1,721,017 research outputs found
Identification de cibles moléculaires mitochondriales communes à différents stresses dans le dysfonctionnement des cellules bêta
No full textIntroduction : Une exposition chronique des cellules ancréatiques bêta à des stresses aussi variés qu’une concentration élevée de glucose, d’acides gras ou
d’oxydants induit un dysfonctionnement du couplage entre le métabolisme du
glucose et la sécrétion d’insuline, généralement lié au métabolisme mitochondrial
et entraîne l’apoptose. Afin de déterminer les mécanismes moléculaires respectifs de ces différents stresses associés au diabète, nous avons voulu identifier
de nouvelles cibles en combinant une analyse sélective de l’expression de
gènes impliqués dans la fonction mitochondriale avec des outils bio-informatiques
récemment développés. Un intérêt particulier a été porté sur l’expression des
transporteurs mitochondriaux de la famille Slc25a.
Matériels et méthodes : Les cellules INS-1E ont été cultivées pendant trois jours en présence de stresses variés : concentrations de glucose basse (5,5 mM), intermédiaire (11,6 mM) et élevée (25mM), de 0,4 mM palmitate ou d’oléate ou à un stress oxydatif 200microM H2O2) transitoire de 10 minutes. Après isolation de l’ARN total et synthèse de l’ADNc, le profil d’expression des 57 gènes d’intérêt sélectionnés a été
obtenu par RT-PCR au moyen d’une carte micro-fluide (Mito-array). Les résultats
ont été intégrés dans le programme bio-informatique Cytoscape, afin d’établir un
réseau compréhensible des interactions géniques et de visualiser les changements
complexes d’expression d’ARNm suite aux différents stresses.
Résultats : Parmi les 49 gènes détectés dans les cellules INS-1E, nous avons
identifié l’expression de 22 transporteurs mitochondriaux de la famille Slc25a.
Une concentration élevée de glucose induit l’expression de plusieurs transporteurs
(Slc25a1, Slc25a10, Slc25a13) ; résultats compatibles avec une activité anaplérotique/
cataplérotique élevée. Le degré d’insaturation des acides gras modifie l’expression des nombreux gènes de manière opposée, suggérant des effets spécifiques sur la cellule bêta tels que toxicité versus dysfonctionnement.
Conclusion : Une meilleure compréhension des réseaux métaboliques altérés
dans le dysfonctionnement des cellules bêta suite à différentes stresses sera utile dans l’approche thérapeutique du diabète
A fourth ADP/ATP carrier isoform in man: identification, bacterial expression, functional characterization and tissue distribution
No full textIdentification and functional characterization of the yeast peroxisomal adenine nucleotide transporter
No full textTHE HUMAN GENE SLC25A17 ENCODES A PEROXISOMAL TRANSPORTER OF COENZYME A, FAD AND NAD+.
No full textThe essential cofactors coenzyme A (CoA), FAD and NAD+ are synthesized outside the peroxisomes and therefore must be transported into the peroxisomal matrix where they are required for important processes. In this work we have functionally identified and characterized SLC25A17, which is the only member of the mitochondrial carrier family that has previously been shown to be localized in the peroxisomal membrane. Herein, recombinant and purified SLC25A17 was reconstituted into liposomes. Its transport properties and kinetic parameters demonstrate that SLC25A17 is a transporter of CoA, FAD, FMN, AMP and to a lesser extent of NAD+, adenosine 3',5'-diphosphate (PAP) and ADP. SLC25A17 functioned almost exclusively by a counter-exchange mechanism, was saturable and inhibited by pyridoxal-5'-phosphate and other mitochondrial carrier inhibitors. It was expressed to various degrees in all the human tissues examined. Its main function is probably to transport free CoA, FAD and NAD+ into peroxisomes in exchange for intraperoxisomally generated PAP, FMN and AMP. This is the first report describing the identification and characterization of a transporter for multiple free cofactors in peroxisomes
Diseases Caused by Mutations in Mitochondrial Carrier Genes SLC25: A Review
Full text linkIn the 1980s, after the mitochondrial DNA (mtDNA) had been sequenced, several diseases resulting from mtDNA mutations emerged. Later, numerous disorders caused by mutations in the nuclear genes encoding mitochondrial proteins were found. A group of these diseases are due to defects of mitochondrial carriers, a family of proteins named solute carrier family 25 (SLC25), that transport a variety of solutes such as the reagents of ATP synthase (ATP, ADP, and phosphate), tricarboxylic acid cycle intermediates, cofactors, amino acids, and carnitine esters of fatty acids. The disease-causing mutations disclosed in mitochondrial carriers range from point mutations, which are often localized in the substrate translocation pore of the carrier, to large deletions and insertions. The biochemical consequences of deficient transport are the compartmentalized accumulation of the substrates and dysfunctional mitochondrial and cellular metabolism, which frequently develop into various forms of myopathy, encephalopathy, or neuropathy. Examples of diseases, due to mitochondrial carrier mutations are: combined D-2- and L-2-hydroxyglutaric aciduria, carnitine-acylcarnitine carrier deficiency, hyperornithinemia-hyperammonemia-homocitrillinuria (HHH) syndrome, early infantile epileptic encephalopathy type 3, Amish microcephaly, aspartate/glutamate isoform 1 deficiency, congenital sideroblastic anemia, Fontaine progeroid syndrome, and citrullinemia type II. Here, we review all the mitochondrial carrier-related diseases known until now, focusing on the connections between the molecular basis, altered metabolism, and phenotypes of these inherited disorders
Changes in mitochondrial carriers exhibit stress-Specific signatures in INS-1E ß-cells exposed to glucose versus fatty acids
No full textChronic exposure of ß-cells to metabolic stresses impairs their function and potentially induces apoptosis. Mitochondria play a central role in coupling glucose metabolism to insulin secretion. However, little is known on mitochondrial responses to specific stresses; i.e. low versus high glucose,saturated versus unsaturated fatty acids, or oxidative stress. INS-1E cells were exposed for 3 days to 5.6 mM glucose, 25 mM glucose, 0.4 mM palmitate, and 0.4 mM oleate. Culture at standard 11.1 mM glucose served as no-stress control and transient oxidative stress (200 μM H2O2 for 10 min at day 0) served as positive stressful condition. Mito-array analyzed transcripts of 60
mitochondrion-associated genes with special focus on members of the Slc25 family. Transcripts of interest were evaluated at the protein level by immunoblotting. Bioinformatics analyzed the
expression profiles to delineate comprehensive networks. Chronic exposure to the different metabolic stresses impaired glucose-stimulated insulin secretion; revealing glucotoxicity and
lipo-dysfunction. Both saturated and unsaturated fatty acids increased expression of the
carnitine/acylcarnitine carrier CAC, whereas the citrate carrier CIC and energy sensor SIRT1 were
specifically upregulated by palmitate and oleate, respectively. High glucose upregulated CIC, the
dicarboxylate carrier DIC and glutamate carrier GC1. Conversely, it reduced expression of energy
sensors (AMPK, SIRT1, SIRT4), metabolic genes,transcription factor PDX1, and anti-apoptotic Bcl2.
This was associated with caspase-3 cleavage and cell death. Expression levels of GC1 and SIRT4 exhibited positive and negative glucose dose-response, respectively. Expression profiles of energy
sensors and mitochondrial carriers were selectively modified by the different conditions, exhibiting
stress-specific signatures
Three mitochondrial transporters of Saccharomyces cerevisiae are essential for ammonium fixation and lysine biosynthesis in synthetic minimal medium
No full textThe nuclear genes of Saccharomyces cerevisiae YHM2, ODC1 and ODC2 encode three transporters that are localized in the inner mitochondrial membrane. In this study, the roles of YHM2, ODC1 and ODC2 in the assimilation of nitrogen and in the biosynthesis of lysine have been investigated. Both the odc1Î ́. odc2Î ́ double knockout and the yhm2Î ́ mutant grew similarly as the YPH499 wild-type strain on synthetic minimal medium (SM) containing 2% glucose and ammonia as the main nitrogen source. In contrast, the yhm2Î ́. odc1Î ́. odc2Î ́ triple knockout exhibited a marked growth defect under the same conditions. This defect was fully restored by the individual expression of YHM2, ODC1 or ODC2 in the triple deletion strain. Furthermore, the lack of growth of yhm2Î ́. odc1Î ́. odc2Î ́ on 2% glucose SM was rescued by the addition of glutamate, but not glutamine, to the medium. Using lysine-prototroph YPH499-derived strains, the yhm2Î ́. odc1Î ́. odc2Î ́ knockout (but not the odc1Î ́. odc2Î ́ and yhm2Î ́ mutants) also displayed a growth defect in lysine biosynthesis on 2% glucose SM, which was rescued by the addition of lysine and, to a lesser extent, by the addition of 2-aminoadipate. Additional analysis of the triple mutant showed that it is not respiratory-deficient and does not display mitochondrial DNA instability. These results provide evidence that only the simultaneous absence of YHM2, ODC1 and ODC2 impairs the export from the mitochondrial matrix of i) 2-oxoglutarate which is necessary for the synthesis of glutamate and ammonium fixation in the cytosol and ii) 2-oxoadipate which is required for lysine biosynthesis in the cytosol. Finally, the data presented allow one to suggest that the yhm2Î ́. odc1Î ́. odc2Î ́ triple knockout is suitable in complementation studies aimed at assessing the pathogenic potential of human SLC25A21 (ODC) mutations
Altered calcium homeostasis and mitochondrial dysfunction in autism
No full textAims: Genetic variants of the brain isoform of the mitochondrial asp/glu carrier (AGC1), were previously found associated with autism. Our initial aim was to investigate asp/glu transport rates and AGC content in post-mortem brains of autistic patients. Methods: Temporocortical gray matter from matched patient-control pairs was used to measure reconstituted AGC1 activity from tissue homogenates or isolated mitochondria.. Protein content and oxidative damage were evaluated by western blotting.and oxyblotting, respectively. The activity of respiratory chain complexes was determined by standard diagnostic procedures. Results: AGC1 transport rates were significantly higher in tissue homogenates from autistic patients, including those with no history of seizures and with normal EEGs prior to death. This increase was consistently blunted by the Ca2+ chelator EGTA; no difference in AGC1 transport rates was found in isolated mitochondria from patients and controls; control mitochondria showed increased AGC1 activity when exposed to the post-mitochondrial supernatant of his/her matched patient than to his/her own supernatant. Complex IV and complex V activities were both increased in autistic patients but no significant difference in their protein abundance as well as in AGC1 content was detected by western blotting; oxidized mitochondrial proteins were markedly increased in the majority but not all of the patients tested. Interestingly, oxidative damage correlated with the reduction of complex I activity. Conclusions: Excessive Ca2+ levels boosts AGC activity in neurons and, to a more variable degree, oxidative stress and OXPHOS dysfunction in autistic brains. The modulation of AGC1 and/or Ca2+ homeostasis could provide new preventive and therapeutic strategies
Identification of a novel mitochondrial carrier involved in a citrate-oxoglutarate NADPH redox shuttle in Saccharomyces cerevisiae
No full textCitrate trafficking supports rewiring of mitochondrial metabolism via RTG signaling in yeast osmoadaptation
No full textInter-organellar cross-talk is an important component of cellular stress response enabling adaptation and survival. We have demonstrated the activation of RTG retrograde signaling to sustain the peroxisomesmitochondria- nucleus axis in a model of osmostressed Saccharomyces cerevisiae yeast cells. In this work, we aimed to gain insight into the molecular mechanisms regulating the communication between these organelles upon NaCl treatment. A metabolomic analysis revealed that the homeostasis of citrate is a pivotal factor in the osmoadaptive response. Gene expression analysis and citrate synthase activity showed that the synthesis of citrate mainly derives from peroxisomes, as indicated by the up-regulation of CIT2, and not CIT1 and CIT3, under the control of the RTG pathway. Furthermore, the involvement of the mitochondrial citrate transporter, encoded by YHM2, in the osmoadaptive response, as judged by gene and protein expression analysis together with growth assay, is demonstrated. In the absence of YHM2, alternative pathways relying on ODC2 and ACO1 are activated, indicating possible compensatory mechanisms for osmoadaptation. We propose a model in which peroxisome-derived citrate is converted to cytosolic 2-oxoglutarate to replenish TCA cycle and promote its rewiring. This work reveals a new layer of metabolic co-ordination among organelles and identifies citrate shuttling as a crucial adaptive mechanism to osmotic stress
- …
