Amino acid transport in schistosomes: Characterization of the permeaseheavy chain SPRM1hc

Schistosomes are human parasitic flatworms that constitute an important public health problem globally. Adult parasites live in the bloodstream where they import nutrients such as amino acids across their body surface (the tegument). One amino acid transporter, Schistosome Permease 1 light chain, SPRM1lc, a member of the glycoprotein-associated family of transporters (gpaAT), has been characterized in schistosomes. Only a single member of the SLC3 family of glycoproteins that associate with gpaATs is found following extensive searching of the genomes of Schistosoma mansoni and S. japonicum. In this report, we characterize this schistosome permease heavy chain (SPRM1hc) gene and protein. The 72-kDa gene product is predicted to possess a single transmembrane domain, a (betaalpha)(8) (TIM barrel) conformation and a catalytic triad. Xenopus oocytes functionally expressing SPRM1hc with SPRM1lc import phenylalanine, arginine, lysine, alanine, glutamine, histidine, tryptophan, and leucine. Biochemical characterization demonstrates that in Xenopus extracts and in schistosome extracts SPRM1hc is associated into a high molecular weight complex with SPRM1lc that is disrupted by reducing agents. Quantitative real-time PCR and Western analysis demonstrate that SPRM1hc is expressed in each schistosome life stage examined (eggs, cercariae, schistosomula, adult males and females). SPRM1hc is widely distributed throughout adult male and female worms as determined by immunolocalization. Consistent with the hypothesis that SPRM1hc functions to facilitate nutrient uptake from host blood, immunogold electron microscopy confirms that the protein is distributed on the host-interactive tegumental membranes. We propose that surface-exposed, host-interactive, nutrient-transporting proteins like the SPRM1 heterodimer are promising vaccine candidates

Thyroid hormone transport by the heterodimeric human system L amino acid transporter

Transport of thyroid hormone across the cell membrane is required for thyroid hormone action and metabolism. We have investigated the possible transport of iodothyronines by the human system L amino acid transporter, a protein consisting of the human 4F2 heavy chain and the human LAT1 light chain. Xenopus oocytes were injected with the cRNAs coding for human 4F2 heavy chain and/or human LAT1 light chain, and after 2 d were incubated at 25 C with 0.01-10 microM [(125)I]T(4), [(125)I]T(3), [(125)I]rT(3), or [(125)I]3,3'-diiodothyronine or with 10-100 microM [(3)H]arginine, [(3)H]leucine, [(3)H]phenylalanine, [(3)H]tyrosine, or [(3)H]tryptophan. Injection of human 4F2 heavy chain cRNA alone stimulated the uptake of leucine and arginine due to dimerization of human 4F2 heavy chain with an endogenous Xenopus light chain, but did not affect the uptake of other ligands. Injection of human LAT1 light chain cRNA alone did not stimulate the uptake of any ligand. Coinjection of cRNAs for human 4F2 heavy chain and human LAT1 light chain stimulated the uptake of phenylalanine > tyrosine > leucine > tryptophan (100 microM) and of 3,3'-diiodothyronine > rT(3) approximately T(3) > T(4) (10 nM), which in all cases was Na(+) independent. Saturation analysis provided apparent Michaelis constant (K(m)) values of 7.9 microM for T(4), 0.8 microM for T(3), 12.5 microM for rT(3), 7.9 microM for 3,3'-diiodothyronine, 46 microM for leucine, and 19 microM for tryptophan. Uptake of leucine, tyrosine, and tryptophan (10 microM) was inhibited by the different iodothyronines (10 microM), in particular T(3). Vice versa, uptake of 0.1 microM T(3) was almost completely blocked by coincubation with 100 microM leucine, tryptophan, tyrosine, or phenylalanine. Our results demonstrate stereospecific Na(+)-independent transport of iodothyronines by the human heterodimeric system L amino acid transporter

Endothelial mineralocorticoid receptor activation mediates endothelial dysfunction in diet-induced obesity

Received 22 July 2012; revised 29 January 2013; accepted 4 March 2013Aims Aldosterone plays a crucial role in cardiovascular disease. ‘Systemic' inhibition of its mineralocorticoid receptor (MR) decreases atherosclerosis by reducing inflammation and oxidative stress. Obesity, an important cardiovascular risk factor, is an inflammatory disease associated with increased plasma aldosterone levels. We have investigated the role of the ‘endothelial' MR in obesity-induced endothelial dysfunction, the earliest stage in atherogenesis. Methods and results C57BL/6 mice were exposed to a normal chow diet (ND) or a high-fat diet (HFD) alone or in combination with the MR antagonist eplerenone (200 mg/kg/day) for 14 weeks. Diet-induced obesity impaired endothelium-dependent relaxation in response to acetylcholine, whereas eplerenone treatment of obese mice prevented this. Expression analyses in aortic endothelial cells isolated from these mice revealed that eplerenone attenuated expression of pro-oxidative NADPH oxidase (subunits p22phox, p40phox) and increased expression of antioxidative genes (glutathione peroxidase-1, superoxide dismutase-1 and -3) in obesity. Eplerenone did not affect obesity-induced upregulation of cyclooxygenase (COX)-1 or prostacyclin synthase. Endothelial-specific MR deletion prevented endothelial dysfunction in obese (exhibiting high ‘endogenous' aldosterone) and in ‘exogenous' aldosterone-infused lean mice. Pre-incubation of aortic rings from aldosterone-treated animals with the COX-inhibitor indomethacin restored endothelial function. Exogenous aldosterone administration induced endothelial expression of p22phox in the presence, but not in the absence of the endothelial MR. Conclusion Obesity-induced endothelial dysfunction depends on the ‘endothelial' MR and is mediated by an imbalance of oxidative stress-modulating mechanisms. Therefore, MR antagonists may represent an attractive therapeutic strategy in the increasing population of obese patients to decrease vascular dysfunction and subsequent atherosclerotic complication

The cataract and glucosuria associated monocarboxylate transporter MCT12 is a new creatine transporter

Creatine transport has been assigned to creatine transporter 1 (CRT1), encoded by mental retardation associated SLC6A8. Here, we identified a second creatine transporter (CRT2) known as monocarboxylate transporter 12 (MCT12), encoded by the cataract and glucosuria associated gene SLC16A12. A non-synonymous alteration in MCT12 (p.G407S) found in a patient with age-related cataract (ARC) leads to a significant reduction of creatine transport. Furthermore, Slc16a12 knockout (KO) rats have elevated creatine levels in urine. Transport activity and expression characteristics of the two creatine transporters are distinct. CRT2 (MCT12)-mediated uptake of creatine was not sensitive to sodium and chloride ions or creatine biosynthesis precursors, breakdown product creatinine or creatine phosphate. Increasing pH correlated with increased creatine uptake. Michaelis-Menten kinetics yielded a Vmax of 838.8 pmol/h/oocyte and a Km of 567.4 µm. Relative expression in various human tissues supports the distinct mutation-associated phenotypes of the two transporters. SLC6A8 was predominantly found in brain, heart and muscle, while SLC16A12 was more abundant in kidney and retina. In the lens, the two transcripts were found at comparable levels. We discuss the distinct, but possibly synergistic functions of the two creatine transporters. Our findings infer potential preventive power of creatine supplementation against the most prominent age-related vision impaired conditio

Продукты химической переработки окисленных углей

Hartnup disorder, an autosomal recessive defect named after an English family described in 1956 (ref. 1), results from impaired transport of neutral amino acids across epithelial cells in renal proximal tubules and intestinal mucosa. Symptoms include transient manifestations of pellagra (rashes), cerebellar ataxia and psychosis(1,2). Using homozygosity mapping in the original family in whom Hartnup disorder was discovered, we confirmed that the critical region for one causative gene was located on chromosome 5p15 (ref. 3). This region is homologous to the area of mouse chromosome 13 that encodes the sodium-dependent amino acid transporter B(0)AT1 (ref. 4). We isolated the human homolog of B(0)AT1, called SLC6A19, and determined its size and molecular organization. We then identified mutations in SLC6A19 in members of the original family in whom Hartnup disorder was discovered and of three Japanese families. The protein product of SLC6A19, the Hartnup transporter, is expressed primarily in intestine and renal proximal tubule and functions as a neutral amino acid transporter

Control of amino-acid transport coordinates metabolic reprogramming in T cell malignancy

This study explores the regulation and importance of System L amino acid transport in a murine model of T cell acute lymphoblastic leukemia (T-ALL) caused by deletion of phosphatase and tensin homologue deleted on chromosome 10 (PTEN). There has been a strong focus on glucose transport in leukemias but the present data show that primary T-ALL cells have increased transport of multiple nutrients. Specifically, increased leucine transport in T-ALL fuels mammalian target of rapamycin complex 1 (mTORC1) activity which then sustains expression of hypoxia inducible factor-1α (HIF1α) and c-Myc; drivers of glucose metabolism in T cells. A key finding is that PTEN deletion and phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) accumulation is insufficient to initiate leucine uptake, mTORC1 activity, HIF1α or c-Myc expression in T cells and hence cannot drive T-ALL metabolic reprogramming. Instead, a key regulator for leucine transport in T-ALL is identified as NOTCH. Mass spectrometry based proteomics identifies SLC7A5 as the predominant amino acid transporter in primary PTEN(-/-) T-ALL cells. Importantly, expression of SLC7A5 is critical for the malignant transformation induced by PTEN deletion. These data reveal the importance of regulated amino acid transport for T cell malignancies, highlighting how a single amino acid transporter can play a key role.Leukemia accepted article preview online, 26 May 2017. doi:10.1038/leu.2017.160.</p

Aromatic amino acid transporter AAT-9 of Caenorhabditis elegans localizes to neurons and muscle cells.

The Caenorhabditis elegans genome encodes nine homologues of mammalian glycoprotein-associated amino acid transporters. Two of these C. elegans proteins (AAT-1 and AAT-3) have been shown to function as catalytic subunits (light chains) of heteromeric amino acid transporters. These proteins need to associate with a glycoprotein heavy chain subunit (ATG-2) to reach the cell surface in a manner similar to that of their mammalian homologues. AAT-1 and AAT-3 contain a cysteine residue in the second putative extracellular loop through which a disulfide bridge can form with a heavy chain. In contrast, six C. elegans members of this family (AAT-4 to AAT-9) lack such a cysteine residue. We show here that one of these transporter proteins, AAT-9, reaches the cell surface in Xenopus oocytes without an exogenous heavy chain and that it functions as an exchanger of aromatic amino acids. Two-electrode voltage clamp experiments demonstrate that AAT-9 displays a substrate-activated conductance. Immunofluorescence shows that it is expressed close to the pharyngeal bulbs within C. elegans neurons. The selective expression of an aat-9 promoter-green fluorescent protein construct in several neurons of this region and in wall muscle cells around the mouth supports and extends these localization data. Taken together, the results show that AAT-9 is expressed in excitable cells of the nematode head and pharynx in which it may provide a pathway for aromatic amino acid transport

Dysfunctional LAT2 Amino Acid Transporter Is Associated With Cataract in Mouse and Humans.

Cataract, the loss of ocular lens transparency, accounts for ∼50% of worldwide blindness and has been associated with water and solute transport dysfunction across lens cellular barriers. We show that neutral amino acid antiporter LAT2 (Slc7a8) and uniporter TAT1 (Slc16a10) are expressed on mouse ciliary epithelium and LAT2 also in lens epithelium. Correspondingly, deletion of LAT2 induced a dramatic decrease in lens essential amino acid levels that was modulated by TAT1 defect. Interestingly, the absence of LAT2 led to increased incidence of cataract in mice, in particular in older females, and a synergistic effect was observed with simultaneous lack of TAT1. Screening SLC7A8 in patients diagnosed with congenital or age-related cataract yielded one homozygous single nucleotide deletion segregating in a family with congenital cataract. Expressed in HeLa cells, this LAT2 mutation did not support amino acid uptake. Heterozygous LAT2 variants were also found in patients with cataract some of which showed a reduced transport function when expressed in HeLa cells. Whether heterozygous LAT2 variants may contribute to the pathology of cataract needs to be further investigated. Overall, our results suggest that defects of amino acid transporter LAT2 are implicated in cataract formation, a situation that may be aggravated by TAT1 defects

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