Mitochondrial Adenine Nucleotide Research Articles

Mitochondrial Proteins and Molecular Interaction with Cardiolipin at Atomistic Level

Functional coupling between mitochondrial creatine kinase (MtCK) and adenine nucleotide translocase (ANT) can play an important role in determining energy transfer pathways in the cell. MtCK, which is an octameric enzyme, catalyzes the conversion of creatine (Cr) and consumes adenosine triphosphate (ATP) to create phosphocreatine (PCr) and adenosine diphosphate (ADP). Mitochondrial CK is located partly in contact sites of outer and inner mitochondrial membranes and partly in the cristae region, but always firmly attached to the inner membrane and in the vicinity of transmembrane ANTs. It is shown that MtCK binds electrostatically to the negatively charged cardiolipins (CL) of the inner membrane. CL is a kind of diphosphatidylglycerol lipid and is found almost exclusively in the inner mitochondrial membrane where it is essential for keeping spatially close and functional CK, ANT and other respiratory complexes. The formation of contact sites of CK-ANT by CL patches is a dynamic process and important to understand at atomistic level, which is the main goal of this work.The molecular dynamics simulation technique is used to study the atomistic details of the functional role of CL close to mitochondrial CK and ANT complexes. The thermodynamics-based coarse-grained force-field, called MARTINI (version 2.0), has been used together with the GROMACS molecular dynamics package (version 4.5.4) for a model of a patch of the mitochondrial inner membrane containing several transmembrane ANTs, and a single CK above the membrane. The membrane model consists of three major type of lipids ­ phosphatidylcholine, phosphatidylethanolamine and cardiolipin in roughly 2:1:1 molar ratio.

Characterization of the Intracellular Distribution of Adenine Nucleotide Translocase (ANT) in Drosophila Indirect Flight Muscles

Background: The high power output necessary for insect flight has driven the evolution of muscles with large myofibrils (primary energy consumers) and abundant mitochondria (primary energy suppliers). The intricate functional interrelationship between these two organelles remains largely unknown despite its fundamental importance in understanding insect flight bioenergetics. Unlike vertebrate muscle that relies on a phosphagen (creatine phosphate/creatine kinase) system to regulate high energy phosphate flux, insect flight muscle has been reported to lack mitochondrial arginine kinase (analogous to creatine kinase), a key enzyme that enables intracellular energy transport. Creatine kinase is known to interact with mitochondrial adenine nucleotide translocase (ANT) in the transfer of ADP and ATP into and out of the mitochondria. Results: Here, we use quantitative immunogold transmission electron microscopy to show that in Drosophila melanogaster indirect flight muscles (IFM), ANT is present in the mitochondria as well as throughout the myofibril. To confirm this unexpected result, we created a transgenic line that expresses a chimeric GFP-ANT protein and used an anti-GFP antibody to determine the intracellular distribution of the fusion protein in the IFM. Similar to results obtained with anti-ANT, the fusion GFP-ANT protein is detected in myofibrils and mitochondria. We confirmed the absence of arginine kinase from IFM mitochondria and show that its sarcomeric (i.e., intramyofibrillar) distribution is similar to that of ANT. Conclusions: These results raise the possibility that direct channeling of nucleotides between mitochondria and myofibrils is assisted by an ANT protein thereby circumventing the need for a phosphagen shuttle in the IFM. The myofibrillar ANT may represent a unique adaptation in the muscles that require efficient exchange of nucleotides between mitochondria and myofibrils.

B-cell translocation gene 2: Expression in the rat ovary and potential association with adenine nucleotide translocase 2 in mitochondria

The B-cell translocation gene 2 (Btg2) is an anti-proliferative tumor suppressor gene that behaves as a transcriptional regulator. The present study investigated gonadotropin induction of Btg2 in the rat ovary and the mechanism of Btg2 action as a partner of mitochondrial protein adenine nucleotide translocase 2 (Ant2). Transient induction of Btg2 as well as Btg1 mRNA levels by LH/hCG was observed in ovarian granulosa cells. Btg2 protein levels were also stimulated by LH/hCG. LH-induced gene expression of Btg2 required ERK signal pathway. Studies of deletion mutants in HeLa cells showed that deletion of Btg2 C-terminus (Btg2/ΔC) abolished the interaction with Ant2. In fact, the expression levels of Btg2/ΔC construct were decreased in mitochondrial fraction. Btg2 was also expressed in mitochondria and interacted with Ant2 in preovulatory granulosa cells. Interestingly, a Btg2/ΔC construct inhibited an action of Btg2 wild-type on ATP and H2O2 production. These findings demonstrate the gonadotropin stimulation of Btg2 in the ovary and, the physical interaction of Btg2 with Ant2 in mitochondria.

Formation of disulfide-linked high molecular protein complex by glutathione transferase in the mitochondrial inner membrane and adenine nucleotide translocator through peroxynitrite

Formation of disulfide-linked high molecular protein complex by glutathione transferase in the mitochondrial inner membrane and adenine nucleotide translocator through peroxynitrite

A Biophysical Model of the Mitochondrial ATP-Mg/Pi Carrier

Mitochondrial adenine nucleotide (AdN) content is regulated through the Ca2+-activated, electroneutral ATP-Mg/Pi carrier (APC). The APC is a protein in the mitochondrial carrier super family that localizes to the inner mitochondrial membrane (IMM). It is known to modulate a number of processes that depend on mitochondrial AdN content, such as gluconeogenesis, protein synthesis, and citrulline synthesis. Despite this critical role, a kinetic model of the underlying mechanism has not been developed and validated. Here, a biophysical model of the APC is developed that is thermodynamically balanced and accurately reproduces a number of reported data sets from isolated rat liver and rat kidney mitochondria. The model is based on an ordered bi-bi mechanism for heteroexchange of ATP and Pi and includes homoexchanges of ATP and Pi to explain both the initial rate and time course data on ATP and Pi transport via the APC. The model invokes seven kinetic parameters regarding the APC mechanism and three parameters related to matrix pH regulation by external Pi. These parameters are estimated based on 19 independent data curves; the estimated parameters are validated using six additional data curves. The model takes into account the effects of pH, Mg2+, and Ca2+ on ATP and Pi transport via the APC, and supports the conclusion that the pH gradient across the IMM serves as the primary driving force for AdN uptake or efflux. Moreover, computer simulations demonstrate that extramatrix Ca2+ modulates the turnover rate of the APC and not the binding affinity of ATP, as previously suggested.

Rotaviral Enterotoxin Nonstructural Protein 4 Targets Mitochondria for Activation of Apoptosis during Infection

Viruses have evolved to encode multifunctional proteins to control the intricate cellular signaling pathways by using very few viral proteins. Rotavirus is known to express six nonstructural and six structural proteins. Among them, NSP4 is the enterotoxin, known to disrupt cellular Ca(2+) homeostasis by translocating to endoplasmic reticulum. In this study, we have observed translocation of NSP4 to mitochondria resulting in dissipation of mitochondrial membrane potential during virus infection and NSP4 overexpression. Furthermore, transfection of the N- and C-terminal truncated NSP4 mutants followed by analyzing NSP4 localization by immunofluorescence microscopy identified the 61-83-amino acid region as the shortest mitochondrial targeting signal. NSP4 exerts its proapoptotic effect by interacting with mitochondrial proteins adenine nucleotide translocator and voltage-dependent anion channel, resulting in dissipation of mitochondrial potential, release of cytochrome c from mitochondria, and caspase activation. During early infection, apoptosis activation by NSP4 was inhibited by the activation of cellular survival pathways (PI3K/AKT), because PI3K inhibitor results in early induction of apoptosis. However, in the presence of both PI3K inhibitor and NSP4 siRNA, apoptosis was delayed suggesting that the early apoptotic signal is initiated by NSP4 expression. This proapoptotic function of NSP4 is balanced by another virus-encoded protein, NSP1, which is implicated in PI3K/AKT activation because overexpression of both NSP4 and NSP1 in cells resulted in reduced apoptosis compared with only NSP4-expressing cells. Overall, this study reports on the mechanism by which enterotoxin NSP4 exerts cytotoxicity and the mechanism by which virus counteracts it at the early stage for efficient infection.

Absence of Ca2+-induced mitochondrial permeability transition but presence of bongkrekate-sensitive nucleotide exchange in C. crangon and P. serratus

Mitochondria from the embryos of brine shrimp (Artemia franciscana) do not undergo Ca2+-induced permeability transition in the presence of a profound Ca2+ uptake capacity. Furthermore, this crustacean is the only organism known to exhibit bongkrekate-insensitive mitochondrial adenine nucleotide exchange, prompting the conjecture that refractoriness to bongkrekate and absence of Ca2+-induced permeability transition are somehow related phenomena. Here we report that mitochondria isolated from two other crustaceans, brown shrimp (Crangon crangon) and common prawn (Palaemon serratus) exhibited bongkrekate-sensitive mitochondrial adenine nucleotide transport, but lacked a Ca2+-induced permeability transition. Ca2+ uptake capacity was robust in the absence of adenine nucleotides in both crustaceans, unaffected by either bongkrekate or cyclosporin A. Transmission electron microscopy images of Ca2+-loaded mitochondria showed needle-like formations of electron-dense material strikingly similar to those observed in mitochondria from the hepatopancreas of blue crab (Callinectes sapidus) and the embryos of Artemia franciscana. Alignment analysis of the partial coding sequences of the adenine nucleotide translocase (ANT) expressed in Crangon crangon and Palaemon serratus versus the complete sequence expressed in Artemia franciscana reappraised the possibility of the 208-214 amino acid region for conferring sensitivity to bongkrekate. However, our findings suggest that the ability to undergo Ca2+-induced mitochondrial permeability transition and the sensitivity of adenine nucleotide translocase to bongkrekate are not necessarily related phenomena.

The effect of glycerol on mRNA expression of growth hormone, insulin-like growth factor, and mitochondrial breast muscle genes of Japanese quail

1. A study was carried out to evaluate the expression of growth hormone (GH), insulin-like growth factor I (IGF-I), mitochondrial adenine nucleotide translocase (ANT), cytochrome oxidase III (COX III) and avian uncoupling protein (avUCP) genes in 14-d-old Japanese quail that were fed different levels of glycerol (0%, 4%, and 12% dietary glycerol) which replaced maize as an energy source. 2. Total RNA was extracted from the breast muscle, and cDNA was amplified using real-time PCR with primers specific to the examined genes. 3. Quail fed the diet with 12% glycerol supplementation presented higher growth hormone (GH) mRNA expression than did those fed 0% glycerol. Supplementation with 12% glycerol negatively influenced IGF-I mRNA expression and reduced ANT mRNA expression in comparison with the treatment with no glycerol. COX III mRNA expression in the pectoralis superficialis muscle was decreased by 26% in quail fed 12% glycerol compared with those fed 0 and 4% glycerol. 4. There was no difference in UCP mRNA expression between quail fed 0 and 4% glycerol; however, UCP expression was reduced (73%) in birds fed the 12% glycerol diet compared with the level in those fed the 4% glycerol diet. 5. The inclusion of 4% glycerol in the diet produced results similar to those of the diet with no glycerol. Based on quail performance and the expression of the GH, IGF-I, ANT, COX III and UCP genes, 4% glycerol can be used in quail feeding without any harmful effects.

Application of Flow Cytometry to Determine Differential Redistribution of Cytochrome c and Smac/DIABLO from Mitochondria during Cell Death Signaling

Mitochondrially mediated apoptosis is characterized by redistribution of proteins from mitochondria to cytoplasm following permeabilization of the outer mitochondrial membrane. We applied flow cytometry to quantify simultaneously the redistribution of two apoptogenic proteins, cytochrome c (cyt c) and Smac/DIABLO (Smac). Mammalian cells were treated with digitonin that selectively permeabilizes the plasma membrane. Following fixation, treated cells were infused successively with primary and secondary antibodies (the latter fluorescently tagged) enabling independent detection of cyt c and Smac. Digitonin-treated cells that retain cyt c or Smac in mitochondria generate strong fluorescence signals in flow cytometry. Cells in which cyt c or Smac have transited the outer mitochondrial membrane show greatly reduced fluorescence because the proteins are lost from the digitonin-permeabilized cells. Quantitative flow cytometry revealed that in 143B TK- cells treated with staurosporine, cyt c and Smac exit mitochondria asymmetrically, with cyt c redistribution preceding that of Smac. However, in HeLa cells likewise treated, cyt c and Smac exit mitochondria concurrently. Under other conditions of apoptotic induction, for example, 143B TK- cells treated with MT-21 (an apoptotic inducer that binds to the mitochondrial adenine nucleotide transporter), redistribution of Smac precedes that of cyt c. The various patterns of redistribution of these proteins were confirmed by immunocytochemical analysis and confocal microscopy. We conclude that flow cytometry can be employed effectively to quantify simultaneously the redistribution of cyt c and Smac from mitochondria to the cytosol. Moreover, differential redistribution of cyt c and Smac occurs under various conditions, thereby reflecting constraints on availability of these proteins to exit mitochondria after permeabilization of the outer membrane.

Absence of Ca2+-Induced Mitochondrial Permeability Transition but Presence of Bongkrekate-Sensitive Nucleotide Exchange in C. crangon and P. serratus

Mitochondria from the embryos of brine shrimp (Artemia franciscana) do not undergo Ca2+-induced permeability transition in the presence of a profound Ca2+ uptake capacity. Furthermore, this crustacean is the only organism known to exhibit bongkrekate-insensitive mitochondrial adenine nucleotide exchange, prompting the conjecture that refractoriness to bongkrekate and absence of Ca2+-induced permeability transition are somehow related phenomena. Here we report that mitochondria isolated from two other crustaceans, brown shrimp (Crangon crangon) and common prawn (Palaemon serratus) exhibited bongkrekate-sensitive mitochondrial adenine nucleotide transport, but lacked a Ca2+-induced permeability transition. Ca2+ uptake capacity was robust in the absence of adenine nucleotides in both crustaceans, unaffected by either bongkrekate or cyclosporin A. Transmission electron microscopy images of Ca2+-loaded mitochondria showed needle-like formations of electron-dense material strikingly similar to those observed in mitochondria from the hepatopancreas of blue crab (Callinectes sapidus) and the embryos of Artemia franciscana. Alignment analysis of the partial coding sequences of the adenine nucleotide translocase (ANT) expressed in Crangon crangon and Palaemon serratus versus the complete sequence expressed in Artemia franciscana reappraised the possibility of the 208-214 amino acid region for conferring sensitivity to bongkrekate. However, our findings suggest that the ability to undergo Ca2+-induced mitochondrial permeability transition and the sensitivity of adenine nucleotide translocase to bongkrekate are not necessarily related phenomena.

The tumour metabolism inhibitors GSAO and PENAO react with cysteines 57 and 257 of mitochondrial adenine nucleotide translocase

BackgroundGSAO (4-(N-(S-glutathionylacetyl)amino) phenylarsonous acid) and PENAO (4-(N-(S-penicillaminylacetyl)amino) phenylarsonous acid) are tumour metabolism inhibitors that target adenine nucleotide translocase (ANT) of the inner-mitochondrial membrane. Both compounds are currently being trialled in patients with solid tumours. The trivalent arsenical moiety of GSAO and PENAO reacts with two matrix facing cysteine residues of ANT, inactivating the transporter. This leads to proliferation arrest and death of tumour and tumour-supporting cells.ResultsThe two reactive ANT cysteine residues have been identified in this study by expressing cysteine mutants of human ANT1 in Saccharomyces cerevisiae and measuring interaction with the arsenical moiety of GSAO and PENAO. The arsenic atom of both compounds cross-links cysteine residues 57 and 257 of human ANT1.ConclusionsThe sulphur atoms of these two cysteines are 20 Å apart in the crystal structures of ANT and the optimal spacing of cysteine thiolates for reaction with As (III) is 3-4 Å. This implies that a significant conformational change in ANT is required for the organoarsenicals to react with cysteines 57 and 257. This conformational change may relate to the selectivity of the compounds for proliferating cells.

Molecular Dynamics Simulations of Creatine Kinase and Adenine Nucleotide Translocase in Mitochondrial Membrane Patch

Interaction between mitochondrial creatine kinase (MtCK) and adenine nucleotide translocase (ANT) can play an important role in determining energy transfer pathways in the cell. Although the functional coupling between MtCK and ANT has been demonstrated, the precise mechanism of the coupling is not clear. To study the details of the coupling, we turned to molecular dynamics simulations. We introduce a new coarse-grained molecular dynamics model of a patch of the mitochondrial inner membrane containing a transmembrane ANT and an MtCK above the membrane. The membrane model consists of three major types of lipids (phosphatidylcholine, phosphatidylethanolamine, and cardiolipin) in a roughly 2:1:1 molar ratio. A thermodynamics-based coarse-grained force field, termed MARTINI, has been used together with the GROMACS molecular dynamics package for all simulated systems in this work. Several physical properties of the system are reproduced by the model and are in agreement with known data. This includes membrane thickness, dimension of the proteins, and diffusion constants. We have studied the binding of MtCK to the membrane and demonstrated the effect of cardiolipin on the stabilization of the binding. In addition, our simulations predict which part of the MtCK protein sequence interacts with the membrane. Taken together, the model has been verified by dynamical and structural data and can be used as the basis for further studies.

The splicing mutant of the human tumor suppressor protein DFNA5 induces programmed cell death when expressed in the yeast Saccharomyces cerevisiae

DFNA5 was first identified as a gene responsible for autosomal dominant deafness. Different mutations were found, but they all resulted in exon 8 skipping during splicing and premature termination of the protein. Later, it became clear that the protein also has a tumor suppression function and that it can induce apoptosis. Epigenetic silencing of the DFNA5 gene is associated with different types of cancers, including gastric and colorectal cancers as well as breast tumors. We introduced the wild-type and mutant DFNA5 allele in the yeast Saccharomyces cerevisiae. The expression of the wild-type protein was well tolerated by the yeast cells, although the protein was subject of degradation and often deposited in distinct foci when cells entered the diauxic shift. In contrast, cells had problems to cope with mutant DFNA5 and despite an apparent compensatory reduction in expression levels, the mutant protein still triggered a marked growth defect, which in part can be ascribed to its interaction with mitochondria. Consistently, cells with mutant DFNA5 displayed significantly increased levels of ROS and signs of programmed cell death. The latter occurred independently of the yeast caspase, Mca1, but involved the mitochondrial fission protein, Fis1, the voltage-dependent anion channel protein, Por1 and the mitochondrial adenine nucleotide translocators, Aac1 and Aac3. Recent data proposed DFNA5 toxicity to be associated to a globular domain encoded by exon 2–6. We confirmed these data by showing that expression of solely this domain confers a strong growth phenotype. In addition, we identified a point mutant in this domain that completely abrogated its cytotoxicity in yeast as well as human Human Embryonic Kidney 293T cells (HEK293T). Combined, our data underscore that the yeast system offers a valuable tool to further dissect the apoptotic properties of DFNA5.

The hepatoprotective effects of adenine nucleotide translocator-2 against aging and oxidative stress

Mitochondrial adenine nucleotide translocator (ANT) plays important roles in the regulation of mitochondrial permeability transition and cell bioenergetics. The mouse has three ANT isoforms (1, 2 and 4) showing tissue-specific expression patterns. Although ANT1 is known to have a pro-apoptotic property, the specific functions of ANT2 have not been well determined. In the present study, ANT2 expression was significantly lower in the aged rat liver and in a liver fibrosis model. To explore the protective role of ANT2 in the liver, we established a hepa1c1c7 cell line overexpressing ANT2. Overexpression of ANT2 caused hepa1c1c7 cells to be more resistant to oxidative stress, and mitochondrial membrane potential (MMP, ∆Ψm) was relatively intact in ANT2-overexpressing cells under oxidative stress. In addition, ANT2 was found to increase ATP production by influencing mitochondrial bioenergetics. These results imply that the hepatoprotective effect of ANT2 is due to the stabilization of MMP and enhanced ATP production, and thus, maintaining ANT2 levels in the liver might be important to enhance resistance to aging and oxidative stress.

Interaction between NH2-tau fragment and Aβ in Alzheimer's disease mitochondria contributes to the synaptic deterioration

Although amyloid beta (Aβ) peptide can promote tau pathology and its toxicity is concurrently tau-dependent, the underlying mechanisms of the in vivo interplay of these proteins remain unsolved. Structural and functional mitochondrial alterations play an early, precipitating role in synaptic failure of Alzheimer's disease (AD) pathogenesis and an aggravated mitochondrial impairment has been described in triple APP/PS/tau transgenic mice carrying both plaques and tangles, if compared with mice overexpressing tau or amyloid precursor protein (APP) alone. Here, we show that a neurotoxic aminoterminal (NH2)-derived tau fragment mapping between 26 and 230 amino acids of the human tau40 isoform (441 amino acids)—but not the physiological full-length protein—preferentially interacts with Aβ peptide(s) in human AD synapses in association with mitochondrial adenine nucleotide translocator-1 (ANT-1) and cyclophilin D. The two peptides—Aβ 1–42 and the smaller and more potent NH2-26-44 peptide of the longest 20–22 kDa NH2-tau fragment—inhibit the ANT–1-dependent adenosine diphosphate-adenosine triphosphate (ADP/ATP) exchange in a noncompetitive and competitive manner, respectively, and together further aggravate the mitochondrial dysfunction by exacerbating the ANT-1 impairment. Taken together, these data establish a common, direct and synergistic toxicity of pathological APP and tau products on synaptic mitochondria and suggest potential, new pathway(s) and target(s) for a combined, more efficient therapeutic intervention of early synaptic dysfunction in AD.

Evolutionary Genomics Implies a Specific Function of Ant4 in Mammalian and Anole Lizard Male Germ Cells

Most vertebrates have three paralogous genes with identical intron-exon structures and a high degree of sequence identity that encode mitochondrial adenine nucleotide translocase (Ant) proteins, Ant1 (Slc25a4), Ant2 (Slc25a5) and Ant3 (Slc25a6). Recently, we and others identified a fourth mammalian Ant paralog, Ant4 (Slc25a31), with a distinct intron-exon structure and a lower degree of sequence identity. Ant4 was expressed selectively in testis and sperm in adult mammals and was indeed essential for mouse spermatogenesis, but it was absent in birds, fish and frogs. Since Ant2 is X-linked in mammalian genomes, we hypothesized that the autosomal Ant4 gene may compensate for the loss of Ant2 gene expression during male meiosis in mammals. Here we report that the Ant4 ortholog is conserved in green anole lizard (Anolis carolinensis) and demonstrate that it is expressed in the anole testis. Further, a degenerate DNA fragment of putative Ant4 gene was identified in syntenic regions of avian genomes, indicating that Ant4 was present in the common amniote ancestor. Phylogenetic analyses suggest an even more ancient origin of the Ant4 gene. Although anole lizards are presumed male (XY) heterogametic, like mammals, copy numbers of the Ant2 as well as its neighboring gene were similar between male and female anole genomes, indicating that the anole Ant2 gene is either autosomal or located in the pseudoautosomal region of the sex chromosomes, in contrast to the case to mammals. These results imply the conservation of Ant4 is not likely simply driven by the sex chromosomal localization of the Ant2 gene and its subsequent inactivation during male meiosis. Taken together with the fact that Ant4 protein has a uniquely conserved structure when compared to other somatic Ant1, 2 and 3, there may be a specific advantage for mammals and lizards to express Ant4 in their male germ cells.

Computational analysis of the transcriptional regulation of the adenine nucleotide translocator isoform 4 gene and its role in spermatozoid glycolytic metabolism

Computational phylogenetic analysis coupled to promoter sequence alignment was used to understand mechanisms of transcriptional regulation and to identify potentially coregulated genes. Our strategy was validated on the human ANT4 gene which encodes the fourth isoform of the mitochondrial adenine nucleotide translocator specifically expressed during spermatogenesis. The movement of sperm flagella is driven mainly by ATP generated by glycolytic pathways, and the specific induction of the mitochondrial ANT4 protein presented an interesting puzzle. We analysed the sequences of the promoters, introns and exons of 30 mammalian ANT4 genes and constructed regulatory models. The whole human genome and promoter database were screened for genes that were potentially regulated by the generated models. 80% of the identified co-regulated genes encoded proteins with specific roles in spermatogenesis and with functions linked to male reproduction. Our in silico study enabled us to precise the specific role of the ANT4 isoform in spermatozoid bioenergetics.

Identification and characterization of interactions between abscisic acid and mitochondrial adenine nucleotide translocators

ABA (abscisic acid) is a plant hormone involved in important processes including development and stress responses. Recent reports have identified a number of plant ABA receptors and transporters, highlighting novel mechanisms of ABA action. In the present paper we describe application of a chemical proteomics approach leading to the identification of mitochondrial ANTs (adenine nucleotide translocators) as ABA-interacting proteins. Initial in vitro studies confirmed inhibition of ANT-dependent ATP translocation by ABA. Further analysis demonstrated ANT-dependent uptake of ABA into both recombinant Arabidopsis thaliana ANT2-containing proteoliposomes and native isolated spinach mitochondria; the latter with a Km of 3.5μM and a Vmax of 2.5 nmol/min per g of protein. ATP was found to inhibit ANT-dependent ABA translocation. Specificity profiles highlight the possibility of mechanistic differences in translocation of ABA and ATP. Finally, ABA was shown to stimulate ATPase activity in spinach mitochondrial extracts. ABA concentrations in plant cells are estimated to reach the low micromolar range during stress responses, supporting potential physiological relevance of these in vitro findings. Overall, the present in vitro work suggests the possibility of as yet uncharacterized mechanisms of ABA action in planta related to inhibition of mitochondrial ATP translocation and functional localization of ABA in the mitochondrial matrix.

Carnitine regulates myocardial metabolism by Peroxisome Proliferator-Activated Receptor-alpha (PPARalpha) in alcoholic cardiomyopathy

SummaryBackgroundChronic alcohol intake exerts myocardial damage en route to the development of alcoholic cardiomyopathy (ACM), although the precise pathogenesis of ACM is unknown. Carnitine is known to participate in the regulation of metabolism in a number of heart diseases. This study was designed to examine the interplay between myocardial metabolism and carnitine in the development of ACM.Material/MethodsExperimental animals were divided into 3 groups: (i) group A: alcohol-fed. (ii) group B: alcohol/carnitine: (200mg/kg/d, p.o. by mixing carnitine in rat chow). (iii) group C: control. Blood levels of free fatty acid (FFA), total carnitine (TC) and free carnitine (FC) were monitored in rats receiving alcohol with or without carnitine. Mitochondrial adenine nucleotide translocator-1 (ANT1) activity, ATPase activity, high energy phosphate concentration, peroxisome proliferator-activated receptor-α (PPARα), carnitine-palmitoyl transferase I (CPT-I), medium-chain acyl-coenzyme A dehydrogenase (MCAD), ANT1 and ATPase mRNA and protein expression were also monitored in myocardial tissue.ResultsExperimental animals received alcohol with or without carnitine for six6 months. Our results indicated that FFA increased abruptly. TC and FC were significantly decreased in groups receiving alcohol at 4 months. The concentration of ATP, ADP and AMP in the myocardium decreased following 2 months of alcohol administration. mRNA and protein expression of PPARα, CPT-I, MCAD, ANT1 and ATPase expressions were gradually altered in groups following alcohol feeding.ConclusionsThese observations suggest that abnormal metabolism is present in the myocardium during the development of ACM. Carnitine may improve myocardial metabolism by elevating the content of PPARα, CPT-I and MCAD.

Proteomic alterations of distinct mitochondrial subpopulations in the type 1 diabetic heart: contribution of protein import dysfunction

Diabetic cardiomyopathy is associated with increased risk of heart failure in type 1 diabetic patients. Mitochondrial dysfunction is suggested as an underlying contributor to diabetic cardiomyopathy. Cardiac mitochondria are characterized by subcellular spatial locale, including mitochondria located beneath the sarcolemma, subsarcolemmal mitochondria (SSM), and mitochondria situated between the myofibrils, interfibrillar mitochondria (IFM). The goal of this study was to determine whether type 1 diabetic insult in the heart influences proteomic make-up of spatially distinct mitochondrial subpopulations and to evaluate the role of nuclear encoded mitochondrial protein import. Utilizing multiple proteomic approaches (iTRAQ and two-dimensional-differential in-gel electrophoresis), IFM proteomic make-up was impacted by type 1 diabetes mellitus to a greater extent than SSM, as evidenced by decreased abundance of fatty acid oxidation and electron transport chain proteins. Mitochondrial phosphate carrier and adenine nucleotide translocator, as well as inner membrane translocases, were decreased in the diabetic IFM (P < 0.05 for both). Mitofilin, a protein involved in cristae morphology, was diminished in the diabetic IFM (P < 0.05). Posttranslational modifications, including oxidations and deamidations, were most prevalent in the diabetic IFM. Mitochondrial heat shock protein 70 (mtHsp70) was significantly decreased in diabetic IFM (P < 0.05). Mitochondrial protein import was decreased in the diabetic IFM with no change in the diabetic SSM (P < 0.05). Taken together, these results indicate that mitochondrial proteomic alterations in the type 1 diabetic heart are more pronounced in the IFM. Further, proteomic alterations are associated with nuclear encoded mitochondrial protein import dysfunction and loss of an essential mitochondrial protein import constituent, mtHsp70, implicating this process in the pathogenesis of the diabetic heart.