Heptahelical protein PQLC2 is a lysosomal cationic amino acid exporter underlying the action of cysteamine in cystinosis therapy.

Cystinosin, the lysosomal cystine exporter defective in cystinosis, is the founding member of a family of heptahelical membrane proteins related to bacteriorhodopsin and characterized by a duplicated motif termed the PQ loop. PQ-loop proteins are more frequent in eukaryotes than in prokaryotes; except for cystinosin, their molecular function remains elusive. In this study, we report that three yeast PQ-loop proteins of unknown function, Ypq1, Ypq2, and Ypq3, localize to the vacuolar membrane and are involved in homeostasis of cationic amino acids (CAAs). We also show that PQLC2, a mammalian PQ-loop protein closely related to yeast Ypq proteins, localizes to lysosomes and catalyzes a robust, electrogenic transport that is selective for CAAs and strongly activated at low extracytosolic pH. Heterologous expression of PQLC2 at the yeast vacuole rescues the resistance phenotype of an ypq2 mutant to canavanine, a toxic analog of arginine efficiently transported by PQLC2. Finally, PQLC2 transports a lysine-like mixed disulfide that serves as a chemical intermediate in cysteamine therapy of cystinosis, and PQLC2 gene silencing trapped this intermediate in cystinotic cells. We conclude that PQLC2 and Ypq1-3 proteins are lysosomal/vacuolar exporters of CAAs and suggest that small-molecule transport is a conserved feature of the PQ-loop protein family, in agreement with its distant similarity to SWEET sugar transporters and to the mitochondrial pyruvate carrier. The elucidation of PQLC2 function may help improve cysteamine therapy. It may also clarify the origin of CAA abnormalities in Batten disease.

Structural insights on pathogenic effects of novel mutations causing pyruvate carboxylase deficiency.

Pyruvate carboxylase (PC), a key enzyme for gluconeogenesis and anaplerotic pathways, consists of four domains, namely, biotin carboxylase (BC), carboxyltransferase (CT), pyruvate carboxylase tetramerization (PT), and biotin carboxyl carrier protein (BCCP). PC deficiency is a rare metabolic disorder inherited in an autosomal recessive way. The most severe form (form B) is characterized by neonatal lethal lactic acidosis, whereas patients with form A suffer chronic lactic acidosis with psychomotor retardation. Diagnosis of PC deficiency relies on enzymatic assay and identification of the PC gene mutations. To date, six mutations of the PC gene have been identified. We report nine novel mutations of the PC gene, in five unrelated patients: three being affected with form B, and the others with form A. Three of them were frameshift mutations predicted to introduce a premature termination codon, the remaining ones being five nucleotide substitutions and one in frame deletion. Impact of these mutations on mRNA was assessed by RT-PCR. Evidence for a deleterious effect of the missense mutations was achieved using protein alignments and three-dimensional structural prediction, thanks to our modeling of the human PC structure. Altogether, our data and those previously reported indicate that form B is consistently associated with at least one truncating mutation, mostly lying in CT (C-terminal part) or BCCP domains, whereas form A always results from association of two missense mutations located in BC or CT (N-terminal part) domains. Finally, although most PC mutations are suggested to interfere with biotin metabolism, none of the PC-deficient patients was biotin-responsive.

Plasma homocysteine levels are not increased in murine models of Alzheimer's disease.

Alzheimer's disease (AD) patients tend to have increased plasma levels of homocysteine. However, it is unclear whether abnormality in homocysteine levels is a primary cause of Alzheimer's disease or a disease marker. In order to investigate the relative impact of Alzheimer's disease on plasma homocysteine levels, total plasma homocysteine levels were evaluated in transgenic mouse models that exhibit abnormalities in their brains that are similar to Alzheimer's patients. No significant difference was observed in blood of murine models compared to control mice, indicating that elevated plasma homocysteine level seems to be a risk marker at the most.

The neuronal SAPK/JNK pathway is altered in a murine model of hyperhomocysteinemia.

Deficiency in cystathionine beta synthase (CBS) leads to high plasma homocysteine concentrations and causes hyperhomocysteinemia, a common risk factor for vascular disease, stroke and possibly neurodegenerative diseases. Various neuronal diseases have been associated with hyperhomocysteinemia, but the molecular mechanisms of homocysteine toxicity are unknown. We investigated the pathways involved in the pathological process, by analyzing differential gene expression in neuronal tissues. We used a combination of differential display and cDNA arrays to identify genes differentially expressed during hyperhomocysteinemia in brain of CBS-deficient mice. In this murine model of hyperhomocysteinemia, both plasma and brain homocysteine concentrations were high. Several genes were found to be differentially expressed in the brains of CBS-deficient mice, and the identities of some of these genes suggested that the SAPK/JNK pathway was altered in the brains of CBS-deficient mice. We therefore investigated the activation of proteins involved in the SAPK/JNK cascade. JNK and c-Jun were activated in the hippocampal neurones of CBS-deficient mice, suggesting that the SAPK/JNK pathway may play an important role in the development of neuronal defects associated with hyperhomocysteinemia.

Prenatal diagnosis of carnitine palmitoyltransferase 2 deficiency in chorionic villi: a novel approach.

Carnitine palmitoyltransferase 2 (CPT2) deficiency, the most common autosomal recessive inherited disease of the mitochondrial long-chain fatty acid (LCFA) beta-oxidation, may result in three distinct clinical phenotypes, namely, a mild adult muscular form, a severe infantile hepatocardiomuscular disease, and a neonatal form, which includes dysmorphic features in addition to hepatocardiomuscular symptoms. Both the latter forms are life-threatening diseases, and prenatal diagnosis (PND) can be offered to couples at a one-fourth risk of having an affected child. PND of CPT2 deficiency hitherto relied mostly on mutation detection from fresh chorionic villi (10 weeks' gestation), since CPT2 activity could be assayed on cultured amniocytes only (16-17 weeks' gestation).We devised a CPT2 activity assay from 10 mg of chorionic villi sampling (CVS). Combining this enzymatic assay to haplotype study using polymorphic markers linked to the CPT2 gene, we were able to carry out within 2 days, CPT2 deficiency PND, in two unrelated families, using a CVS performed at the 11th week of gestation.

Altered gene expression in liver from a murine model of hyperhomocysteinemia.

Cystathionine beta-synthase (CBS) deficiency causes severe hyperhomocysteinemia and other signs of homocystinuria syndrome, in particular a premature atherosclerosis with multiple thrombosis. However, the molecular mechanisms by which homocysteine could interfere with normal cell function are poorly understood in a whole organ like the liver, which is central to the catabolism of homocysteine. We used a combination of differential display and cDNA arrays to analyze differential gene expression in association with elevated hepatic homocysteine levels in CBS-deficient mice, a murine model of hyperhomocysteinemia. Expression of several genes was found to be reproducibly abnormal in the livers of heterozygous and homozygous CBS-deficient mice. We report altered expression of genes encoding ribosomal protein S3a and methylthioadenosine phosphorylase, suggesting such cellular growth and proliferation perturbations may occur in homozygous CBS-deficient mice liver. Many up- or down-regulated genes encoded cytochromes P450, evidence of perturbations of the redox potential in heterozygous and homozygous CBS-deficient mice liver. The expression of various genes involved in severe oxidative processes was also abnormal in homozygous CBS-deficient mice liver. Among them, the expression of heme oxygenase 1 gene was increased, concomitant with overexpression of heme oxygenase 1 at the protein level. Commensurate with the difference in hepatic mRNA paraoxonase 1 abundance, the mean hepatic activity of paraoxonase 1, an enzyme that protects low density lipoprotein from oxidation, was 3-fold lower in homozygous CBS-deficient mice. Heterozygous CBS-deficient mice, when fed a hyperhomocysteinemic diet, have also reduced PON1 activity, which demonstrates the effect of hyperhomocysteinemia in the paraoxonase 1 activity.