Differential Expression of NME4 in Trophoblast Stem-Like Cells and Peripheral Blood Mononuclear Cells of Normal Pregnancy and Preeclampsia.

BACKGROUND: Preeclampsia (PE) is known to arise from insufficient trophoblast invasion as uterine spiral arteries lack remodeling. A significant reduction in placental perfusion induces an ischemic placental microenvironment due to reduced oxygen delivery to the placenta and fetus, leading to oxidative stress. Mitochondria are involved in the regulation of cellular metabolism and the production of reactive oxygen species (ROS). NME/NM23 nuceloside diphosphate kinase 4 (NME4) gene is known to have the ability to supply nucleotide triphosphate and deoxynucleotide triphosphate for replication and transcription of mitochondria. Our study aimed to investigate changes in NME4 expression in PE using trophoblast stem-like cells (TSLCs) from induced pluripotent stem cells (iPSCs) as a model of early pregnancy and peripheral blood mononuclear cells (PBMNCs) as a model of late preterm pregnancy. METHODS: Transcriptome analysis using TSLCs was performed to identify the candidate gene associated with the possible pathophysiology of PE. Then, the expression of NME4 associated with mitochondrial function, p53 associated with cell death, and thioredoxin (TRX) linked to ROS were investigated through qRT-PCR, western blotting and deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end labelling (TUNEL) assay. RESULTS: In patients with PE, NME4 was significantly downregulated in TSLCs but upregulated in PBMNCs. p53 was shown to be upregulated in TSLCs and PBMNCs of PE. In addition, western blot analysis confirmed that TRX expression had the tendency to increase in TSLCs of PE. Similarly, TUNEL analysis confirmed that the dead cells were higher in PE than in normal pregnancy. CONCLUSION: Our study showed that the expression of the NME4 differed between models of early and late preterm pregnancy of PE, and suggests that this expression pattern may be a potential biomarker for early diagnosis of PE.

The mitochondrially-localized nucleoside diphosphate kinase D (NME4) is a novel metastasis suppressor.

BACKGROUND: Mitochondrial nucleoside diphosphate kinase (NDPK-D, NME4, NM23-H4) is a multifunctional enzyme mainly localized in the intermembrane space, bound to the inner membrane. RESULTS: We constructed loss-of-function mutants of NDPK-D, lacking either NDP kinase activity or membrane interaction and expressed mutants or wild-type protein in cancer cells. In a complementary approach, we performed depletion of NDPK-D by RNA interference. Both loss-of-function mutations and NDPK-D depletion promoted epithelial-mesenchymal transition and increased migratory and invasive potential. Immunocompromised mice developed more metastases when injected with cells expressing mutant NDPK-D as compared to wild-type. This metastatic reprogramming is a consequence of mitochondrial alterations, including fragmentation and loss of mitochondria, a metabolic switch from respiration to glycolysis, increased ROS generation, and further metabolic changes in mitochondria, all of which can trigger pro-metastatic protein expression and signaling cascades. In human cancer, NME4 expression is negatively associated with markers of epithelial-mesenchymal transition and tumor aggressiveness and a good prognosis factor for beneficial clinical outcome. CONCLUSIONS: These data demonstrate NME4 as a novel metastasis suppressor gene, the first localizing to mitochondria, pointing to a role of mitochondria in metastatic dissemination.

A genome-wide screen uncovers multiple roles for mitochondrial nucleoside diphosphate kinase D in inflammasome activation.

Noncanonical inflammasome activation by cytosolic lipopolysaccharide (LPS) is a critical component of the host response to Gram-negative bacteria. Cytosolic LPS recognition in macrophages is preceded by a Toll-like receptor (TLR) priming signal required to induce transcription of inflammasome components and facilitate the metabolic reprograming that fuels the inflammatory response. Using a genome-scale arrayed siRNA screen to find inflammasome regulators in mouse macrophages, we identified the mitochondrial enzyme nucleoside diphosphate kinase D (NDPK-D) as a regulator of both noncanonical and canonical inflammasomes. NDPK-D was required for both mitochondrial DNA synthesis and cardiolipin exposure on the mitochondrial surface in response to inflammasome priming signals mediated by TLRs, and macrophages deficient in NDPK-D had multiple defects in LPS-induced inflammasome activation. In addition, NDPK-D was required for the recruitment of TNF receptor-associated factor 6 (TRAF6) to mitochondria, which was critical for reactive oxygen species (ROS) production and the metabolic reprogramming that supported the TLR-induced gene program. NDPK-D knockout mice were protected from LPS-induced shock, consistent with decreased ROS production and attenuated glycolytic commitment during priming. Our findings suggest that, in response to microbial challenge, NDPK-D-dependent TRAF6 mitochondrial recruitment triggers an energetic fitness checkpoint required to engage and maintain the transcriptional program necessary for inflammasome activation.

The let-7f-5p-Nme4 pathway mediates tumor necrosis factor α-induced impairment in osteogenesis of bone marrow-derived mesenchymal stem cells.

Although tumor necrosis factor α (TNF-α)-mediated inflammation significantly impacts osteoporosis, the mechanisms underlying the osteogenic differentiation defects of bone marrow-derived mesenchymal stem cells (BM-MSCs) caused by TNF-α remain poorly understood. We found that TNF-α stimulation of murine BM-MSCs significantly upregulated the expression levels of several microRNAs (miRNAs), including let-7f-5p, but this increase was significantly reversed by treatment with the kinase inhibitor BAY 11-7082. To study gain- or loss of function, we transfected cells with an miRNA inhibitor or miRNA mimic. We then demonstrated that let-7f-5p impaired osteogenic differentiation of BM-MSCs in the absence and presence of TNF-α, as evidenced by alkaline phosphatase and alizarin red staining as well as quantitative assays of the mRNA levels of bone formation marker genes in differentiated BM-MSCs. Moreover, let-7f-5p targets the 3' untranslated region of Nucleoside diphosphate kinase 4 (Nme4) mRNA and negatively regulates Nme4 expression in mouse BM-MSCs. Ectopic expression of Nme4 completely reversed the inhibitory effects of the let-7f-5p mimic on osteogenic differentiation of mouse BM-MSCs. Furthermore, inhibition of let-7f-5p or overexpression of Nme4 in BM-MSCs restored in-vivo bone formation in an ovariectomized animal model. Collectively, our work indicates that let-7f-5p is involved in TNF-α-mediated reduction of BM-MSC osteogenesis via targeting Nme4.

Emerging Molecular Connections between NM23 Proteins, Telomeres and Telomere-Associated Factors: Implications in Cancer Metastasis and Ageing.

The metastasis suppressor function of NM23 proteins is widely understood. Multiple enzymatic activities of NM23 proteins have also been identified. However, relatively less known interesting aspects are being revealed from recent developments that corroborate the telomeric interactions of NM23 proteins. Telomeres are known to regulate essential physiological events such as metastasis, ageing, and cellular differentiation via inter-connected signalling pathways. Here, we review the literature on the association of NM23 proteins with telomeres or telomere-related factors, and discuss the potential implications of emerging telomeric functions of NM23 proteins. Further understanding of these aspects might be instrumental in better understanding the metastasis suppressor functions of NM23 proteins.

Widely targeted metabolomic analyses unveil the metabolic variations after stable knock-down of NME4 in esophageal squamous cell carcinoma cells.

NME4, also designated nm23-H4 or NDPK-D, has been known for years for its well-established roles in the synthesis of nucleoside triphosphates, though; little has been known regarding the differential metabolites involved as well as the biological roles NME4 plays in proliferation and invasion of esophageal squamous cell carcinoma (ESCC) cells. To understand the biological roles of NME4 in ESCC cells, lentiviral-based short hairpin RNA interference (shRNA) vectors were constructed and used to stably knock down NME4. Then, the proliferative and invasive variations were assessed using MTT, Colony formation and Transwell assays. To understand the metabolites involved after silencing of NME4 in ESCC cells, widely targeted metabolomic screening was taken. It was discovered that silencing of NME4 can profoundly suppress the proliferation and invasion in ESCC cells in vitro. Metabolically, a total of 11 differential metabolites were screened. KEGG analyses revealed that Tryptophan, Riboflavin, Purine, Nicotinate, lysine degradation, and Linoleic acid metabolism were also involved in addition to the well-established nucleotides metabolism. Some of these differential metabolites, say, 2-Picolinic Acid, Nicotinic Acid and Pipecolinic Acid were suggested to be associated with tumor immunomodulation. The data we described here support the idea that metabolisms occurred in mitochondrial was closely related to tumor immunity.

NME4 modulates PD-L1 expression via the STAT3 signaling pathway in squamous cell carcinoma.

NME4, also named Nm23-H4, is a contraction of NME/NM23 Nucleoside Diphosphate Kinase 4, whose major role is the synthesis of nucleoside triphosphates. However, its association with programmed death ligand 1 (PD-L1) remains far from understood. Herein, it was discovered that silencing NME4 can lead to the marked downregulation of PD-L1, with phosphorylated STAT3 at the 705th serine being inactivated in vitro in esophageal squamous cell carcinoma (ESCC) cell lines. To further validate the association between NME4 and PD-L1 that was observed in cell lines, Pearson correlation analysis was performed on the data regarding the transcriptomic RNA sequencing of NME4 and PD-L1 in cervical squamous cell carcinoma (CSCC), which pathologically highly resembles ESCC in terms of tumor origin, obtained from the GEPIA database. It was demonstrated that their correlation was significant but negative between NME4 and PD-L1 in CSCC. To the best of our knowledge, this is the first report describing a modulation exerted by NME4 over PD-L1 in the background of squamous cell carcinoma, strongly suggestive of the underlying role of NME4 working to exclude CD8 T cells from infiltrating into the squamous cell carcinoma microenvironment.

Genome-wide In Vivo CNS Screening Identifies Genes that Modify CNS Neuronal Survival and mHTT Toxicity.

Unbiased in vivo genome-wide genetic screening is a powerful approach to elucidate new molecular mechanisms, but such screening has not been possible to perform in the mammalian central nervous system (CNS). Here, we report the results of the first genome-wide genetic screens in the CNS using both short hairpin RNA (shRNA) and CRISPR libraries. Our screens identify many classes of CNS neuronal essential genes and demonstrate that CNS neurons are particularly sensitive not only to perturbations to synaptic processes but also autophagy, proteostasis, mRNA processing, and mitochondrial function. These results reveal a molecular logic for the common implication of these pathways across multiple neurodegenerative diseases. To further identify disease-relevant genetic modifiers, we applied our screening approach to two mouse models of Huntington's disease (HD). Top mutant huntingtin toxicity modifier genes included several Nme genes and several genes involved in methylation-dependent chromatin silencing and dopamine signaling, results that reveal new HD therapeutic target pathways.

NME4 may enhance non­small cell lung cancer progression by overcoming cell cycle arrest and promoting cellular proliferation.

Nucleoside diphosphate kinase 4 (NME4) is abnormally expressed in a variety of cancer types. However, the function of the NME4 gene in non­small cell lung cancer (NSCLC) remains to be elucidated. In order to investigate the role of NME4 in NSCLC, the present study detected the expression of the NME4 gene in the Cancer Genome Atlas database, and in BEAS­2B, NCI­H1299 and A549 cell lines. NME4 was significantly overexpressed in NSCLC tissues and NSCLC cell lines. Furthermore, lentivirus­mediated knockdown vector infection, cell proliferation, cell cycle, apoptosis, colony formation and MTT assays were conducted to explore the effect of NME4 on NSCLC in vitro. After knockdown of NME4 with short hairpin RNA, the cell cycle was arrest at the G1 phase, and proliferation and colony formation were inhibited in the NCI­H1299 and A549 cell lines. The present results suggested that NME4 may serve as a novel tumor promoter, capable of enhancing NSCLC progression by overcoming cell cycle arrest and promoting proliferation.

The mitochondrial nucleoside diphosphate kinase (NDPK-D/NME4), a moonlighting protein for cell homeostasis.

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.

NME4/nucleoside diphosphate kinase D in cardiolipin signaling and mitophagy.

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.

NDPK-D (NM23-H4)-mediated externalization of cardiolipin enables elimination of depolarized mitochondria by mitophagy.

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.

Acetylation of NDPK-D Regulates Its Subcellular Localization and Cell Survival.

Nucleoside diphosphate kinases (NDPK) are ubiquitous enzymes that catalyze the reversible phosphotransfer of γ-phosphates between di- and triphosphonucleosides. NDPK-D (Nm23-H4) is the only member of the NDPK family with a mitochondrial targeting sequence. Despite the high expression of NDPK-D in the developing central nervous system, its function remains to be determined. In this study, we show that NDPK-D knockdown induces apoptosis in neuroblastoma cells as well as in mouse cortex, suggesting that NDPK-D is required for neuronal survival. We identified NDPK-D as a binding partner of NAD+-dependent histone deacetylase, SIRT1, by yeast two-hybrid screening. NDPK-D co-localized with SIRT1, and the association of these molecules was confirmed by co-immunoprecipitation. Inhibition of SIRT1 increases the acetylation of NDPK-D. Overexpression of NDPK-D along with SIRT1, or mutation in the acetylated lysine residues in NDPK-D, increases its nuclear accumulation. Furthermore, the NDPK-D acetylation-mimic mutant increased apoptosis in N1E-115 cells. Our data demonstrate that acetylation regulates the shuttling of NDPK-D between nucleus and cytoplasm, and increased acetylation of NDPK-D causes apoptosis.

Mitochondrial NM23-H4/NDPK-D: a bifunctional nanoswitch for bioenergetics and lipid signaling.

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.

OncomiR-196 promotes an invasive phenotype in oral cancer through the NME4-JNK-TIMP1-MMP signaling pathway.

BACKGROUND: MicroRNA-196 (miR-196), which is highly up-regulated in oral cancer cells, has been reported to be aberrantly expressed in several cancers; however, the significance of miR-196 in oral cancer has not yet been addressed. METHODS: Cellular functions in response to miR-196 modulation were examined, including cell growth, migration, invasion and radio/chemosensitivity. Algorithm-based studies were used to identify the regulatory target of miR-196. The miR-196 target gene and downstream molecular mechanisms were confirmed by RT-qPCR, western blot, luciferase reporter and confocal microscopy analyses. miR-196 expression was determined in paired cancer and adjacent normal tissues from oral cancer patients. RESULTS: Both miR-196a and miR-196b were highly over-expressed in the cancer tissue and correlated with lymph node metastasis (P = 0.001 and P = 0.006, respectively). Functionally, miR-196 actively promoted cell migration and invasion without affecting cell growth. Mechanistically, miR-196 performed it's their function by inhibiting NME4 expression and further activating p-JNK, suppressing TIMP1, and augmenting MMP1/9. CONCLUSION: miR-196 contributes to oral cancer by promoting cell migration and invasion. Clinically, miR-196a/b was significantly over-expressed in the cancer tissues and correlated with lymph node metastasis. Thus, our findings provide new knowledge of the underlying mechanism of cancer metastasis. miR-196 may serve as a promising marker for better oral cancer management.

Structural comparison of highly similar nucleoside-diphosphate kinases: Molecular explanation of distinct membrane-binding behavior.

NDPK-A, NDPK-B and NDPK-D are three enzymes which belong to the NDPK group I isoforms and are not only involved in metabolism process but also in transcriptional regulation, DNA cleavage, histidine protein kinase activity and metastasis development. Those enzymes were reported to bind to membranes either in mitochondria where NDPK-D influences cardiolipin lateral organization and is thought to be involved in apoptotic pathway or in cytosol where NDPK-A and NDPK-B membrane association was shown to influence several cellular processes like endocytosis, cellular adhesion, ion transport, etc. However, despite numerous studies, the role of NDPK-membrane association and the molecular details of the binding process are still elusive. In the present work, a comparative study of the three NDPK isoforms allowed us to show that although membrane binding is a common feature of these enzymes, mechanisms differ at the molecular scale. NDPK-A was not able to bind to model membranes mimicking the inner leaflet of plasma membrane, suggesting that its in vivo membrane association is mediated by a non-lipidic partner or other partners than the studied phospholipids. On the contrary, NDPK-B and NDPK-D were shown to bind efficiently to liposomes mimicking plasma membrane and mitochondrial inner membrane respectively but details of the binding mechanism differ between the two enzymes as NDPK-B binding necessarily involved an anionic phospholipid partner while NDPK-D can bind either zwitterionic or anionic phospholipids. Although sharing similar secondary structure and homohexameric quaternary arrangement, tryptophan fluorescence revealed fine disparities in NDPK tertiary structures. Interfacial behavior as well as ANS fluorescence showed further dissimilarities between NDPK isoforms, notably the presence of distinct accessible hydrophobic areas as well as different capacity to form Gibbs monolayers related to their surface activity properties. Those distinct features may contribute to explain the differences in the protein behavior towards membrane binding.

Membrane trafficking. Nucleoside diphosphate kinases fuel dynamin superfamily proteins with GTP for membrane remodeling.

Dynamin superfamily molecular motors use guanosine triphosphate (GTP) as a source of energy for membrane-remodeling events. We found that knockdown of nucleoside diphosphate kinases (NDPKs) NM23-H1/H2, which produce GTP through adenosine triphosphate (ATP)-driven conversion of guanosine diphosphate (GDP), inhibited dynamin-mediated endocytosis. NM23-H1/H2 localized at clathrin-coated pits and interacted with the proline-rich domain of dynamin. In vitro, NM23-H1/H2 were recruited to dynamin-induced tubules, stimulated GTP-loading on dynamin, and triggered fission in the presence of ATP and GDP. NM23-H4, a mitochondria-specific NDPK, colocalized with mitochondrial dynamin-like OPA1 involved in mitochondria inner membrane fusion and increased GTP-loading on OPA1. Like OPA1 loss of function, silencing of NM23-H4 but not NM23-H1/H2 resulted in mitochondrial fragmentation, reflecting fusion defects. Thus, NDPKs interact with and provide GTP to dynamins, allowing these motor proteins to work with high thermodynamic efficiency.

Analysis of sequence variation underlying tissue-specific transcription factor binding and gene expression.

Although mutations causing monogenic disorders most frequently lie within the affected gene, sequence variation in complex disorders is more commonly found in noncoding regions. Furthermore, recent genome- wide studies have shown that common DNA sequence variants in noncoding regions are associated with "normal" variation in gene expression resulting in cell-specific and/or allele-specific differences. The mechanism by which such sequence variation causes changes in gene expression is largely unknown. We have addressed this by studying natural variation in the binding of key transcription factors (TFs) in the well-defined, purified cell system of erythropoiesis. We have shown that common polymorphisms frequently directly perturb the binding sites of key TFs, and detailed analysis shows how this causes considerable (~10-fold) changes in expression from a single allele in a tissue-specific manner. We also show how a SNP, located at some distance from the recognized TF binding site, may affect the recruitment of a large multiprotein complex and alter the associated chromatin modification of the variant regulatory element. This study illustrates the principles by which common sequence variation may cause changes in tissue-specific gene expression, and suggests that such variation may underlie an individual's propensity to develop complex human genetic diseases.

New insights into lipid-Nucleoside Diphosphate Kinase-D interaction mechanism: protein structural changes and membrane reorganisation.

Nucleoside Diphosphate Kinases (NDPKs) have long been considered merely as housekeeping enzymes. The discovery of the NME1 gene, an anti-metastatic gene coding for NDPK-A, led the scientific community to re-evaluate their role in the cell. It is now well established that the NDPK family is more complex than what was first thought, and despite the increasing amount of evidence suggesting the multifunctional role of nm23/NDPKs, the specific functions of each family member are still elusive. Among these isoforms, NDPK-D is the only one to present a mitochondria-targeting sequence. It has recently been shown that this protein is able to bind and cross-link with mitochondrial membranes, suggesting that NDPK-D can mediate contact sites and contributes to the mitochondrial intermembrane space structuring. To better understand the influence of NDPK-D on mitochondrial lipid organisation, we analysed its behaviour in different lipid environments. We found that NDPK-D not only interacts with CL or anionic lipids, but is also able to bind in a non negligible manner to zwitterionic PC. NDPK-D alters membrane organisation in terms of fluidity, hydration and lipid clustering, effects which depend on lipid structure. Changes in the protein structure after lipid binding were evidenced, both by fluorescence and infrared spectroscopy, regardless of membrane composition. Taking into account all these elements, a putative mechanism of interaction between NDPK-D and zwitterionic or anionic lipids was proposed.

Dual function of mitochondrial Nm23-H4 protein in phosphotransfer and intermembrane lipid transfer: a cardiolipin-dependent switch.

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.