The mitochondrial ADP/ATP carrier : functional studies of the prolines in transmembrane helices 1, 3 and 5 and of the conformations associated with the nucleotide transport

Le transporteur mitochondrial de nucléotides adényliques (Ancp), localisé dans la membrane interne mitochondriale, catalyse l'échange ADP/ATP entre le cytoplasme et la matrice mitochondriale. Il lie deux classes d'inhibiteurs naturels avec une grande spécificité et une haute affinité. Ces deux types d'inhibiteurs, BA et CATR, stabilisent Ancp dans deux conformations distinctes impliquées dans le transport des nucléotides. La compréhension des changements conformationnels subits par Ancp est essentielle pour décrire précisément le mécanisme d'échange des nucléotides. La structure atomique du transporteur de bœuf a montré que les hélices transmembranaires 1, 3 et 5 sont coudées par la présence de prolines qui pourraient donc être impliquées dans les changements conformationnels associés au transport.Dans la première partie de ce manuscrit, ces prolines ont été mutées en alanine ou en leucine et les conséquences de ces mutations ont été étudiées au niveau de la cellule (phénotype, morphologie, contenu en protéines) et des mitochondries en examinant le transport lui-même ainsi que toutes les fonctions mitochondriales (respiration, contenu en protéines, importation des protéines, morphologie…). Il peut-être conclu de ces études que ces prolines jouent un rôle dans le transport mais également dans la biogenèse mitochondriale (import d'Ancp, équilibre fusion/fission mitochondriale).Dans la deuxième partie, ont été étudiées des mutations qui stabilisent Ancp dans la conformation BA ou CATR. L'objectif était d'obtenir des formes stables du transporteur représentant des états intermédiaires du transport pour en étudier la structure atomique par cristallographie. Les résultats préliminaires sont prometteurs.The mitochondrial ADP/ATP carrier (Ancp), located in the inner mitochondrial membrane, catalyzes the ADP/ATP exchange between the cytoplasm and the mitochondrial membrane. Two classes of natural inhibitors can bind to the carrier with high specificity and affinity. These two families of inhibitors, BA and CATR, stabilize Ancp in two different conformations, which are involved in the nucleotide transport. Understanding the conformational changes undergone by Ancp is essential to describe precisely the nucleotide exchange mechanism. The atomic structure of the Beef Ancp unveiled kinks in transmembrane helices 1, 3 and 5 induced by the prolines, which therefore could be involved in the conformational changes associated with the nucleotide transport. In the first part of this manuscript, the three prolines were mutated into alanine or leucine and the results of these mutations were studied at the level of the cell (phenotype, morphology, protein content) and of the mitochondria by examining the transport itself and various mitochondria functions (respiration, protein content, protein import, morphology...). It can be concluded from these studies that these prolines play a key role in the transport but also in mitochondria biogenesis (Ancp import, mitochondrial fusion/fission balance).In the second part were studied mutations that stabilize Ancp in BA or CATR conformation. The goal was to obtain stable forms of Ancp that would correspond to intermediate steps of the transport to study their atomic structure by crystallography. The preliminary results are promising

The mitochondrial ADP/ATP carrier : functional studies of the prolines in transmembrane helices 1, 3 and 5 and of the conformations associated with the nucleotide transport

Le transporteur mitochondrial de nucléotides adényliques (Ancp), localisé dans la membrane interne mitochondriale, catalyse l'échange ADP/ATP entre le cytoplasme et la matrice mitochondriale. Il lie deux classes d'inhibiteurs naturels avec une grande spécificité et une haute affinité. Ces deux types d'inhibiteurs, BA et CATR, stabilisent Ancp dans deux conformations distinctes impliquées dans le transport des nucléotides. La compréhension des changements conformationnels subits par Ancp est essentielle pour décrire précisément le mécanisme d'échange des nucléotides. La structure atomique du transporteur de bœuf a montré que les hélices transmembranaires 1, 3 et 5 sont coudées par la présence de prolines qui pourraient donc être impliquées dans les changements conformationnels associés au transport.Dans la première partie de ce manuscrit, ces prolines ont été mutées en alanine ou en leucine et les conséquences de ces mutations ont été étudiées au niveau de la cellule (phénotype, morphologie, contenu en protéines) et des mitochondries en examinant le transport lui-même ainsi que toutes les fonctions mitochondriales (respiration, contenu en protéines, importation des protéines, morphologie…). Il peut-être conclu de ces études que ces prolines jouent un rôle dans le transport mais également dans la biogenèse mitochondriale (import d'Ancp, équilibre fusion/fission mitochondriale).Dans la deuxième partie, ont été étudiées des mutations qui stabilisent Ancp dans la conformation BA ou CATR. L'objectif était d'obtenir des formes stables du transporteur représentant des états intermédiaires du transport pour en étudier la structure atomique par cristallographie. Les résultats préliminaires sont prometteurs.The mitochondrial ADP/ATP carrier (Ancp), located in the inner mitochondrial membrane, catalyzes the ADP/ATP exchange between the cytoplasm and the mitochondrial membrane. Two classes of natural inhibitors can bind to the carrier with high specificity and affinity. These two families of inhibitors, BA and CATR, stabilize Ancp in two different conformations, which are involved in the nucleotide transport. Understanding the conformational changes undergone by Ancp is essential to describe precisely the nucleotide exchange mechanism. The atomic structure of the Beef Ancp unveiled kinks in transmembrane helices 1, 3 and 5 induced by the prolines, which therefore could be involved in the conformational changes associated with the nucleotide transport. In the first part of this manuscript, the three prolines were mutated into alanine or leucine and the results of these mutations were studied at the level of the cell (phenotype, morphology, protein content) and of the mitochondria by examining the transport itself and various mitochondria functions (respiration, protein content, protein import, morphology...). It can be concluded from these studies that these prolines play a key role in the transport but also in mitochondria biogenesis (Ancp import, mitochondrial fusion/fission balance).In the second part were studied mutations that stabilize Ancp in BA or CATR conformation. The goal was to obtain stable forms of Ancp that would correspond to intermediate steps of the transport to study their atomic structure by crystallography. The preliminary results are promising

Molecular mechanism and physiological role of active-deactive transition of mitochondrial complex I

Abstract The unique feature of mitochondrial complex I is the so-called A/D transition (active-deactive transition). The A-form catalyses rapid oxidation of NADH by ubiquinone (k ∼10 4 min − 1 ) and spontaneously converts into the D-form if the enzyme is idle at physiological temperatures. Such deactivation occurs in vitro in the absence of substrates or in vivo during ischaemia, when the ubiquinone pool is reduced. The D-form can undergo reactivation given both NADH and ubiquinone availability during slow (k ∼1-10 min − 1 ) catalytic turnover(s). We examined known conformational differences between the two forms and suggested a mechanism exerting A/D transition of the enzyme. In addition, we discuss the physiological role of maintaining the enzyme in the D-form during the ischaemic period. Accumulation of the D-form of the enzyme would prevent reverse electron transfer from ubiquinol to FMN which could lead to superoxide anion generation. Deactivation would also decrease the initial burst of respiration after oxygen reintroduction. Therefore the A/D transition could be an intrinsic protective mechanism for lessening oxidative damage during the early phase of reoxygenation. Exposure of Cys 39 of mitochondrially encoded subunit ND3 makes the Dform susceptible for modification by reactive oxygen species and nitric oxide metabolites which arrests the reactivation of the D-form and inhibits the enzyme. The nature of thiol modification defines deactivation reversibility, the reactivation timescale, the status of mitochondrial bioenergetics and therefore the degree of recovery of the ischaemic tissues after reoxygenation

Characterisation of the active/de-active transition of mitochondrial complex I

AbstractOxidation of NADH in the mitochondrial matrix of aerobic cells is catalysed by mitochondrial complex I. The regulation of this mitochondrial enzyme is not completely understood. An interesting characteristic of complex I from some organisms is the ability to adopt two distinct states: the so-called catalytically active (A) and the de-active, dormant state (D). The A-form in situ can undergo de-activation when the activity of the respiratory chain is limited (i.e. in the absence of oxygen).The mechanisms and driving force behind the A/D transition of the enzyme are currently unknown, but several subunits are most likely involved in the conformational rearrangements: the accessory subunit 39kDa (NDUFA9) and the mitochondrially encoded subunits, ND3 and ND1. These three subunits are located in the region of the quinone binding site.The A/D transition could represent an intrinsic mechanism which provides a fast response of the mitochondrial respiratory chain to oxygen deprivation. The physiological role of the accumulation of the D-form in anoxia is most probably to protect mitochondria from ROS generation due to the rapid burst of respiration following reoxygenation. The de-activation rate varies in different tissues and can be modulated by the temperature, the presence of free fatty acids and divalent cations, the NAD+/NADH ratio in the matrix, the presence of nitric oxide and oxygen availability.Cysteine-39 of the ND3 subunit, exposed in the D-form, is susceptible to covalent modification by nitrosothiols, ROS and RNS. The D-form in situ could react with natural effectors in mitochondria or with pharmacological agents. Therefore the modulation of the re-activation rate of complex I could be a way to ameliorate the ischaemia/reperfusion damage. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference. Guest Editors: Manuela Pereira and Miguel Teixeira

Le transporteur ADP/ATP mitochondrial (études fonctionnelles des prolines des hélices transmembranaires 1, 3 et 5 et étude des conformations associées au transport de nucléotides)

Le transporteur mitochondrial de nucléotides adényliques (Ancp), localisé dans la membrane interne mitochondriale, catalyse l'échange ADP/ATP entre le cytoplasme et la matrice mitochondriale. Il lie deux classes d'inhibiteurs naturels avec une grande spécificité et une haute affinité. Ces deux types d'inhibiteurs, BA et CATR, stabilisent Ancp dans deux conformations distinctes impliquées dans le transport des nucléotides. La compréhension des changements conformationnels subits par Ancp est essentielle pour décrire précisément le mécanisme d'échange des nucléotides. La structure atomique du transporteur de bœuf a montré que les hélices transmembranaires 1, 3 et 5 sont coudées par la présence de prolines qui pourraient donc être impliquées dans les changements conformationnels associés au transport.Dans la première partie de ce manuscrit, ces prolines ont été mutées en alanine ou en leucine et les conséquences de ces mutations ont été étudiées au niveau de la cellule (phénotype, morphologie, contenu en protéines) et des mitochondries en examinant le transport lui-même ainsi que toutes les fonctions mitochondriales (respiration, contenu en protéines, importation des protéines, morphologie ). Il peut-être conclu de ces études que ces prolines jouent un rôle dans le transport mais également dans la biogenèse mitochondriale (import d'Ancp, équilibre fusion/fission mitochondriale).Dans la deuxième partie, ont été étudiées des mutations qui stabilisent Ancp dans la conformation BA ou CATR. L'objectif était d'obtenir des formes stables du transporteur représentant des états intermédiaires du transport pour en étudier la structure atomique par cristallographie. Les résultats préliminaires sont prometteurs.The mitochondrial ADP/ATP carrier (Ancp), located in the inner mitochondrial membrane, catalyzes the ADP/ATP exchange between the cytoplasm and the mitochondrial membrane. Two classes of natural inhibitors can bind to the carrier with high specificity and affinity. These two families of inhibitors, BA and CATR, stabilize Ancp in two different conformations, which are involved in the nucleotide transport. Understanding the conformational changes undergone by Ancp is essential to describe precisely the nucleotide exchange mechanism. The atomic structure of the Beef Ancp unveiled kinks in transmembrane helices 1, 3 and 5 induced by the prolines, which therefore could be involved in the conformational changes associated with the nucleotide transport. In the first part of this manuscript, the three prolines were mutated into alanine or leucine and the results of these mutations were studied at the level of the cell (phenotype, morphology, protein content) and of the mitochondria by examining the transport itself and various mitochondria functions (respiration, protein content, protein import, morphology...). It can be concluded from these studies that these prolines play a key role in the transport but also in mitochondria biogenesis (Ancp import, mitochondrial fusion/fission balance).In the second part were studied mutations that stabilize Ancp in BA or CATR conformation. The goal was to obtain stable forms of Ancp that would correspond to intermediate steps of the transport to study their atomic structure by crystallography. The preliminary results are promising.BORDEAUX2-Bib. électronique (335229905) / SudocSudocFranceF

The mitochondrial ADP/ATP carrier (SLC25 family): pathological implications of its dysfunction.

In aerobic eukaryotic cells, the high energy metabolite ATP is generated mainly within the mitochondria following the process of oxidative phosphorylation. The mitochondrial ATP is exported to the cytoplasm using a specialized transport protein, the ADP/ATP carrier, to provide energy to the cell. Any deficiency or dysfunction of this membrane protein leads to serious consequences on cell metabolism and can cause various diseases such as muscular dystrophy. Described as a decisive player in the programmed cell death, it was recently shown to play a role in cancer. The objective of this review is to summarize the current knowledge of the involvement of the ADP/ATP carrier, encoded by the SLC25A4, SLC25A5, SLC25A6 and SLC25A31 genes, in human diseases and of the efforts made at designing different model systems to study this carrier and the associated pathologies through biochemical, genetic, and structural approaches

Cadmium and Copper Cross-Tolerance. Cu+^+ Alleviates Cd2+^{2+} Toxicity, and Both Cations Target Heme and Chlorophyll Biosynthesis Pathway in Rubrivivax gelatinosusRubrivivax\ gelatinosus

International audienceCadmium, although not redox active is highly toxic. Yet, the underlying mechanisms driving toxicity are still to be characterized. In this study, we took advantage of the purple bacterium Rubrivivax gelatinosus strain with defective Cd2+-efflux system to identify targets of this metal. Exposure of the 1cadA strain to Cd2+ causes a decrease in the photosystem amount and in the activity of respiratory complexes. As in case of Cu+ toxicity, the data indicated that Cd2+ targets the porphyrin biosynthesis pathway at the level of HemN, a S-adenosylmethionine and CxxxCxxC coordinated [4Fe-4S] containing enzyme. Cd2+ exposure therefore results in a deficiency in heme and chlorophyll dependent proteins and metabolic pathways. Given the importance of porphyrin biosynthesis, HemN represents a key metal target to account for toxicity. In the environment, microorganisms are exposed to mixture of metals. Nevertheless, the biological effects of such mixtures, and the toxicity mechanisms remain poorly addressed. To highlight a potential cross-talk between Cd2+ and Cu+ -efflux systems, we show (i) that Cd2+ induces the expression of the Cd2+-efflux pump CadA and the Cu+ detoxification system CopA and CopI; and (ii) that Cu+ ions improve tolerance towards Cd2+, demonstrating thus that metal mixtures could also represent a selective advantage in the environment