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International audience; ATAD3 (ATPase family AAA-Domain containing protein 3) is a mitochondrial inner membrane ATPase with unknown but vital functions. Initial researches have focused essentially on the major p66-ATAD3 isoform, but other proteins and mRNAs are described in the data banks. Using a set of anti-peptide antibodies and by the use of rodent and human cell lines and organs, we tried to detail ATAD3 gene expression profiles and to verify the existence of the various ATAD3 isoforms. In rodent, the single ATAD3 gene is expressed as a major isoform of 67 kDa, (ATAD3I; long), in all cells and organs studied. A second isoform, p57-ATAD3s (small), is expressed specifically throughout brain development and in adult, and overexpressed around the pen-natal period. p57-ATAD3s is also expressed in neuronal and glial rodent cell lines, and during in vitro differentiation of primary cultured rat oligodendrocytes. Other smaller isoforms were also detected in a tissue-specific manner. In human and primates, ATAD3 paralogues are encoded by three genes (ATAD3A, 3B and 3C), each of them presenting several putative variants. Analyzing the expression of ATAD3A and ATAD3B with four specific anti-peptide antibodies, and comparing their expressions with in vitro expressed ATAD3 cDNAs, we were able to observe and define five isoforms. In particular, the previously described p72-ATAD3B is confirmed to be in certain cases a phosphorylated form of ATAD3As. Moreover, we observed that the ATAD3As phosphorylation level is regulated by insulin and serum. Finally, exploring ATAD3 mRNA expression, we confirmed the existence of an alternative splicing in rodent and of several mRNA isoforms in human. Considering these observations, we propose the development of a uniform denomination for ATAD3 isoforms in rodent and human. Crown Copyright (C) 2013 Published by Elsevier B.V. All rights reserved.

The well-established function of the hexameric intermembrane space protein, NDPK-D/NM23-H4, is phosphotransfer activity as a nucleoside diphosphate kinase. However, recent data revealed a second function in lipid signaling that is involved in mitophagy, a critical process for cell homeostasis. Externalization of the inner mitochondrial membrane phospholipid, cardiolipin (CL), to the mitochondrial surface was identified as a mitophageal signal, recognized by the microtubule-associated protein 1 light chain 3. Here we demonstrate that NDPK-D binds CL and facilitates its re-distribution to the outer mitochondrial membrane. We found that mitophagy induced by a protonophoric uncoupler, CCCP, caused externalization of CL to the surface of mitochondria in murine lung epithelial MLE-12 cells and human cervical adenocarcinoma HeLa cells. RNAi knockdown of endogenous NDPK-D decreased CCCP-induced CL externalization and mitochondrial degradation. A R90D NDPK-D mutant which does not bind CL was inactive in promoting mitophagy. In situ proximity ligation assay showed that mitophagy-inducing CL transfer activity of NDPK-D is closely associated with the dynamin-like GTPase OPA1, and OPA1 silencing favored NDPK-D supported CL transfer, implicating fission-fusion dynamics in mitophagy regulation. Support: FRM DPM20121125557, ANR France, NIH PO1HL114453, U19AIO68021, NS076511, ES020693, NS065789, AG026389, NIOSH OH008282, HFSP, and Barth Syndrome Foundation of Canada.

International audience; AMP-activated protein kinase (AMPK) is emerging as a central cellular signaling hub involved in energy homeostasis and proliferation. The kinase is considered as a suitable target for pharmacological intervention in several energy-related pathologies like diabetes type II and cancer, although its signaling network is still incompletely understood. Here we apply an original two-dimensional in vitro screening approach for AMPK substrates that combines biophysical interaction based on surface plasmon resonance with in vitro phosphorylation. By enriching for proteins that interact with a specific AMPK isoform, we aimed to identify substrates that are also preferentially phosphorylated by this specific AMPK isoform. Application of this screen to full-length AMPK α2β2γ1 and soluble rat liver proteins identified the tumor suppressor fumarate hydratase (FH). FH was confirmed to interact with and to be preferentially phosphorylated by the AMPKα2 isoform by using yeast-two-hybrid and in vitro phosphorylation assays. AMPK-mediated phosphorylation of FH significantly increased enzyme activity in vitro and in vivo, suggesting that it is a bona fide AMPK substrate. In vivo, AMPKα2 is supposed to target the cytosolic/nuclear pools of FH, whose tumor suppressor function relies on DNA damage repair and inhibition of HIF-1α-signaling.

International audience; This review describes the recent experimental data on the importance of the VDAC-cytoskeleton interactions in determining the mechanisms of energy and metabolite transfer between mitochondria and cytoplasm in cardiac cells. In the intermembrane space mitochondrial creatine kinase connects VDAC with adenine nucleotide translocase and ATP synthase complex, on the cytoplasmic side VDAC is linked to cytoskeletal proteins. Applying immunofluorescent imaging and Western blot analysis we have shown that β2-tubulin coexpressed with mitochondria is highly important for cardiac muscle cells mitochondrial metabolism. Since it has been shown by Rostovtseva et al. that αβ-heterodimer of tubulin binds to VDAC and decreases its permeability, we suppose that the β-tubulin subunit is bound on the cytoplasmic side and α-tubulin C-terminal tail is inserted into VDAC. Other cytoskeletal proteins, such as plectin and desmin may be involved in this process. The result of VDAC-cytoskeletal interactions is selective restriction of the channel permeability for adenine nucleotides but not for creatine or phosphocreatine that favors energy transfer via the phosphocreatine pathway. In some types of cancer cells these interactions are altered favoring the hexokinase binding and thus explaining the Warburg effect of increased glycolytic lactate production in these cells. This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism.

The well-established function of the mitochondrial intermembrane space protein NM23-H4/NDPK-D is phosphotransfer activity as a nucleoside diphosphate kinase (NDPK). However, recent data have revealed a second function in lipid signaling that triggers mitophagy, a critical process for cell homeostasis (1,2). This latter function involves NM23-H4-mediated intermembrane transfer of cardiolipin (CL) from the mitochondrial inner membrane to the mitochondrial surface. Interestingly, both functions seem to involve an interaction of NM23-H4 with OPA1, a dynamin-like GTPase of the mitochondrial inner membrane. First, NM23-H4 directly fuels OPA1 with GTP via its NDPK bioenergetic function (3). In addition, also the CL transfer activity of NM23-H4 seems to depend on OPA1, since knock-down of OPA1 in HeLa cells reduces CL transfer in NM23-H4 WT expressing cells as compared to those expressing CL-transfer incompetent NM23-H4 mutants. Thus, OPA1 seems to be a negative regulator of the CL transfer function of NM23-H4. Our current model suggests that NM23-H4/OPA1 complexes exist in healthy mitochondria at the inner membrane to maintain OPA1 functions in membrane fusion and dynamics. Upon OPA1 cleavage, an early step during mitophagy, NM23-H4 may be released from these complexes, allowing simultaneous interaction of the hexameric NM23-H4 complex with inner and outer mitochondrial membrane and CL transfer.(1) Schlattner et al. (2015) Naunyn Schmiedebergs Arch. Pharmacol. 388, 271-8.(2) Kagan et al. (2016) Cell Death Diff. 23, 1140-51.(3) Boissan et al. (2014) Science 344, 1510-5.

Heterotrimeric AMP-activated protein kinase (AMPK) is crucial for energy homeostasis of eukaryotic cells and organisms. Here we report on (i) bacterial expression of untagged mammalian AMPK isoform combinations, all containing gamma(1), (ii) an automated four-dimensional purification protocol, and (iii) biophysical characterization of AMPK heterotrimers by small angle x-ray scattering in solution (SAXS), transmission and scanning transmission electron microscopy (TEM, STEM), and mass spectrometry (MS). AMPK in solution at low concentrations (~1 mg/ml) largely consisted of individual heterotrimers in TEM analysis, revealed a precise 1:1:1 stoichiometry of the three subunits in MS, and behaved as an ideal solution in SAXS. At higher AMPK concentrations, SAXS revealed concentration-dependent, reversible dimerization of AMPK heterotrimers and formation of higher oligomers, also confirmed by STEM mass measurements. Single particle reconstruction and averaging by SAXS and TEM, respectively, revealed similar elongated, flat AMPK particles with protrusions and an indentation. In the lower AMPK concentration range, addition of AMP resulted in a significant decrease of the radius of gyration by approximately 5% in SAXS, which indicates a conformational switch in AMPK induced by ligand binding. We propose a structural model involving a ligand-induced relative movement of the kinase domain resulting in a more compact heterotrimer and a conformational change in the kinase domain that protects AMPK from dephosphorylation of Thr(172), thus positively affecting AMPK activity.

International audience; Doxorubicin (DXR) belongs to the most efficient anticancer drugs. However, its use is limited by a risk of cardiotoxicity, which is not completely understood. Recently, we have shown that DXR impairs essential properties of purified mitochondrial creatine kinase (MtCK), with cardiac isoenzyme (sMtCK) being particularly sensitive. In this study we assessed the effects of DXR on respiration of isolated structurally and functionally intact heart mitochondria, containing sMtCK, in the presence and absence of externally added creatine (Cr), and compared these effects with the response of brain mitochondria expressing uMtCK, the ubiquitous, non-muscle MtCK isoenzyme. DXR impaired respiration of isolated heart mitochondria already after short-term exposure (minutes), affecting both ADP- and Cr-stimulated respiration. During a first short time span (minutes to 1 h), detachment of MtCK from membranes occurred, while a decrease of MtCK activity related to oxidative damage was only observed after longer exposure (several hours). The early inhibition of Cr-stimulated respiration, in addition to impairment of components of the respiratory chain involves a partial disturbance of functional coupling between MtCK and ANT, likely due to interaction of DXR with cardiolipin leading to competitive inhibition of MtCK/membrane binding. The relevance of these findings for the regulation of mitochondrial energy production in the heart, as well as the obvious differences of DXR action in the heart as compared to brain tissue, is discussed.

International audience; Doxorubicin and other anthracyclines are among the most potent chemotherapeutic drugs for the treatment of acute leukaemia, lymphomas and different types of solid tumours such as breast, liver and lung cancers. Their clinical use is, however, limited by the risk of severe cardiotoxicity, which can lead to irreversible congestive heart failure. There is increasing evidence that essential components of myocardial energy metabolism are among the highly sensitive and early targets of doxorubicin-induced damage. Here we review doxorubicin-induced detrimental changes in cardiac energetics, with an emphasis on the emerging importance of defects in energy-transferring and -signalling systems, like creatine kinase and AMP-activated protein kinase.

International audience; The ecdysteroids (Ec), invertebrate steroid hormones, elicit genomic but also non-genomic effects. By analogy to vertebrates, non-genomic responses towards Ec may be mediated not only by distinct membrane-integrated but also by membrane-associated receptors like the classical nuclear ecdysteroid receptor (EcR) of arthropods. This is supported by a comparison of physiological properties between invertebrate and vertebrate steroid hormone systems and recent findings on the subcellular localization of EcR. The measured or predicted high degree of conformational flexibility of both Ec and the ligand binding domain (LBD) of EcR give rise to a conformational compatibility model: the compatibility between conformations of the cognate receptor's ligand binding domain and structures or conformations of the ligand would determine their interaction and eventually the initiation of genomic versus non-genomic pathways. This model could also explain why specific non-genomic effects are generally not observed with non-steroidal agonists of the bisacylhydrazine group.

International audience; Mitochondrial creatine kinase (MtCK), together with cytosolic creatine kinase isoenzymes and the highly diffusible CK reaction product, phosphocreatine, provide a temporal and spatial energy buffer to maintain cellular energy homeostasis. Mitochondrial proteolipid complexes containing MtCK form microcompartments that are involved in channeling energy in form of phosphocreatine rather than ATP into the cytosol. Under situations of compromised cellular energy state, which are often linked to ischemia, oxidative stress and calcium overload, two characteristics of mitochondrial creatine kinase are particularly relevant: its exquisite susceptibility to oxidative modifications and the compensatory up-regulation of its gene expression, in some cases leading to accumulation of crystalline MtCK inclusion bodies in mitochondria that are the clinical hallmarks for mitochondrial cytopathies. Both of these events may either impair or reinforce, respectively, the functions of mitochondrial MtCK complexes in cellular energy supply and protection of mitochondria form the so-called permeability transition leading to apoptosis or necrosis.

International audience; Parkinson's disease is a disabling neurodegenerative disorder of unknown etiology characterized by a predominant and progressive loss of dopaminergic neurons in the substantia nigra. Recent findings suggest that impaired energy metabolism plays an important role in the pathogenesis of this disorder. The endogenously occurring guanidino compound creatine is a substrate for mitochondrial and cytosolic creatine kinases. Creatine supplementation improves the function of the creatine kinase/phosphocreatine system by increasing cellular creatine and phosphocreatine levels and the rate of ATP resynthesis. In addition, mitochondrial creatine kinase together with high cytoplasmic creatine levels inhibit mitochondrial permeability transition, a major step in early apoptosis. In the present study, we analyzed the effects of externally added creatine on the survival and morphology of dopaminergic neurons and also addressed its neuroprotective properties in primary cultures of E14 rat ventral mesencephalon. Chronic administration of creatine [5 mM] for 7 days significantly increased survival (by 1.32-fold) and soma size (by 1.12-fold) of dopaminergic neurons, while having no effect on other investigated morphological parameters. Most importantly, concurrent creatine exerted significant neuroprotection for dopaminergic neurons against neurotoxic insults induced by serum and glucose deprivation (P < 0.01), 1-methyl-4-phenyl pyridinium ion (MPP+) [15 microM] and 6-hydroxydopamine (6-OHDA) [90 microM] exposure (P < 0.01). In addition, creatine treatment significantly protected dopaminergic cells facing MPP+-induced deterioration of neuronal morphology including overall process length/neuron (by 60%), number of branching points/neuron (by 80%) and area of influence per individual neuron (by 60%). Less pronounced effects on overall process length/neuron and number of branching points/neuron were also found after 6-OHDA exposure (P < 0.05) and serum/glucose deprivation (P < 0.05). In conclusion, our findings identify creatine as a rather potent natural survival- and neuroprotective factor for developing nigral dopaminergic neurons, which is of relevance for therapeutic approaches in Parkinson's disease and for the improvement of cell replacement strategies.

Metformin, one of the most commonly used drugs for the treatment of type II diabetes, was recently found to exert its therapeutic effects, at least in part, by activating the AMP-activated protein kinase (AMPK). However, the site of its action, as well as the mechanism to activate AMPK, remains elusive. Here we report how metformin activates AMPK. In cultured bovine aortic endothelial cells, metformin dose-dependently activated AMPK in parallel with increased detection of reactive nitrogen species (RNS). Further, either depletion of mitochondria or adenoviral overexpression of superoxide dismutases, as well as inhibition of nitric-oxide synthase, abolished the metformin-enhanced phosphorylations and activities of AMPK, implicating that activation of AMPK by metformin might be mediated by the mitochondria-derived RNS. Furthermore, administration of metformin, which increased 3-nitrotyrosine staining in hearts of C57BL6, resulted in parallel activation of AMPK in the aorta and hearts of C57BL6 mice but not in those of endothelial nitric-oxide synthase (eNOS) knockout mice in which metformin had no effect on 3-nitrotyrosine staining. Because the eNOS knockout mice expressed normal levels of AMPK-α that was activated by 5-aminoimidazole-4-carboxamide riboside, an AMPK agonist, these data indicate that RNS generated by metformin is required for AMPK activation in vivo. In addition, metformin significantly increased the co-immunoprecipitation of AMPK and its upstream kinase, LKB1, in C57BL6 mice administered to metformin in vivo. Using pharmacological and genetic inhibitors, we found that inhibition of either c-Src or PI3K abolished AMPK that was enhanced by metformin. We conclude that activation of AMPK by metformin might be mediated by mitochondria-derived RNS, and activation of the c-Src/PI3K pathway might generate a metabolite or other molecule inside the cell to promote AMPK activation by the LKB1 complex.

International audience; Inverse metabolic engineering attempts to identify or construct desired phenotypes of applied interest to endow them on appropriate host organisms. A particular desirable phenotype is the ATP homeostasis exhibited by animal cells with high and variable ATP turnover through temporal and spatial energy buffering. This buffering is achieved by phosphagen kinase systems that consist of a specific kinase and its cognate phosphagen, which functions as a large pool of 'high-energy phosphates' that are used to replenish ATP during periods of high energetic demand. This review discusses recent advances and potentials of inverse metabolic engineering of cell types that do not normally contain such systems--bacteria, yeast, plants, and liver--with creatine or arginine kinase systems. Examples are discussed that illustrate how microbial metabolism can be tailored for large-scale industrial processes with imperfect mixing and how the liver can be protected from metabolic insults or stimulated for better regeneration.

Mitochondria from transgenic mice, expressing enzymatically active mitochondrial creatine kinase in liver, were analyzed for opening of the permeability transition pore in the absence and presence of creatine kinase substrates but with no external adenine nucleotides added. In mitochondria from these transgenic mice, cyclosporin A-inhibited pore opening was delayed by creatine or cyclocreatine but not by β-guanidinopropionic acid. This observation correlated with the ability of these substrates to stimulate state 3 respiration in the presence of extramitochondrial ATP. The dependence of transition pore opening on calcium and magnesium concentration was studied in the presence and absence of creatine. If mitochondrial creatine kinase activity decreased (i.e. by omitting magnesium from the medium), protection of permeability transition pore opening by creatine or cyclocreatine was no longer seen. Likewise, when creatine kinase was added externally to liver mitochondria from wild-type mice that do not express mitochondrial creatine kinase in liver, no protective effect on pore opening by creatine and its analog was observed. All these findings indicate that mitochondrial creatine kinase activity located within the intermembrane and intercristae space, in conjunction with its tight functional coupling to oxidative phosphorylation, via the adenine nucleotide translocase, can modulate mitochondrial permeability transition in the presence of creatine. These results are of relevance for the design of creatine analogs for cell protection as potential adjuvant therapeutic tools against neurodegenerative diseases.

Creatine kinase isoenzymes are very susceptible to free radical damage and are inactivated by superoxide radicals and peroxynitrite. In this study, we have analyzed the effects of peroxynitrite on enzymatic activity and octamer stability of the two human mitochondrial isoenzymes (ubiquitous mitochondrial creatine kinase (uMtCK) and sarcomeric mitochondrial creatine kinase (sMtCK)), as well as of chicken sMtCK, and identified the involved residues. Inactivation by peroxynitrite was concentration-dependent and similar for both types of MtCK isoenzymes. Because peroxynitrite did not lower the residual activity of a sMtCK mutant missing the active site cysteine (C278G), oxidation of this residue is sufficient to explain MtCK inactivation. Mass spectrometric analysis confirmed oxidation of Cys-278 and further revealed oxidation of the C-terminal Cys-358, possibly involved in MtCK/membrane interaction. Peroxynitrite also led to concentration-dependent dissociation of MtCK octamers into dimers. In this study, ubiquitous uMtCK was much more stable than sarcomeric sMtCK. Mass spectrometric analysis revealed chemical modifications in peptide Gly-263-Arg-271 located at the dimer/dimer interface, including oxidation of Met-267 and nitration of Trp-268 and/or Trp-264, the latter being a very critical residue for octamer stability. These data demonstrate that peroxynitrite affects the octameric state of MtCK and confirms human sMtCK as the generally more susceptible isoenzyme. The results provide a molecular explanation of how oxidative damage can lead to inactivation and decreased octamer/dimer ratio of MtCK, as seen in neurodegenerative diseases and heart pathology, respectively.

A soluble isoform of adenylate kinase (AK, EC 2.7.4.3) from tobacco leaves ( Nicotiana tabacum L.) was purified about 60-fold by a protocol using ammonium sulphate fractionation, anion exchange chromatography, affinity chromatography and gel filtration. The purified protein was homogeneous, as judged by SDS-PAGE, IEF-PAGE and Mono Q ion exchange chromatography, and had a specific activity of 500 nkat mg −1 . Its M r was determined as 28 000 and 30 000 by SDS-PAGE and gel filtration, respectively. It is therefore monomeric and belongs to the long-variant-type adenylate kinases. The isoelectric point of ca 4.45, as measured by IEF-PAGE and the elution profile of the Mono Q column, is characteristic for a chloroplast AK isoform. Like the chloroplast AK of maize, the activity with ATP/AMP as substrates was about two times higher than with ADP and the apparent K m was about 10-times higher for ATP/AMP than for ADP. In contrast to the maize enzyme and many other eukaryotic AKs, both substrate binding sites showed an exceptionally high specificity for all three adenylate substrates, together with a rather low affinity, as judged by the apparent K m -values. These differences at the substrate-binding sites are confirmed by a low sensitivity of the enzyme to the competitive AK inhibitor diadenosine pentaphosphate, i.e. high K i -values.

Summary Specific activity and isoform patterns of adenylate kinase (AK) were investigated in tissue cultures of cloned tobacco cell lines that differed mainly in hormone requirement, proximate cause of hormoneautotrophy and neoplastic growth characteristics. Variations in the AK isoform pattern were related to growth and ageing processes, differentiation state of tissues, differences between culture cycles as well as the neoplastic characteristics of the cell lines. High specific AK activity and isoform patterns with dominating chloroplastic activities appeared as typical of green, photosynthetic and actively growing tissues (calli in growth phase, callus outer tissues). Lower specific AK activity and isoform patterns with dominating cytosolic activity were typical for senescent or non-photosynthetic tissue (calli in stationary phase, callus core tissues), but sometimes also occurred in entire young calli, apparently as a consequence of stress. This isoform pattern is therefore proposed as a stress marker in plant cell cultures. The isoform patterns of the different cell lines were analyzed in detail for the variations during different culture cycles and the differences appearing under optimal growth conditions. The observed characteristics were related to proximate cause of hormone-autotrophy and neoplastic state. The main differences appeared between normal and neoplastic, transformed tissues. Changes in compartmentation of AK activities under different metabolic situations are discussed with respect to the physiological characteristics of the cell lines, energy metabolism and ATP/ADP ratios of cellular compartments and the presumed role of AK in ageing and stress.

Mitochondrial isoforms of creatine kinase (MtCK) and nucleoside diphosphate kinase (NDPK-D) have critical functions in bioenergetics, membrane topology and organelle morphology with roles in human health and disease. X-ray structural analysis, electron microscopy, surface plasmon resonance (SPR) and scanning calorimetry revealed that both kinases form large oligomers that bind to and cross-link mitochondrial membranes via anionic phospholipids, mainly cardiolipin; at least MtCK can also induce cardiolipin-rich membrane domains. First we used surface plasmon resonance combined with thermodynamic analysis to study kinase/cardiolipin interaction. The two kinases differed in their membrane binding mechanism: (i) NDPK-D showed monophasic binding due to electrostatic interactions of a triad of basic amino acids, while binding of MtCK was biphasic, with only the main component depending on electrostatic interactions of C-terminal basic amino acids. (ii) Rising temperature increased cardiolipin affinity of MtCK, in particular in the second binding component, but not of NDPK-D, indicating hydrophobic interactions in case of MtCK. (iv) Kinase/membrane interaction occurred to be an entropy-driven binding process, in particular for MtCK, possibly due to charge neutralization, release of bound water, and effects on membrane order. Second, we studied the effect of membrane-association on kinase enzyme activity. While basic cardiolipin had no effect on MtCK, NDPK-D was strongly inhibited. This inhibition was relieved by doxorubicin that strongly competes for cardiolipin binding. We propose a model for MtCK and NDPK-D interaction with cardiolipin-containing lipid membranes. For NDPK-D, a single phase, purely electrostatic binding would lead to a partial shielding of the enzymes’ active sites and thus catalytic inhibition. For MtCK, a two-phase binding model of rapid electrostatic docking and slower anchoring via hydrophobic stretches is proposed, which does not affect the active sites.

Oxidative modifications of creatine kinase (CK) isoenzymes are thought to play a critical role during pathologies involving oxidative stress. Reactive oxygen and nitrogen species (ROS, RNS) not only induce enzymatic inactivation, which occurs with all CK isoenzymes, but also specific damage to the mitochondrial CK isoforms, namely interference with their oligomeric state and membrane binding capacity. Using purified recombinant proteins, cell homogenates and mitochondria isolated from rat heart and brain, we have compared the two isoforms of mitochondrial CK (sarcomeric sMtCK expressed in heart and skeletal muscle, ubiquitous uMtCK expressed in other tissues) in respect to their sensitivity to oxidative inactivation induced by the drug doxorubicin or occurring spontaneously after extraction under non-reducing condition. We confirmed that sarcomeric sMtCK shows significantly higher sensitivity to oxidation and that the loss of total CK activity in heart extracts upon storage under non-reducing condition is mainly due to the inactivation of sMtCK. We could also show that the sMtCK dimer is particularly easily inactivated and that solubilization of sMtCK from membrane (promoting dimerization) makes the protein an especially vulnerable substrate for inactivation. The differential susceptibility of the two MtCK isoenzymes has been related to some differences in their molecular structures (e.g. number and surface exposure of cysteine residues).

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Mitochondria-specific cardiolipin (CL) is mostly confined to the site of its biosynthesis, the inner mitochondrial membrane. Some CL remodeling occurs in the ER and thus implies the necessity of CL trafficking across mitochondrial membranes. The involved mechanisms are unknown. However, we have recently identified mitochondrial nucleoside diphosphate kinase (NDPK-D) to be capable in vitro to simultaneously bind two CL-containing membranes and to facilitate transfer of model lipids between them. This property relies on the symmetrical hexameric structure of NDPK-D which exposes positively charged residues that bind with high affinity to anionic phospholipids, in particular CL. In this work, we have studied the role of NDPK-D for mitochondrial membrane asymmetry in vivo. We used Hela cells that are devoid of immuno-detectable amounts of NDPK-D to stably express wild-type and membrane-binding incompetent mutant forms NDPK-D. Analysis of purified mitochondrial inner and outer membranes by electro-spray ionization mass spectrometry revealed that the presence of NDPK-D wild-type, but not of membrane-binding incompetent mutant, leads to a partial collapse of CL asymmetry. These changes are CL-specific as the distribution of phosphatidylcholine remains unchanged in both HeLa cell lines. The pattern of different CL species in the membranes is also similar in both cases. We conclude that NDPK-D is a part of machinery that is involved in CL trafficking within mitochondria.

Mitochondria are vulnerable to damage, particularly by oxidative stress-induced injury imposed by many genetic and environmental factors. These malfunctioning mitochondria have to be eliminated to prevent an increasing generation of reactive oxygen species (ROS) that could trigger cell injury and death. This selective elimination of damaged mitochondria is executed via initiation of a specific type of mitochondrial autophagy - mitophagy. Our previous work has established that a mitochondria-specific phospholipid, cardiolipin (CL) - normally asymmetrically distributed between the mitochondrial inner (IMM) and outer (OMM) membranes - undergoes translocation to the OMM where it becomes externalized to the mitochondrial surface. This externalized CL serves as recognition signal for the autophageal machinery leading to the elimination of these mitochondria. The recognition is achieved through the selective binding of externalized CL with microtubule-associated protein light chain 3 (LC3). The mechanisms driving CL redistribution/externalization remain unknown. By using LC-MS analysis of mono-lyso-cardiolipins (mL-CL) formed by phospholipase A2 exogenously added and impermeable to mitochondria, we established that treatment of HeLa cells with a protonophoric uncoupler, CCCP, triggers mitophagy of depolarized mitochondria accompanied by CL externalization. We further found that CL externalization is dependent on an intermembrane space enzyme, nucleoside diphosphate kinase, NDPK-D. The latter, upon interaction with CL, loses its kinase function and turns into a CL-translocase. CL externalization and mitophagy were stimulated by transfecting HeLa cells with w/type but not mutant R90A NDPK-D, incapable of CL binding. Identification of NDPK-D as a pro-mitophageal CL translocase may be used in drug discovery paradigms for regulation of “mitochondrial health.” Supported by NIH U19AIO6802, Fondation pour la Recherche Medicale France (US, DPM20121125557) and Fulbright scholarship program (VEK).

Molecular functions of mitochondrial nucleoside diphosphate kinase (NDPK-D) were studied using different biophysical and biochemical techniques. Subfractionation of rat liver and HEK 293 cell mitochondria revealed that NDPK-D is essentially bound to the inner membrane. The kinase interacted electrostatically with anionic phospholipids, showing highest affinity for cardiolipin as quantified by surface plasmon resonance. NDPK-D was also able to cross-link anionic phospholipid-containing liposomes as seen in light scattering assays, suggesting that the hexameric kinase could promote intermembrane contacts. Mutation of the central arginine (R90) in a surface exposed basic RRK motif unique to NDPK-D strongly reduced these membrane interactions. In a model using HeLa cells naturally almost devoid of NDPK-D, wt protein and R90D mutant were stably expressed, but only wt protein was found attached to membranes. Respiration was significantly stimulated by the NDPK substrate TDP only in mitochondria containing wt NDPK-D, but not in those expressing R90D mutant that is catalytically equally active. This indicates local ADP regeneration in the mitochondrial intermembrane space and a tight functional coupling of NDPK-D with oxidative phosphorylation that depends on the membrane-bound state of the kinase. A model is proposed for a mitochondrial NDPK microcompartment.

Fig. 2. Comparison between the differential surface representations of the ligand binding domains of the ecdysteroid receptor (EcR–LBD) of Heliothis virescens and of the human Vitamin D receptor (VDR–LBD). (a) The surfaces of EcR–LBD co-crystallized either with the natural ecdysteroid ponasterone A or with the non-steroidal ecdysone agonist BY-106830 were determined by X-ray crystallography and superimposed. Degree of deviation between the two surfaces is indicated by the colour code (according to Billas et al. (2003); figure reproduced by the courtesy of the authors and the publisher). H1, H2, H3, H7: designation of -helices; L7–8, L9–10: loops between -helices; : -sheet; S3: third strand of the -sheet. Faint labels are from original picture. (c) Same for VDR–LBD, except that the two holo-conformations compared arise from binding with either 1 ,25(OH)2-Vitamin D3 or 1 ,25(OH)2-lumisterol to two conformationally different pockets which were related to genomic or non-genomic effects, respectively. The structure of the Vitamin D3-containing LBD was determined by X-ray crystallography (Rochel et al., 2000). The structure of the lumisterol-containing LBD was generated in silico (Mizwicki et al., 2004). The colour code corresponds to deviations in the peptide backbone as published by Mizwicki et al. (2004). Note the different colour codes in (a) and (c). (c) Prepared withWebLab Viewer Pro on the basis of X-ray data (PDB code 1DB1). “S3”: region homologous to -strand 3 of EcR–LBD. H3n: hVDR-specific -helix lying between -helices 2 and 3. (b) and (d): Structural models with -helices shown as red ribbons and -strands as blue arrows.