Structure-function studies of human SLC36 amino acid transporters

The Solute Linked Carrier family 36 (SLC36) of integral membrane transport proteins has four members which are also known as the human Proton coupled Amino acid Transporters (hPATs).This is due to hPATs 1 and 2 having been functionally characterized as proton coupled transporters that bring about a 1:1 symport of H+ and an amino acid across the membrane (with optimum uptake at an extracellular pH of 5.5). Their main substrates were found to be the small neutral amino acids glycine, alanine and proline; however, in addition to these, PATs 1 and 2 were also observed to transport a range of other substrates including some pharmacologically active compounds. hPATs 3 and 4 were considered orphan transporters, with no information available except their mRNA localization in various tissues. Additionally there is no structural information available about the PAT family of transport proteins. The thesis has two broad aims. The first aim was to study the orphan PAT transporters and to investigate whether these proteins are also amino acids transporters and, if so, what their substrates are. The second aim was to use transmembrane domain software to predict the secondary structure of the PAT transporters using hPAT1 as the model protein. Epitope tags, luminometry and functional assays were used to test these predictions, and homology modelling then employed to gain a better understanding of the structure-function relationship of this transporter family. The results of both these areas of investigation are reported in this thesis. The orphan transporter hPAT4 was found to be a very high affinity electroneutral proline and tryptophan transporter when expressed in the Xenopus laevis oocyte system. Preliminary data for hPAT3 suggested uptake of proline at extracellular pH 7.4 in Xenopus laevis oocytes and in the mouse Sertoli cell line TM4. Membrane topology studies on hPAT1 provided experimental support for the prediction of 11 transmembrane domains for this transporter protein. Homology modelling indicates a region which might play a role in the substrate specificity of PAT proteins

Differential impact of dietary branched chain and aromatic amino acids on chronic kidney disease progression in rats

The metabolism of dietary proteins generates waste products that are excreted by the kidney, in particular nitrogen-containing urea, uric acid, ammonia, creatinine, and other metabolites such as phosphates, sulfates, and protons. Kidney adaptation includes an increase in renal plasma flow (RPF) and glomerular filtration rate (GFR) and represents a burden for diseased kidneys increasing the progression rate of CKD. The present study aimed at identifying potential differences between amino acid (AA) groups constituting dietary proteins regarding their impact on RPF, GFR, and CKD progression. We utilized the well-established 5/6 nephrectomy (5/6 Nx) CKD model in rats and submitted the animals for 5 weeks to either the control diet (18% casein protein) or to diets containing 8% casein supplemented with 10% of a mix of free amino acids, representing all or only a subset of the amino acids contained in casein. Whereas the RPF and GFR measured in free moving animals remained stable during the course of the diet in rats receiving the control mix, these parameters decreased in animals receiving the branched chain amino acid (BCAA) supplementation and increased in the ones receiving the aromatic amino acids (AAAs). In animals receiving essential amino acids (EAAs) containing both BCAAs and AAAs, there was only a small increase in RPF. The kidneys of the 5/6 Nx rats receiving the BCAA diet showed the strongest increase in smooth muscle actin and collagen mRNA expression as a result of higher level of inflammation and fibrosis. These animals receiving BCAAs also showed an increase in plasma free fatty acids pointing to a problem at the level of energy metabolism. In contrast, the animals under AAA diet showed an activation of AMPK and STAT3. Taken together, our results demonstrate that subsets of EAAs contained in dietary proteins, specifically BCAAs and AAAs, exert contrasting effects on kidney functional parameters and CKD progression

Kidney mass reduction leads to L‐arginine metabolism‐dependent blood pressure increase in mice

Background Uninephrectomy (UNX) is performed for various reasons, including kidney cancer or donation. Kidneys being the main site of L‐arginine production in the body, we tested whether UNX mediated kidney mass reduction impacts L‐arginine metabolism and thereby nitric oxide production and blood pressure regulation in mice. Methods and Results In a first series of experiments, we observed a significant increase in arterial blood pressure 8 days post‐UNX in female and not in male mice. Further experimental series were performed in female mice, and the blood pressure increase was confirmed by telemetry. L‐citrulline, that is used in the kidney to produce L‐arginine, was elevated post‐UNX as was also asymmetric dimethylarginine, an inhibitor of nitric oxide synthase that competes with L‐arginine and is a marker for renal failure. Interestingly, the UNX‐induced blood pressure increase was prevented by supplementation of the diet with 5% of the L‐arginine precursor, L‐citrulline. Because L‐arginine is metabolized in the kidney and other peripheral tissues by arginase‐2, we tested whether the lack of this metabolic pathway also compensates for decreased L‐arginine production in the kidney and/or for local nitric oxide synthase inhibition and consecutive blood pressure increase. Indeed, upon uninephrectomy, arginase‐2 knockout mice (Arg‐2−/−) neither displayed an increase in asymmetric dimethylarginine and L‐citrulline plasma levels nor a significant increase in blood pressure. Conclusions UNX leads to a small increase in blood pressure that is prevented by L‐ citrulline supplementation or arginase deficiency, 2 measures that appear to compensate for the impact of kidney mass reduction on L‐arginine metabolism

Brain catecholamine depletion and motor impairment in a Th knock-in mouse with type B tyrosine hydroxylase deficiency

Tyrosine hydroxylase catalyses the hydroxylation of L-tyrosine to l-DOPA, the rate- limiting step in the synthesis of catecholamines. Mutations in the TH gene encoding tyrosine hydroxylase are associated with the autosomal recessive disorder tyrosine hydroxylase deficiency, which manifests phenotypes varying from infantile parkinsonism and DOPA-responsive dystonia, also termed type A, to complex encephalopathy with perinatal onset, termed type B. We generated homozygous Th knock-in mice with the mutation Th-p.R203H, equivalent to the most recurrent human mutation associated with type B tyrosine hydroxylase deficiency (TH-p.R233H), often unresponsive to l-DOPA treatment. The Th knock-in mice showed normal survival and food intake, but hypotension, hypokinesia, reduced motor coordination, wide-based gate and catalepsy. This phenotype was associated with a gradual loss of central catecholamines and the serious manifestations of motor impairment presented diurnal fluctuation but did not improve with standard l-DOPA treatment. The mutant tyrosine hydroxylase enzyme was unstable and exhibited deficient stabilization by catecholamines, leading to decline of brain tyrosine hydroxylase-immunoreactivity in the Th knock-in mice. In fact the substantia nigra presented an almost normal level of mutant tyrosine hydroxylase protein but distinct absence of the enzyme was observed in the striatum, indicating a mutation-associated mislocalization of tyrosine hydroxylase in the nigrostriatal pathway. This hypomorphic mouse model thus provides understanding on pathomechanisms in type B tyrosine hydroxylase deficiency and a platform for the evaluation of novel therapeutics for movement disorders with loss of dopaminergic input to the striatum

Evaluation of in-situ thermal transmittance of innovative building integrated photovoltaic modules: Application to thermal performance assessment for green mark certification in the tropics

10.1016/j.energy.2021.121316Energy235121316-12131