants. Under conditions preventing transport by amino acid exchangers, all amino acids appearing in the fetal circulation were substrates of TAT1, LAT3 or LAT4. Western blots demonstrated the presence of TAT1, LAT3 and LAT4 in placental BM preparations. Placental TAT1 and LAT3 mRNA expression were positively associated with measures of fetal growth in SWS infants (P < 0.05). We provide evidence that the efflux transporters TAT1, LAT3 and LAT4 are present in the human placental BM, and may play an important role in the net efflux of amino acids to the fetus. Unlike other transporters they can increase fetal amino acid concentrations. Consistent with a role in placental amino acid transfer capacity and fetal growth TAT1 and LAT3 mRNA expression showed positive associations with infant size at birth.

Does modulation of organic cation transporters improve pralidoxime activity in an animal model of organophosphate poisoning?

OBJECTIVES: Pralidoxime is an organic cation used as an antidote in addition to atropine to treat organophosphate poisoning. Pralidoxime is rapidly eliminated by the renal route and thus has limited action. The objectives of this work were as follows. 1) Study the role of organic cation transporters in the renal secretion of pralidoxime using organic cation transporter substrates (tetraethylammonium) and knockout mice (Oct1/2⁻/⁻; Oct3⁻/⁻). 2) Assess whether sustained high plasma concentrations increase pralidoxime antidotal activity toward paraoxon-induced respiratory toxicity. SETTING: INSERM U705, Faculté de Pharmacie, Université Paris Descartes, 4 Avenue de l'Observatoire, 75006 Paris, France. SUBJECTS: Rodents: Knockout mice (Oct1/2⁻/⁻; Oct3⁻/⁻) and Sprague-Dawley rats. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: In rats, the renal clearance of pralidoxime was 3.6-fold higher than the creatinine clearance. Pretreatment with tetraethylammonium (75 mg/kg) in rats or deficiencies in organic cation transporters 1 and 2 in mice (Oct1/2⁻/⁻) resulted in a significant increase in plasma pralidoxime concentrations. Lack of Oct3 did not alter plasma pralidoxime concentrations. The antidotal activity of pralidoxime (50 mg/kg intramuscularly) was longer and with greater effect, resulting in a return to normal values when administered to rats pretreated with tetraethylammonium. CONCLUSIONS: Pralidoxime is secreted in rats and mice by renal Oct1 and/or Oct2 but not by Oct3. Modulation of organic cation transporter activity increased the plasma pralidoxime concentrations and the antidotal effect of pralidoxime with sustained return within the normal range of respiratory variables in paraoxon-poisoned rats. These results suggest a promising approach in an animal model toward the increase in efficiency of pralidoxime. However, further studies are needed before these results are extended to human poisoning.

Cationic amino acid transporters and Salmonella Typhimurium ArgT collectively regulate arginine availability towards intracellular Salmonella growth.

Cationic amino acid transporters (mCAT1 and mCAT2B) regulate the arginine availability in macrophages. How in the infected cell a pathogen can alter the arginine metabolism of the host remains to be understood. We reveal here a novel mechanism by which Salmonella exploit mCAT1 and mCAT2B to acquire host arginine towards its own intracellular growth within antigen presenting cells. We demonstrate that Salmonella infected bone marrow derived macrophages and dendritic cells show enhanced arginine uptake and increased expression of mCAT1 and mCAT2B. We show that the mCAT1 transporter is in close proximity to Salmonella containing vacuole (SCV) specifically by live intracellular Salmonella in order to access the macrophage cytosolic arginine pool. Further, Lysosome associated membrane protein 1, a marker of SCV, also was found to colocalize with mCAT1 in the Salmonella infected cell. The intra vacuolar Salmonella then acquire the host arginine via its own arginine transporter, ArgT for growth. The argT knockout strain was unable to acquire host arginine and was attenuated in growth in both macrophages and in mice model of infection. Together, these data reveal survival strategies by which virulent Salmonella adapt to the harsh conditions prevailing in the infected host cells.

Regulation of amino acid/carnitine transporter B 0,+ (ATB 0,+) in astrocytes by protein kinase C: independent effects on raft and non-raft transporter subpopulations.

Neutral and basic amino acid transporter B(0,+) belongs to a Na,Cl-dependent superfamily of proteins transporting neurotransmitters, amino acids and osmolytes, known to be regulated by protein kinase C (PKC). The present study demonstrates an increased phosphorylation of B(0,+) on serine moiety after treatment of rat astrocytes with phorbol 12-myristate 13-acetate, a process correlated with an augmented activity of l-leucine transport and an enhanced presence of the transporter at the cell surface. After solubilization with Triton X-100 and sucrose gradient centrifugation, B(0,+) was detected in non-raft as well as in detergent-resistant raft fractions under control conditions, while phorbol 12-myristate 13-acetate treatment resulted in a complete disappearance of the transporter from the raft fraction. B(0,+) was observed to interact with caveolin-1 and flotillin-1 (reggie-2) proteins, the markers of detergent-resistant microdomains of plasma membrane. As verified in immunocytochemistry and immunoprecipitation experiments, modification of PKC activity did not affect these interactions. It is proposed that PKC reveals different effects on raft and non-raft subpopulations of B(0,+). Phorbol ester treatment results in trafficking of the transporter from the intracellular pool to non-raft microdomains and increased activity, while B(0,+) present in raft microdomains undergoes either internalization or is transferred laterally to non-raft domains.

Cationic amino acid transporters and beta-defensins in dry eye syndrome.

Several diseases concomitant with L-arginine deficiency (diabetes, chronic kidney failure, psoriasis) are significantly associated with dry eye syndrome. One important factor that has so far been neglected is the y(+) transporter. In humans, y(+) accounts for nearly 80% of arginine transport, exclusively carrying the cationic amino acids L-arginine, L-lysine and L-ornithine. y(+) is represented by CAT(cationic amino acid transporter) proteins. L-arginine is a precursor of the moisturizer urea, which has been used in the treatment of dry skin diseases. Although urea has also been shown to be part of the tear film, little attention has been paid to it in this role. Moreover, L-arginine and L-lysine are major components contributing to synthesis of the antimicrobially active beta-defensins induced under dry eye conditions. The first results have demonstrated that transport of L-arginine and L-lysine into epithelial cells is limited by the y(+) transporter at the ocular surface.

Arginine transport in human erythroid cells: discrimination of CAT1 and 4F2hc/y+LAT2 roles.

Since arginine metabolites, such as nitric oxide and polyamines, influence the expression of genes involved in erythroid differentiation, the transport of the cationic amino acid may play an important role in erythroid cells. However, available data only concern the presence in these cells of CAT1 transporter (system y(+)), while no information exists on the role of the heterodimeric transporters of system y(+)L (4F2hc/y(+)LAT1 and 4F2hc/y(+)LAT2) which operates transmembrane arginine fluxes cis-inhibited by neutral amino acids in the presence of sodium. Using erythroleukemia K562 cells and normal erythroid precursors, we demonstrate here that arginine transport in human erythroid cells is due to the additive contributions of a leucine-sensitive and leucine-insensitive component. In both cell types, leucine inhibition of arginine influx is much less evident in the absence of sodium, a hallmark of system y(+)L. In K562 cells, N-ethylmaleimide, a known inhibitor of CAT transporters (system y(+)), suppresses only a fraction of arginine influx corresponding to leucine-insensitive uptake. Moreover, in Xenopus oocytes coexpressing 4F2hc and y(+)LAT2, leucine exerts a marked inhibition of arginine transport, partially dependent on sodium, while no inhibition is seen in oocytes expressing CAT1. Lastly, silencing of SLC7A6, the gene for y(+)LAT2, lowers arginine transport and doubles the intracellular content of the cationic amino acid in K562 cells. We conclude that arginine transport in human erythroid cells is due to both system y(+) (CAT1 transporter) and system y(+)L (4F2hc/y(+)LAT2 isoform), which mainly contribute, respectively, to the influx and to the efflux of the cationic amino acid.

Absence of Btn1p in the yeast model for juvenile Batten disease may cause arginine to become toxic to yeast cells.

Lymphoblast cell lines established from individuals with juvenile Batten disease (JNCL) bearing mutations in CLN3 and yeast strains lacking Btn1p (btn1-Delta), the homolog to CLN3, have decreased intracellular levels of arginine and defective lysosomal/vacuolar transport of arginine. It is important to establish the basis for this decrease in arginine levels and whether restoration of arginine levels would be of therapeutic value for Batten disease. Previous studies have suggested that synthesis and degradation of arginine are unaltered in btn1-Delta. Using the yeast model for the Batten disease, we have determined that although btn1-Delta results in decreased intracellular arginine levels, it does not result from altered arginine uptake, arginine efflux or differences in arginine incorporation into peptides. However, expression of BTN1 is dependent on arginine and Gcn4p, the master regulator of amino acid biosynthesis. Moreover, deletion of GCN4 (gcn4-Delta), in combination with btn1-Delta, results in a very specific growth requirement for arginine. In addition, increasing the intracellular levels of arginine through overexpression of Can1p, the plasma membrane basic amino acid permease, results in increased cell volume and a severe growth defect specific to basic amino acid availability for btn1-Delta, but not wild-type cells. Therefore, elevation of intracellular levels of arginine in btn1-Delta cells is detrimental and is suggestive that btn1-Delta and perhaps mutation of CLN3 predispose cells to keep arginine levels lower than normal.

Equilibrative nucleoside (ENTs) and cationic amino acid (CATs) transporters: implications in foetal endothelial dysfunction in human pregnancy diseases.

Gestational diabetes (GD, characterized by abnormal D-glucose metabolism), intrauterine growth restriction (IUGR, a disease associated with reduced oxygen delivery (hypoxia) to the foetus), and preeclampsia (PE, a pregnancy complication characterized by high blood pressure, proteinuria and increased vascular resistance), induce foetal endothelial dysfunction with implications in adult life and increase the risk of vascular diseases. Synthesis of nitric oxide (NO) and uptake of L-arginine (the NO synthase (NOS) substrate) and adenosine (a vasoactive endogenous nucleoside) by the umbilical vein endothelium is altered in pregnancies with GD, IUGR or PE. Mechanisms underlying these alterations include differential expression of equilibrative nucleoside transporters (ENTs), cationic amino acid transporters (CATs), and NOS. Modulation of ENTs, CATs, and NOS expression and activity in endothelium involves protein kinase C (PKC), mitogen-activated protein kinases p42 and p44 (p42/44(mapk)), calcium, and phosphatidyl inositol 3 kinase (PI3k), among others. Elevated extracellular D-glucose and hypoxia alter human endothelial function. However, information regarding the transcriptional modulation of ENTs, CATs, and NOS is limited. This review focuses on the effect of transcriptional and post-transcriptional regulatory mechanisms involved in the modulation of ENTs and CATs, and NOS expression and activity, and the consequences for foetal endothelial function in GD, IUGR and PE. The available information will contribute to a better understanding of the cell and molecular basis of the altered vascular endothelial function in these pregnancy diseases and will emphasize the key role of this type of epithelium in placental function and the normal foetal development and growth.

Identification of two cationic amino acid transporters required for nutritional signaling during mosquito reproduction.

The defining characteristic of anautogenous mosquitoes is their requirement for a blood meal to initiate reproduction. The need for blood drives the association of vector and host, and is the primary reason why anautogenous mosquitoes are effective disease vectors. During mosquito vitellogenesis, a key process in reproduction, yolk protein precursor (YPP) gene expression is activated specifically in the fat body, the insect analogue of the vertebrate liver. We have demonstrated that blood meal derived amino acids (AAs) activate YPP genes via the target of rapamycin (TOR)-signal transduction pathway. Here we show, by stimulating fat bodies with balanced AA solutions lacking individual AAs, that specific cationic and branched AAs are essential for activation of the vitellogenin (vg) gene, the major YPP gene. Treatment of fat bodies with AA uptake inhibitors results in a strong inhibition of AA-induced vg gene expression proving that an active transport mechanism is necessary to transduce the AA signal. We identified two cationic AA transporters (CATs) in the fat body of Aedes aegypti females--Aa slimfast and iCAT2. RNAi knockdown of slimfast and iCAT2 results in a strong decrease in the response to AAs by the vg gene similar to that seen due to TOR inhibition. These data demonstrate that active uptake of specific AAs plays a key role in nutritional signaling during the onset of vitellogenic gene expression in mosquitoes and it is mediated by two cationic AA transporters.