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Analysis of LPI-causing mutations on y+LAT1 function and localization.

ABSTRACT: BACKGROUND:y+LAT1, encoded by SCL7A7, is the protein mutated in Lysinuric Protein Intolerance (LPI), a rare metabolic disease caused by a defective cationic amino acid (CAA, arginine, lysine, ornithine) transport at the basolateral membrane of intestinal and renal tubular cells. The disease is characterized by protein-rich food intolerance with secondary urea cycle disorder, but symptoms are heterogeneous with lung and immunological complications that are not explainable by the CAA transport defect. With the exception of the Finnish founder mutation (c.895-2A?>?T, LPIFin), LPI-causative mutations are heterogeneous and genotype-phenotype correlations have not been found. Here we addressed system y+L-mediated arginine uptake in monocytes from three LPI Italian patients and in lymphoblasts carrying the same mutations; in parallel, the genetic defects carried by the patients were reproduced as eGFP-tagged y+LAT1 mutants in transfected CHO cells to define the function and localization protein. RESULTS:System y+L activity is impaired in monocytes isolated from all LPI patients, and in CHO cells transfected with the three eGFP-y+LAT1 mutants, but not in lymphoblasts bearing the same mutations. The analysis of protein localization with confocal microscopy revealed that the eGFP-tagged mutants were retained inside the cytosol, with a pattern of expression quite heterogeneous among the mutants. CONCLUSIONS:The three mutations studied of y+LAT1 transporter result in a defective arginine transport both in ex vivo (monocytes) and in vitro (CHO transfected cells) models, likely caused by the retention of the mutated proteins in the cytosol. The different effect of y+LAT1 mutation on arginine transport in monocytes and lymphoblasts is supposed to be due to the different expression of SLC7A7 mRNA in the two models, supporting the hypothesis that the impact of LPI defect largely depends on the relative abundance of LPI target gene in each cell type.

SUBMITTER: Rotoli BM

PROVIDER: S-EPMC6399926 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Analysis of LPI-causing mutations on y+LAT1 function and localization.

Rotoli Bianca Maria BM Barilli Amelia A Ingoglia Filippo F Visigalli Rossana R Bianchi Massimiliano G MG Ferrari Francesca F Martinelli Diego D Dionisi-Vici Carlo C Dall'Asta Valeria V

Orphanet journal of rare diseases 20190304 1

<h4>Background</h4>y+LAT1, encoded by SCL7A7, is the protein mutated in Lysinuric Protein Intolerance (LPI), a rare metabolic disease caused by a defective cationic amino acid (CAA, arginine, lysine, ornithine) transport at the basolateral membrane of intestinal and renal tubular cells. The disease is characterized by protein-rich food intolerance with secondary urea cycle disorder, but symptoms are heterogeneous with lung and immunological complications that are not explainable by the CAA trans ...[more]

PMID: 30832686

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Project description:BackgroundMutations of EFNB1 cause the X-linked malformation syndrome craniofrontonasal syndrome (CFNS). CFNS is characterized by an unusual phenotypic pattern of inheritance, because it affects heterozygous females more severely than hemizygous males. This sex-dependent inheritance has been explained by random X-inactivation in heterozygous females and the consequences of cellular interference of wild type and mutant EFNB1-expressing cell populations. EFNB1 encodes the transmembrane protein ephrin-B1, that forms bi-directional signalling complexes with Eph receptor tyrosine kinases expressed on complementary cells. Here, we studied the effects of patient-derived EFNB1 mutations predicted to give rise to truncated ephrin-B1 protein or to disturb Eph/ephrin-B1 reverse ephrin-B1 signalling. Five mutations are investigated in this work: nonsense mutation c.196C > T/p.R66X, frameshift mutation c.614_615delCT, splice-site mutation c.406 + 2T > C and two missense mutations p.P54L and p.T111I. Both missense mutations are located in the extracellular ephrin domain involved in Eph-ephrin-B1 recognition and higher order complex formation.MethodsNonsense mutation c.196C > T/p.R66X, frameshift mutation c.614_615delCT and splice-site mutation c.406+2T > C were detected in the primary patient fibroblasts by direct sequencing of the DNA and were further analysed by RT-PCR and Western blot analyses.The impact of missense mutations p.P54L and p.T111I on cell behaviour and reverse ephrin-B1 cell signalling was analysed in a cell culture model using NIH 3T3 fibroblasts. These cells were transfected with the constructs generated by in vitro site-directed mutagenesis. Investigation of missense mutations was performed using the Western blot analysis and time-lapse microscopy.Results and discussionNonsense mutation c.196C > T/p.R66X and frameshift mutation c.614_615delCT escape nonsense-mediated RNA decay (NMD), splice-site mutation c.406+2T > C results in either retention of intron 2 or activation of a cryptic splice site in exon 2. However, c.614_615delCT and c.406+2T > C mutations were found to be not compatible with production of a soluble ephrin-B1 protein. Protein expression of the p.R66X mutation was predicted unlikely but has not been investigated.Ectopic expression of p.P54L ephrin-B1 resists Eph-receptor mediated cell cluster formation in tissue culture and intracellular ephrin-B1 Tyr324 and Tyr329 phosphorylation. Cells expressing p.T111I protein show similar responses as wild type expressing cells, however, phosphorylation of Tyr324 and Tyr329 is reduced.ConclusionsPathogenic mechanisms in CFNS manifestation include impaired ephrin-B1 signalling combined with cellular interference.

Project description:Mitochondrial dysfunction and oxidative stress are frequently observed in the early stages of Alzheimer's disease (AD). Studies have shown that presenilin-1 (PS1), the catalytic subunit of γ-secretase whose mutation is linked to familial AD (FAD), localizes to the mitochondrial membrane and regulates its homeostasis. Thus, we investigated how five PS1 mutations (A431E, E280A, H163R, M146V, and Δexon9) observed in FAD affect mitochondrial functions. Methods: We used H4 glioblastoma cell lines genetically engineered to inducibly express either the wild-type PS1 or one of the five PS1 mutants in order to examine mitochondrial morphology, dynamics, membrane potential, ATP production, mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs), oxidative stress, and bioenergetics. Furthermore, we used brains of PS1M146V knock-in mice, 3xTg-AD mice, and human AD patients in order to investigate the role of PS1 in regulating MAMs formation. Results: Each PS1 mutant exhibited slightly different mitochondrial dysfunction. Δexon9 mutant induced mitochondrial fragmentation while A431E, E280A, H163R, and M146V mutants increased MAMs formation. A431E, E280A, M146V, and Δexon9 mutants also induced mitochondrial ROS production. A431E mutant impaired both complex I and peroxidase activity while M146V mutant only impaired peroxidase activity. All PS1 mutants compromised mitochondrial membrane potential and cellular ATP levels were reduced by A431E, M146V, and Δexon9 mutants. Through comparative profiling of hippocampal gene expression in PS1M146V knock-in mice, we found that PS1M146V upregulates Atlastin 2 (ATL2) expression level, which increases ER-mitochondria contacts. Down-regulation of ATL2 after PS1 mutant induction rescued abnormally elevated ER-mitochondria interactions back to the normal level. Moreover, ATL2 expression levels were significantly elevated in the brains of 3xTg-AD mice and AD patients. Conclusions: Overall, our findings suggest that each of the five FAD-linked PS1 mutations has a deleterious effect on mitochondrial functions in a variety of ways. The adverse effects of PS1 mutations on mitochondria may contribute to MAMs formation and oxidative stress resulting in an accelerated age of disease onset in people harboring mutant PS1.

Dataset Information

Analysis of LPI-causing mutations on y+LAT1 function and localization.

Publications

Analysis of LPI-causing mutations on y+LAT1 function and localization.

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