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Background NME6 is a member of the nucleoside diphosphate kinase (NDPK/NME/Nm23) family which has key roles in nucleotide homeostasis, signal transduction, membrane remodeling and metastasis suppression. The well-studied NME1-NME4 proteins are hexameric and catalyze, via a phospho-histidine intermediate, the transfer of the terminal phosphate from (d)NTPs to (d)NDPs (NDP kinase) or proteins (protein histidine kinase). For the NME6, a gene/protein that emerged early in eukaryotic evolution, only scarce and partially inconsistent data are available. Here we aim to clarify and extend our knowledge on the human NME6. Results We show that NME6 is mostly expressed as a 186 amino acid protein, but that a second albeit much less abundant isoform exists. The recombinant NME6 remains monomeric, and does not assemble into homo-oligomers or hetero-oligomers with NME1-NME4. Consequently, NME6 is unable to catalyze phosphotransfer: it does not generate the phospho-histidine intermediate, and no NDPK activity can be detected. In cells, we could resolve and extend existing contradictory reports by localizing NME6 within mitochondria, largely associated with the mitochondrial inner membrane and matrix space. Overexpressing NME6 reduces ADP-stimulated mitochondrial respiration and complex III abundance, thus linking NME6 to dysfunctional oxidative phosphorylation. However, it did not alter mitochondrial membrane potential, mass, or network characteristics. Our screen for NME6 protein partners revealed its association with NME4 and OPA1, but a direct interaction was observed only with RCC1L, a protein involved in mitochondrial ribosome assembly and mitochondrial translation, and identified as essential for oxidative phosphorylation. Conclusions NME6, RCC1L and mitoribosomes localize together at the inner membrane/matrix space where NME6, in concert with RCC1L, may be involved in regulation of the mitochondrial translation of essential oxidative phosphorylation subunits. Our findings suggest new functions for NME6, independent of the classical phosphotransfer activity associated with NME proteins.

Mitochondrial nucleoside diphosphate kinase (NDPK-D; synonyms: NME4, NM23-H4) represents the major mitochondrial NDP kinase. The homohexameric complex emerged as a protein with multiple functions in bioenergetics and phospholipid signaling. It occurs at different but precise mitochondrial locations and can affect among other mitochondrial shapes and dynamics, as well as the specific elimination of defective mitochondria or cells via mitophagy or apoptosis. With these various functions in cell homeostasis, NDPK-D/NME4 adds to the group of so-called moonlighting (or gene sharing) proteins.

Isoforms of creatine kinase (CK) generate and use phosphocreatine, a concentrated and highly diffusible cellular “high energy” intermediate, for the main purpose of energy buffering and transfer in order to maintain cellular energy homeostasis. The mitochondrial CK isoform (mtCK) localizes to the mitochondrial intermembrane and cristae space, where it assembles into peripherally membrane-bound, large cuboidal homooctamers. These are part of proteolipid complexes wherein mtCK directly interacts with cardiolipin and other anionic phospholipids, as well as with the VDAC channel in the outer membrane. This leads to a stabilization and cross-linking of inner and outer mitochondrial membrane, forming so-called contact sites. Also the adenine nucleotide translocator of the inner membrane can be recruited into these proteolipid complexes, probably mediated by cardiolipin. The complexes have functions mainly in energy transfer to the cytosol and stimulation of oxidative phosphorylation, but also in restraining formation of reactive oxygen species and apoptosis. In vitro evidence indicates a putative role of mtCK in mitochondrial phospholipid distribution, and most recently a role in thermogenesis has been proposed. This review summarizes the essential structural and functional data of these mtCK complexes and describes in more detail the more recent advances in phospholipid interaction, thermogenesis, cancer and evolution of mtCK.

International audience; Mitophagy is an emerging paradigm for mitochondrial quality control and cell homeostasis. Dysregulation of mitophagy can lead to human pathologies such as neurodegenerative disorders and contributes to the aging process. Complex protein signaling cascades have been described that regulate mitophagy. We have identified a novel lipid signaling pathway that involves the phospholipid cardiolipin (CL). CL is synthesized and normally confined at the inner mitochondrial membrane. However, upon a mitophagic trigger, ie, collapse of the inner membrane potential, CL is rapidly externalized to the mitochondrial surface with the assistance of the hexameric nucleoside diphosphate kinase D (NME4, NDPK-D, or NM23-H4). In addition to its NDP kinase activity, NME4/NDPK-D shows intermembrane phospholipid transfer activity in vitro and in cellular systems, which relies on NME4/NDPK-D interaction with CL, CL-dependent crosslinking of inner and outer mitochondrial membranes by symmetrical, hexameric NME4/NDPK-D, and a putative NME4/NDPK-D-based CL-transfer pathway. CL exposed at the mitochondrial surface then serves as an 'eat me' signal for the mitophagic machinery; it is recognized by the LC3 receptor of autophagosomes, targeting the dysfunctional mitochondrion to lysosomal degradation. Similar NME4-supported CL externalization is likely also involved in apoptosis and inflammatory reactions.

International audience; For maintaining energy homeostasis, creatine kinase (CK) is present at elevated levels in tissueswith high and/or fluctuating energy requirements such as muscle, brain, and epithelia, while there is very fewCK, if any, in peripheral blood cells. However, an ectopic expression of brain-type creatine kinase (BCK) has been reported for platelets and leukocytes in an autosomal dominant inherited anomaly named CKBE. Here we investigated CK inerythrocytes of CKBE individuals from eight unrelated families. The data revealed a varying but significant increase of CK activity in CKBE individuals as compared to controls, reaching an almost 800-fold increase in two CKBE individuals which also had increased erythrocyte creatine. Immunoblotting with highly specific antibodies confirmed that the expressed CK isoform is BCK. Cell fractionation evidenced soluble BCK, suggesting cytosolic and not membrane localization of erythrocyte CK as reported earlier. These results are discussed in the context of putative CK energy buffering and transfer functions in red blood cells.

Mitophagy is critical for cell homeostasis. Externalization of the inner mitochondrial membrane phospholipid, cardiolipin (CL), to the surface of the outer mitochondrial membrane (OMM) was identified as a mitophageal signal recognized by the microtubule-associated protein 1 light chain 3. However, the CL-translocating machinery remains unknown. Here we demonstrate that a hexameric intermembrane space protein, NDPK-D (or NM23-H4), binds CL and facilitates its redistribution to the OMM. We found that mitophagy induced by a protonophoric uncoupler, carbonyl cyanide m-chlorophenylhydrazone (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 that does not bind CL was inactive in promoting mitophagy. Similarly, rotenone and 6-hydroxydopamine triggered mitophagy in SH-SY5Y cells was also suppressed by knocking down of NDPK-D. In situ proximity ligation assay (PLA) showed that mitophagy-inducing CL-transfer activity of NDPK-D is closely associated with the dynamin-like GTPase OPA1, implicating fission-fusion dynamics in mitophagy regulation.

International audience; Resveratrol is attracting much interest because of its potential to decrease body weight and increase life span, influencing liver and muscle function by increasing mitochondrial mass and energy expenditure. Even though resveratrol was already shown to reduce the adipose tissue mass in animal models, its effects on mitochondrial mass and network structure in adipocytes have not yet been studied. For this purpose, we investigated the effect of resveratrol on mitochondrial mass increase and remodeling during adipogenic differentiation of two in vitro models of adipocyte biology, the murine 3T3-L1 cell line and the human SGBS cell strain. We confirm that resveratrol inhibits lipogenesis in differentiating adipocytes, both mouse and human. We further show that this is linked to inhibition of the normally observed mitochondrial mass increase and mitochondrial remodeling. At the molecular level, the anti-lipogenic effect of resveratrol seems to be mediated by a blunted expression increase and an inhibition of acetyl-CoA carboxylase (ACC). This is one of the consequences of an inhibited insulin-induced signaling via Akt, and maintained signaling via AMP-activated protein kinase. The anti-lipogenic effect of resveratrol is further modulated by expression levels of mitochondrial ATAD3, consistent with the emerging role of this protein as an important regulator of mitochondrial biogenesis and lipogenesis. Our data suggest that resveratrol acts on differentiating preadipocytes by inhibiting insulin signaling, mitochondrial biogenesis, and lipogenesis, and that resveratrol-induced reduction of mitochondrial biogenesis and lipid storage contribute to adipose tissue weight loss in animals and humans.

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.

Development of cardiac hypertrophy and progression to heart failure entails profound changes in myocardial metabolism, characterized by a switch from fatty acid utilization to glycolysis and lipid accumulation. We report that hypoxia-inducible factor (HIF)1alpha and PPARgamma, key mediators of glycolysis and lipid anabolism, respectively, are jointly upregulated in hypertrophic cardiomyopathy and cooperate to mediate key changes in cardiac metabolism. In response to pathologic stress, HIF1alpha activates glycolytic genes and PPARgamma, whose product, in turn, activates fatty acid uptake and glycerolipid biosynthesis genes. These changes result in increased glycolytic flux and glucose-to-lipid conversion via the glycerol-3-phosphate pathway, apoptosis, and contractile dysfunction. Ventricular deletion of Hif1alpha in mice prevents hypertrophy-induced PPARgamma activation, the consequent metabolic reprogramming, and contractile dysfunction. We propose a model in which activation of the HIF1alpha-PPARgamma axis by pathologic stress underlies key changes in cell metabolism that are characteristic of and contribute to common forms of heart disease.

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; 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; AMP-activated protein kinase (AMPK) and cytosolic brain-type creatine kinase (BCK) cooperate under energy stress to compensate for loss of adenosine triphosphate (ATP) by either stimulating ATP-generating and inhibiting ATP-consuming pathways, or by direct ATP regeneration from phosphocreatine, respectively. Here we report on AMPK-dependent phosphorylation of BCK from different species identified by in vitro screening for AMPK substrates in mouse brain. Mass spectrometry, protein sequencing, and site-directed mutagenesis identified Ser6 as a relevant residue with one site phosphorylated per BCK dimer. Yeast two-hybrid analysis revealed interaction of active AMPK specifically with non-phosphorylated BCK. Pharmacological activation of AMPK mimicking energy stress led to BCK phosphorylation in astrocytes and fibroblasts, as evidenced with a highly specific phospho-Ser6 antibody. BCK phosphorylation at Ser6 did not affect its enzymatic activity, but led to the appearance of the phosphorylated enzyme at the endoplasmic reticulum (ER), close to the ER calcium pump, a location known for muscle-type cytosolic creatine kinase (CK) to support Ca2+-pumping.

International audience; A novel paradigm for the function of the mitochondrial nucleoside diphosphate kinase NM23-H4/NDPK-D is proposed: acting as a bifunctional nanoswitch in bioenergetics and cardiolipin (CL) trafficking and signaling. Similar to some other mitochondrial proteins like cytochrome c or AIF, NM23-H4 seems to have dual functions in bioenergetics and apoptotic signaling. In its bioenergetic phosphotransfer mode, the kinase reversibly phosphorylates NDPs into NTPs, driven by mitochondrially generated ATP. Among others, this reaction can locally supply GTP to mitochondrial GTPases as shown for the dynamin-like GTPase OPA1, found in a complex together with NM23-H4. Further, NM23-H4 is functionally coupled to adenylate translocase (ANT) of the mitochondrial inner membrane (MIM), so generated ADP can stimulate respiration to rapidly regenerate ATP. The lipid transfer mode of NM23-H4 can support, dependent on the presence of CL, the transfer of anionic lipids between membranes in vitro and the sorting of CL from its mitochondrial sites of synthesis (MIM) to the mitochondrial outer membrane (MOM) in vivo. Such (partial) collapse of MIM/MOM CL asymmetry results in CL externalization on the mitochondrial surface, where CL can serve as pro-apoptotic or pro-mitophagic “eat me”-signal. The functional state of NM23-H4 depends on its degree of CL-membrane interaction. In vitro assays have shown that only NM23-H4 that fully cross-links two membranes is lipid transfer competent, but at the same time phosphotransfer (kinase) inactive. Thus, the two functions of NM23-H4 seem to be mutually exclusive. This novel mitochondrial regulatory circuit has potential for the development of interventions in various human pathologies.

International audience; Historically, cellular trafficking of lipids has received much less attention than protein trafficking, mostly because its biological importance was underestimated, involved sorting and translocation mechanisms were not known, and analytical tools were limiting. This has changed during the last decade, and we discuss here some progress made in respect to mitochondria and the trafficking of phospholipids, in particular cardiolipin. Different membrane contact site or junction complexes and putative lipid transfer proteins for intra- and intermembrane lipid translocation have been described, involving mitochondrial inner and outer membrane, and the adjacent membranes of the endoplasmic reticulum. An image emerges how cardiolipin precursors, remodeling intermediates, mature cardiolipin and its oxidation products could migrate between membranes, and how this trafficking is involved in cardiolipin biosynthesis and cell signaling events. Particular emphasis in this review is given to mitochondrial nucleoside diphosphate kinase D and mitochondrial creatine kinases, which emerge to have roles in both, membrane junction formation and lipid transfer.

International audience; Integrated mechanisms of regulation of energy metabolism at cellular, tissue, and organ levels are analyzed from a systems biology perspective. These integrated mechanisms comprise the coordinated function of three cycles of mass and energy transfer and conversion: (1) the Randle cycle of substrate supply, (2) the Krebs cycle coupled with energy transformation in mitochondrial oxidative phosphorylation, and (3) the kinase cycles of intracellular energy transfer and signal transduction for regulation of energy fluxes. These cycles are extended and partially governed by information transfer systems like those linked to protein kinase signaling. In the heart, these cycles are closely related to the Ca2+ cycle during excitation–contraction coupling. According to the view of integrated metabolic cycles, the phosphocreatine/creatine kinase system represents a most important subsystem determining the efficiency of regulation of metabolic and energy fluxes in heart, brain, and oxidative skeletal muscles. It carries about 80 % of the energy flux between mitochondria and cytoplasm in heart. The substrate uptake, respiration rate, and energy fluxes are regulated in response to workload via phosphotransfer pathways and Ca2+ cycling. We propose integrated network mechanisms to explain the linear relationship between myocardial oxygen consumption and heart work output under conditions of metabolic stability (metabolic aspect of Frank–Starling’s law of the heart). The efficiency of energy transfer, force of contraction, and metabolic regulation of respiration and energy fluxes depend upon the intracellular concentration of total creatine, which is decreased in heart failure. The role of creatine, creatine kinase, and adenylate kinase phosphotransfer and AMP-activated protein kinase (AMPK) signaling systems and their interrelationship with substrate supply and Ca2+ cycles are analyzed. Finally, an introduction to the AMPK signaling network is provided with a particular emphasis on the heart in health and disease.

International audience; Nm23-H4/NDPK-D forms symmetrical homohexameric complexes in the mitochondrial intermembrane space. The well established function of the enzyme is phosphotransfer activity as a nucleoside diphosphate kinase (NDPK), using mitochondrial ATP to regenerate nucleoside triphosphates. Nm23-H4 is further known to strongly bind in vitro to anionic phospholipids, mainly cardiolipin, and in vivo to inner mitochondrial membranes. We show here that such protein/lipid complexes inhibit NDPK activity but are necessary for Nm23-H4 to function in selective intermembrane lipid transfer. Nm23-H4-deficient HeLa cells expressing either wild-type Nm23-H4 or a membrane-binding deficient mutant were analyzed by membrane fractionation and LC-ESI-MS. Data revealed that wild-type Nm24-H4 increased cardiolipin content in the outer mitochondrial membrane as compared to mutant enzyme. This correlated with higher susceptibility of wild-type enzyme expressing cells to rotenone-induced apoptosis as seen by increased annexin V binding, elevated caspase 3/7 activity and stimulated release of cytochrome c into the cytosol. Molecular modeling of Nm23-H4 binding with cardiolipin reveals potential intermembrane transfer mechanisms. We propose that Nm23-H4 acts as a lipid-dependent mitochondrial switch with dual function, allowing either for phosphotransfer or for cardiolipin transfer, with a role in apoptotic signaling.

The nucleoside diphosphate kinase Nm23-H4/NDPK-D forms symmetrical hexameric complexes in the mitochondrial intermembrane space with phosphotransfer activity using mitochondrial ATP to regenerate nucleoside triphosphates. We demonstrate the complex formation between Nm23-H4 and mitochondrial GTPase OPA1 in rat liver, suggesting its involvement in local and direct GTP delivery. Similar to OPA1, Nm23-H4 is further known to strongly bind in vitro to anionic phospholipids, mainly cardiolipin, and in vivo to the inner mitochondrial membrane. We show here that such protein-lipid complexes inhibit nucleoside diphosphate kinase activity but are necessary for another function of Nm23-H4, selective intermembrane lipid transfer. Mitochondrial lipid distribution was analyzed by liquid chromatography-mass spectrometry using HeLa cells expressing either wild-type Nm23-H4 or a membrane binding-deficient mutant at a site predicted based on molecular modeling to be crucial for cardiolipin binding and transfer mechanism. We found that wild type, but not the mutant enzyme, selectively increased the content of cardiolipin in the outer mitochondrial membrane, but the distribution of other more abundant phospholipids (e.g. phosphatidylcholine) remained unchanged. HeLa cells expressing the wild-type enzyme showed increased accumulation of Bax in mitochondria and were sensitized to rotenone-induced apoptosis as revealed by stimulated release of cytochrome c into the cytosol, elevated caspase 3/7 activity, and increased annexin V binding. Based on these data and molecular modeling, we propose that Nm23-H4 acts as a lipid-dependent mitochondrial switch with dual function in phosphotransfer serving local GTP supply and cardiolipin transfer for apoptotic signaling and putative other functions.

Background Ubiquitous mitochondrial creatine kinase (uMtCK) accumulates as macroenzyme creatine kinase type 2 (macro CK2) in the serum of HIV-infected patients under a tenofovir disoproxil fumarate (TDF)-containing antiretroviral regimen. The genesis and clinical significance of this finding is unclear. Methods A prospective observational 5-year follow-up study was performed on those patients in which macro CK2 appearance was initially described (‘TDF switch study’ cohort). In addition, tenofovir (TFV), its prodrug TDF and its active, intracellular derivative TFV diphosphate (TDP) were tested in vitro for their effects on different key properties of uMtCK to clarify possible interactions of uMtCK with TFV compounds. Results In just under 5 years of continuous TDF treatment, only 4/12 (33%) patients remained macro CK2-positive, whereas 8/12 (66%) originally positive patients were macro CK2-negative at the end of follow-up. Prospective clinical follow-up data indicate that macro CK2 appearance under TDF is not associated with significant cell damage or occurrence of malignancies. A trend towards grade 1 hypophosphataemia suggests subclinical proximal tubular dysfunction in macro-CK2-positive patients, although it was not associated with a significant decrease in estimated glomerular filtration rate. In vitro, TFV, TDF and TDP did not interfere with uMtCK enzyme activity as competitive inhibitors or pseudo-substrates, but TFV and TDF stabilized the native uMtCK octameric structure in dilute solutions. Conclusions Appearance of octameric uMtCK as macro CK2 in the serum of TDF-treated patients is suggested to result from a combination of low-level mitochondrial damage caused by subclinical renal tubular dysfunction together with possible compensatory uMtCK overexpression and a putative concomitant stabilization of uMtCK octamers by higher levels of TFV in proximal tubules.

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.

International audience; Mitochondrial isoforms of creatine kinase (MtCK) and nucleoside diphosphate kinase (NDPK-D) are not phylogenetically related but share functionally important properties. They both use mitochondrially generated ATP with the ultimate goal of maintaining proper nucleotide pools, are located in the intermembrane/cristae space, have symmetrical oligomeric structures, and show high affinity binding to anionic phospholipids, in particular cardiolipin. The structural basis and functional consequences of the cardiolipin interaction have been studied and are discussed in detail in this review. They mainly result in a functional interaction of MtCK and NDPK-D with inner membrane adenylate translocator, probably by forming proteolipid complexes. These interactions allow for privileged exchange of metabolites (channeling) that ultimately regulate mitochondrial respiration. Further functions of the MtCK/membrane interaction include formation of cardiolipin membrane patches, stabilization of mitochondria and a role in apoptotic signaling, as well as in case of both kinases, a role in facilitating lipid transfer between two membranes. Finally, disturbed cardiolipin interactions of MtCK, NDPK-D and other proteins like cytochrome c and truncated Bid are discussed more generally in the context of apoptosis and necrosis.

International audience; Nucleoside diphosphate kinases (NDPKs/Nm23), responsible for intracellular di- and tri-phosphonucleoside homeostasis, play multi-faceted roles in cellular energetic, signaling, proliferation, differentiation and tumor invasion. The mitochondrial NDPK-D, the NME4 gene product, is a peripheral protein of the inner membrane. Several new aspects of the interaction of NDPK-D with the inner mitochondrial membrane have been recently characterized. Surface plasmon resonance analysis using recombinant NDPK-D and different phospholipid liposomes showed that NDPK-D interacts electrostatically with anionic phospholipids, with highest affinity observed for cardiolipin, a phospholipid located mostly in the mitochondrial inner membrane. Mutation of the central arginine (R90) in a surface exposed cationic RRK motif unique to NDPK-D strongly reduced phospholipid interaction in vitro and in vivo. Stable expression of NDPK-D proteins in HeLa cells naturally almost devoid of this isoform revealed a tight functional coupling of NDPK-D with oxidative phosphorylation that depends on the membrane-bound state of the enzyme. Owing to its symmetrical hexameric structure exposing membrane binding motifs on two opposite sides, NDPK-D could bridge liposomes containing anionic phospholipids and promote lipid transfer between them. In vivo, NDPK-D could induce intermembrane contacts and facilitate lipid movements between mitochondrial membranes. Most of these properties are reminiscent to those of the mitochondrial creatine kinase. We review here the common properties of both kinases and we discuss their potential roles in mitochondrial functions such as energy production, apoptosis and mitochondrial dynamics.

International audience; Nucleoside diphosphate kinase (NDPK/Nm23), responsible for intracellular di- and triphosphonucleoside homeostasis, plays multiple roles in cellular energetics, signaling, proliferation, differentiation and tumor invasion. The only human NDPK with a mitochondrial targeting sequence is NDPK-D, the NME4 gene product, which is a peripheral protein of mitochondrial membranes. Subfractionation of rat liver and HEK 293 cell mitochondria revealed that NDPK-D is essentially bound to the inner membrane. Surface plasmon resonance analysis of the interaction using recombinant NDPK-D and model liposomes showed that NDPK-D interacts electrostatically with anionic phospholipids, with highest affinity observed for cardiolipin. Mutation of the central arginine (Arg-90) in a surface-exposed basic RRK motif unique to NDPK-D strongly reduced interaction with anionic phospholipids. Due to its symmetrical hexameric structure, NDPK-D was able to cross-link anionic phospholipid-containing liposomes, suggesting that NDPK-D could promote intermembrane contacts. Latency assays with isolated mitochondria and antibody binding to mitoplasts indicated a dual orientation for NDPK-D. In HeLa cells, stable expression of wild type but not of the R90D mutant led to membrane-bound enzyme in vivo. Respiration was significantly stimulated by the NDPK substrate TDP in mitochondria containing wild-type NDPK-D, but not in those expressing the R90D mutant, which 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 its membrane-bound state.

International audience; Evidence for the important physiological role of the creatine kinase (CK)- phosphocreatine (PCr) system in energy homeostasis in sarcomeric muscle, brain, and other organs of high and fluctuating energy requirements is presented in the context of defined subcellular compartments of CK isoenzymes with processes of energy production, i.e., glycolysis and mitochondrial oxidative phosphorylation, and those of energy utilization, i.e., ATPases, ATP-gated ion channels, and ATP-dependent signaling events. The concepts of functional coupling and metabolite channeling are discussed, and the CK subcompartments that have been characterized in great detail are described. Mitochondrial CK (MtCK) plays a central role (1) in high-energy phosphoryl transfer and channeling, (2) in metabolic feedback regulation of mitochondrial respiration in vivo, (3) in stabilizing the contact sites of inner and outer mitochondrial membranes, (4) in delaying and preventing mitochondrial permeability pore opening, an early event in apoptosis, and (5) in preventing, by efficient ADP recycling inside mitochondria and by optimal coupling of respiration with ATP synthesis, excessive free oxygen radical (ROS) formation. Additional new and exciting findings indicate that octameric MtCK, a highly symmetrical cube-like molecule that is able to cross-link two membranes, is also involved in lipid transfer between mitochondrial membranes and in clustering of cardiolipin (CL) and the formation of membrane patches. Because of the high reactivity of the active-site cysteine of CK isoenzymes, this enzyme is highly susceptible to oxidation that leads to inactivation. Oxidation of other residues at the dimer-dimer interface of MtCK induces dimerization of the MtCK octamer, the functional entity in the mitochondrial intermembrane space, and dissociation of MtCK from the mitochondrial membranes. (PDF) The Phosphocreatine Circuit: Molecular and Cellular Physiology of Creatine Kinases, Sensitivity to Free Radicals, and Enhancement by Creatine Supplementation. Available from: https://www.researchgate.net/publication/200159132_The_Phosphocreatine_Circuit_Molecular_and_Cellular_Physiology_of_Creatine_Kinases_Sensitivity_to_Free_Radicals_and_Enhancement_by_Creatine_Supplementation [accessed Dec 10 2018].

International audience; We show in this study that mitochondrial creatine kinase promotes segregation and clustering of cardiolipin in mixed membranes, a phenomenon that has been proposed to occur at contact sites in the mitochondria. This property of mitochondrial creatine kinase is dependent on the native octameric structure of the protein and does not occur after heat-denaturation or with the native dimeric form of the protein. Cardiolipin segregation was demonstrated by differential scanning calorimetry using membranes containing cardiolipin and either dipalmitoylphosphatidylethanolamine or 1-palmitoyl-2-oleoylphosphatidylethanolamine. Addition of the ubiquitous form of mitochondrial creatine kinase leads to the formation of a phosphatidylethanolamine-rich domain as a result of the protein binding preferentially to the cardiolipin. Such phase separation does not occur if cardiolipin is replaced with dioleoyl phosphatidylglycerol. Lipid phase separation is observed with other cardiolipin-binding proteins, including cytochrome c and, to a very small extent, with truncated Bid (t-Bid), as well as with the cationic polypeptide poly-L-lysine, but among these proteins the octameric form of mitochondrial creatine kinase is by far the most effective in causing segregation and clustering of cardiolipin. The proteins included in this study are found at mitochondrial contact sites where they are known to associate with cardiolipin. Domains in mitochondria enriched in cardiolipin play an important role in apoptosis and in energy flux processes.

International audience; High affinity interaction between octameric mitochondrial creatine kinase (MtCK) and the phospholipid cardiolipin in the inner mitochondrial membrane plays an important role in metabolite channeling between MtCK and inner membrane adenylate translocator, which itself is tightly bound to cardiolipin. Three C-terminal basic residues revealed as putative cardiolipin anchors in the x-ray structures of MtCK and corresponding to lysines in human sarcomeric MtCK (sMtCK) were exchanged by in vitro mutagenesis (K369A/E, K379Q/A/E, K380Q/A/E) to yield double and triple mutants. sMtCK proteins were bacterially expressed, purified to homogeneity, and verified for structural integrity by enzymatic activity, gel filtration chromatography, and CD spectroscopy. Interaction with cardiolipin and other acidic phospholipids was quantitatively analyzed by light scattering, surface plasmon resonance, and fluorescence spectroscopy. All mutant sMtCKs showed a strong decrease in vesicle cross-linking, membrane affinity, binding capacity, membrane ordering capability, and binding-induced changes in protein structure as compared with wild type. These effects did not depend on the nature of the replacing amino acid but on the number of exchanged lysines. They were moderate for Lys-379/Lys-380 double mutants but pronounced for triple mutants, with a 30-fold lower membrane affinity and an entire lack of alterations in protein structure compared with wild-type sMtCK. However, even triple mutants partially maintained an increased order of cardiolipin-containing membranes. Thus, the three C-terminal lysines determine high affinity sMtCK/cardiolipin interaction and its effects on MtCK structure, whereas low level binding and some effect on membrane fluidity depend on other structural components. These results are discussed in regard to MtCK microcompartments and evolution.

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.

International audience; Anthracyclines are among the most efficient drugs of cancer chemotherapy, but their use is limited by a significant risk of cardiotoxicity, which is still far from being understood. This study investigates whether impairment of mitochondrial creatine kinase (MtCK), a key enzyme in cellular energy metabolism, could be involved in anthracycline cardiotoxicity. We have analyzed the effects of three anthracyclines, doxorubicin, daunorubicin, and idarubicin, on two MtCK isoenzymes, sarcomeric/cardiac sMtCK and ubiquitous uMtCK, from human and chicken. Using surface plasmon resonance, gel filtration, and enzyme assays, we have quantified properties that are of basic importance for MtCK functioning in vivo: membrane binding, octameric state, and enzymatic activity. Anthracyclines significantly impaired all three properties with differences in dose-, time-, and drug-dependence. Membrane binding and enzymatic activity were already affected at low anthracycline concentrations (5-100 microM), indicating high clinical relevance. Effects on membrane binding were immediate, probably because of competitive binding of the drug to cardiolipin. In contrast, dissociation of MtCK octamers into dimers, enzymatic inactivation and cross-linking occurred only after hours to days. Different protection assays suggest that the deleterious effects were caused by oxidative damage, mainly affecting the highly susceptible MtCK cysteines, followed by generation of free oxygen radicals at higher drug concentrations. Enzymatic inactivation occurred mainly at the active site and involved Cys278, as indicated by experiments with protective agents and sMtCK mutant C278G. All anthracycline effects were significantly more pronounced for sMtCK than for uMtCK. These in vitro results suggest that sMtCK damage may play a role in anthracycline cardiotoxicity.

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).

International audience; Mitochondrial creatine kinase (MtCK) co-localizes with mitochondrial porin (voltage-dependent anion channel) and adenine nucleotide translocator in mitochondrial contact sites. A specific, direct protein-protein interaction between MtCK and mitochondrial porin was demonstrated using surface plasmon resonance spectroscopy. This interaction was independent of the immobilized binding partner (porin reconstituted in liposomes or MtCK) or the analyzed isoform (chicken sarcomeric MtCK or human ubiquitous MtCK, human recombinant porin, or purified bovine porin). Increased ionic strength reduced the binding of MtCK to porin, suggesting predominantly ionic interactions. By contrast, micromolar concentrations of Ca(2+) increased the amount of bound MtCK, indicating a physiological regulation of complex formation. No interaction of MtCK with reconstituted adenine nucleotide translocator was detectable in our experimental setup. The relevance of these findings for structure and function of mitochondrial contact sites is discussed.

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.

Amino acids, 40 (5), ISSN:0939-4451, ISSN:1438-2199

NM23-H4/NDPK-D forms symmetrical homohexameric complexes in the mitochondrial inter-membrane space. The well-established function of NM23-H4 is phosphotransfer activity as a nucleoside diphosphate kinase, using mitochondrial ATP to regenerate NTPs, especially GTP. NM23-H4 also strongly binds in vitro to anionic phospholipids, mainly cardiolipin (CL), and in vivo to the mitochondrial inner membrane (MIM). Membrane-binding seems to be important for close co-localization of NM23-H4 with mitochondrial OPA1, a dynamin-like GTPase, involved in fusion of MIM. NM23-H4/OPA1 association increases GTP-loading on OPA1. Like OPA1 loss-of-function, silencing of NM23-H4, but not cytosolic NM23-H1/H2, results in mitochondrial fragmentation, reflecting fusion defects. Thus, NM23-H4 interacts with and provides GTP to OPA1, similar to what is observed for cytosolic NM23 isoforms which interact with endocytic dynamin-1 and −2 and provide GTP for efficient dynamin-mediated endocytosis (Boissan et al.2014, Science 344:1510). Such close association allows these motor proteins to work with high thermodynamic efficiency. Earlier, we have shown that NM23-H4, when fully bound simultaneously to MIM and outer membrane (MOM), loses its kinase activity, but becomes competent to support intermembrane lipid transfer. This depends on the presence of the mitochondria-specific CL, and allows CL to move from its site of synthesis, MIM, to the opposed MOM (Schlattner et al.2013, JBC 288:111). Once CL is externalized at the mitochondrial surface, it can serve as a recognition signal for the autophageal machinery, leading to the elimination of damaged mitochondria. In cells treated with a protonophoric uncoupler, CCCP, CL externalization and mitophagy are stimulated only by transfection with NM23-H4 wild-type, but not R90D-mutant, incapable of CL binding. Similarly, in mouse lung epithelial cells, knocking-down NM23-H4 suppresses CL externalization and mitophagy. These findings suggest that NM23-H4 has dual functions in bioenergetics and lipid signaling leading to autophagy. Support: FRM,ARC,GEFLUC, NIH(U19AIO6802/HL114453), HFSP(RGP0013/2014).

Mitochondrial creatine kinase (MtCK) is a key enzyme for bioenergetics, membrane topology and possibly also for general organelle morphology. X-ray structural analysis (1), EM (2) and mutational studies with SPR (3,4) revealed that the large MtCK octamers bind to and “cross-link” mitochondrial membranes by their two identical top or bottom faces (5). These expose expose four C-terminal basic interaction motifs that interact mainly with acidic cardiolipin (4). This interaction induces cardiolipin-rich domains in the membrane (5,6). However, earlier data point to additional hydrophobic interactions (7,8). Using SPR, we have performed a thermodynamic analysis of the MtCK binding process. Main results were: (i) Affinity of the MtCK-cardiolipin interaction increases with temperature, pointing to a participation of hydrophobic interactions. (ii) Rate constants of two MtCK binding sites identified earlier differed in temperature-dependence. (iii) Thermodynamic parameters revealed that the gain in free energy of MtCK binding mainly depends on the contribution of entropy, possibly due to charge neutralization and release of bound water. These data are consistent with a two-phase model of rapid electrostatic docking of MtCK to cardiolipin, and slower anchoring via a C-terminal hydrophobic MtCK stretch. This would reinforce MtCK membrane interaction, allow integration of this bulky enzyme into the narrow mitochondrial intermembrane space, and contribute to its functional coupling with adenine nucleotide translocator.1. Eder M et al (2000) Proteins 39, 216.2. Schlattner et al (2000) Biol Chem 381, 1063.3. Schlattner et al (2000) J Biol Chem 275, 17314.4. Schlattner et al (2004) J Biol Chem 279, 24334.5. Schlattner et al (2009) Biochim Biophys Acta 1788, 2032.6. Epand et al (2007) J Mol Biol 365, 968.7. Rojo et al (1991) FEBS Lett 281, 123.8. Granjon et al (2001) Biochemistry 40, 6016.