ABSTRACT
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.
Subject(s)
Neoplasms , Nucleoside-Diphosphate Kinase , Animals , Intracellular Membranes , Mice , Mitochondria , NM23 Nucleoside Diphosphate Kinases/genetics , NM23 Nucleoside Diphosphate Kinases/metabolism , Neoplasms/genetics , Neoplasms/metabolism , Nucleoside Diphosphate Kinase D/metabolism , Nucleoside-Diphosphate Kinase/genetics , Nucleoside-Diphosphate Kinase/metabolismABSTRACT
Membrane Type 1 Matrix Metalloprotease (MT1-MMP) contributes to the invasive progression of breast cancers by degrading extracellular matrix tissues. Nucleoside diphosphate kinase, NME1/NM23-H1, has been identified as a metastasis suppressor; however, its contribution to local invasion in breast cancer is not known. Here, we report that NME1 is up-regulated in ductal carcinoma in situ (DCIS) as compared to normal breast epithelial tissues. NME1 levels drop in microinvasive and invasive components of breast tumor cells relative to synchronous DCIS foci. We find a strong anti-correlation between NME1 and plasma membrane MT1-MMP levels in the invasive components of breast tumors, particularly in aggressive histological grade III and triple-negative breast cancers. Knockout of NME1 accelerates the invasive transition of breast tumors in the intraductal xenograft model. At the mechanistic level, we find that MT1-MMP, NME1 and dynamin-2, a GTPase known to require GTP production by NME1 for its membrane fission activity in the endocytic pathway, interact in clathrin-coated vesicles at the plasma membrane. Loss of NME1 function increases MT1-MMP surface levels by inhibiting endocytic clearance. As a consequence, the ECM degradation and invasive potentials of breast cancer cells are enhanced. This study identifies the down-modulation of NME1 as a potent driver of the in situ-to invasive transition during breast cancer progression.
Subject(s)
Breast Neoplasms/metabolism , Dynamin II/metabolism , Extracellular Matrix/metabolism , Matrix Metalloproteinase 14/metabolism , NM23 Nucleoside Diphosphate Kinases/metabolism , Animals , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Line , Cell Movement/physiology , Female , Humans , Matrix Metalloproteinase 14/genetics , Mice , Mice, Nude , Middle Aged , Neoplasm Metastasis , Neoplasm Staging , Xenograft Model Antitumor AssaysABSTRACT
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.
Subject(s)
Homeostasis , Nucleoside Diphosphate Kinase D/physiology , Animals , Apoptosis , Humans , Mitophagy , Neoplasms/pathology , Nucleoside Diphosphate Kinase D/analysis , Nucleoside Diphosphate Kinase D/chemistry , Nucleoside Diphosphate Kinase D/genetics , Phospholipids/chemistryABSTRACT
Nucleoside diphosphate kinases (NDPK) are nucleotide metabolism enzymes encoded by NME genes (also called NM23). Given the fact that not all NME-encoded proteins are catalytically active NDPKs and that NM23 generally refers to clinical studies on metastasis, we use here NME/NDPK to denote the proteins. Since their discovery in the 1950's, NMEs/NDPKs have been shown to be involved in multiple physiological and pathological cellular processes, but the molecular mechanisms have not been fully determined. Recent progress in elucidating these underlying mechanisms has been presented by experts in the field at the 10th International Congress on the NDPK/NME/AWD protein family in October 2016 in Dubrovnik, Croatia, and is summarized in review articles or original research in this and an upcoming issue of Laboratory Investigation. Within this editorial, we discuss three major cellular processes that involve members of the multi-functional NME/NDPK family: (i) cancer and metastasis dissemination, (ii) membrane remodeling and nucleotide channeling, and iii) protein histidine phosphorylation.
Subject(s)
Multigene Family , Nucleoside-Diphosphate Kinase/genetics , Animals , Humans , Isoenzymes/genetics , Isoenzymes/metabolism , Neoplasm Metastasis/genetics , Neoplasms/enzymology , Neoplasms/genetics , Neoplasms/pathology , Nucleoside-Diphosphate Kinase/metabolism , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolismABSTRACT
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.
Subject(s)
Cardiolipins/metabolism , Mitophagy , Nucleoside Diphosphate Kinase D/metabolism , Signal Transduction , Animals , Apoptosis , Humans , Mitochondrial Membranes/metabolism , Models, Biological , Protein BindingABSTRACT
NME1 (nonmetastatic expressed 1) gene, which encodes nucleoside diphosphate kinase (NDPK) A [also known as nonmetastatic clone 23 (NM23)-H1 in humans and NM23-M1 in mice], is a suppressor of metastasis, but several lines of evidence-mostly from plants-also implicate it in the regulation of the oxidative stress response. Here, our aim was to investigate the physiologic relevance of NDPK A with respect to the oxidative stress response in mammals and to study its molecular basis. NME1-knockout mice died sooner, suffered greater hepatocyte injury, and had lower superoxide dismutase activity than did wild-type (WT) mice in response to paraquat-induced acute oxidative stress. Deletion of NME1 reduced total NDPK activity and exacerbated activation of the stress-related MAPK, JNK, in the liver in response to paraquat. In a mouse transformed hepatocyte cell line and in primary cultures of normal human keratinocytes, MAPK activation in response to H2O2 and UVB, respectively, was dampened by expression of NM23-M1/NM23-H1, dependent on its NDPK catalytic activity. Furthermore, excess or depletion of NM23-M1/NM23-H1 NDPK activity did not affect the intracellular bulk concentration of nucleoside di- and triphosphates. NME1-deficient mouse embryo fibroblasts grew poorly in culture, were more sensitive to stress than WT fibroblasts, and did not immortalize, which suggested that they senesce earlier than do WT fibroblasts. Collectively, these results indicate that the NDPK activity of NM23-M1/NM23-H1 protects cells from acute oxidative stress by inhibiting activation of JNK in mammal models.-Peuchant, E., Bats, M.-L., Moranvillier, I., Lepoivre, M., Guitton, J., Wendum, D., Lacombe, M.-L., Moreau-Gaudry, F., Boissan, M., Dabernat, S. Metastasis suppressor NM23 limits oxidative stress in mammals by preventing activation of stress-activated protein kinases/JNKs through its nucleoside diphosphate kinase activity.
Subject(s)
MAP Kinase Signaling System , NM23 Nucleoside Diphosphate Kinases/genetics , Oxidative Stress , Animals , Cell Line , Cells, Cultured , Fibroblasts/metabolism , Gene Deletion , Hepatocytes/metabolism , Humans , Keratinocytes/metabolism , Liver/drug effects , Liver/metabolism , Male , Mice , Mice, Inbred C57BL , NM23 Nucleoside Diphosphate Kinases/metabolism , Paraquat/toxicityABSTRACT
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.
Subject(s)
Lipid Metabolism , Nucleoside Diphosphate Kinase D/metabolism , Apoptosis , Cardiolipins/metabolism , Energy Metabolism , Humans , Mitochondria/metabolism , Nucleoside Diphosphate Kinase D/chemistryABSTRACT
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.
Subject(s)
Cell Membrane/metabolism , Dynamins/metabolism , Guanosine Triphosphate/metabolism , NM23 Nucleoside Diphosphate Kinases/metabolism , Adenosine Triphosphate/metabolism , Animals , Cell Line , Coated Pits, Cell-Membrane/metabolism , Endocytosis , GTP Phosphohydrolases/metabolism , Guanosine Diphosphate/metabolism , Humans , Intracellular Membranes/metabolism , Membrane Fusion , Mitochondria/metabolism , NM23 Nucleoside Diphosphate Kinases/genetics , Nucleoside Diphosphate Kinase D/metabolismABSTRACT
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.
Subject(s)
Cardiolipins/metabolism , Carrier Proteins/metabolism , Intracellular Membranes/metabolism , Mitochondria/metabolism , Biological Transport , Mitochondrial Membranes/metabolismABSTRACT
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.
Subject(s)
Cardiolipins/physiology , Intracellular Membranes/metabolism , Lipids/chemistry , Nucleoside Diphosphate Kinase D/chemistry , Nucleoside Diphosphate Kinase D/physiology , Animals , Apoptosis , Cardiolipins/chemistry , GTP Phosphohydrolases/chemistry , Lipid Metabolism , Liver/metabolism , Male , Models, Molecular , Phospholipids/chemistry , Protein Binding , Protein Conformation , Rats , Rats, WistarABSTRACT
Metastasis suppressor genes - unlike tumor suppressor genes - are defined by their capacity to control metastatic dissemination in vivo without affecting growth of the primary tumor. The first of these metastasis suppressor genes, NM23, was identified in 1988. Since then, expression of NM23 has been studied widely in human tumor cohorts, often with contradictory results. Not only is NM23 overexpressed in most human solid tumors when compared to healthy tissues, but also low expression of NM23 correlates with metastasis and poor clinical prognosis in the advanced stages of a number of epithelial cancer types, including melanoma, breast, colon, and liver carcinoma. This does not hold true, however, for other cancer types such as neuroblastoma and hematological malignancies, in which high NM23 expression correlates with more aggressive disease. Genetic alterations in the NM23 gene - loss of heterozygosity, spontaneous mutations and polymorphisms - are rarely found in tumors; thus, the metastatic potential of tumor cells is probably affected by NM23 protein levels. Three lines of evidence demonstrate the anti-metastatic activity of NM23: first, overexpression of NM23 in metastatic cell lines reduces their metastatic potential in xenograft models; second, the incidence of lung metastases is elevated in NM23 knockout mice prone to develop hepatocellular carcinoma, and, third, silencing NM23 by RNA interference confers a "metastatic phenotype" on non-invasive human epithelial liver and colon cancer cell lines. It appears that NM23 is crucial for inhibiting invasive migration, so acting at early stages of metastatic dissemination. The mechanistic basis of the metastasis suppressor function of NM23 and its regulated expression still remains obscure, however. Reactivation of expression of the endogenous NM23 gene in tumor cells, or stimulation of the pathways it controls, constitutes a promising avenue for anti-metastatic therapy.
Subject(s)
Genes, Tumor Suppressor/physiology , NM23 Nucleoside Diphosphate Kinases/genetics , Neoplasm Metastasis/genetics , Carrier Proteins/physiology , Cell Adhesion/physiology , Endocytosis/genetics , Extracellular Matrix/physiology , GTP-Binding Proteins/physiology , Gene Expression Regulation, Neoplastic , Humans , Intercellular Junctions/genetics , Intercellular Junctions/physiology , NM23 Nucleoside Diphosphate Kinases/metabolism , Neoplasm Invasiveness/genetics , Phosphoric Monoester HydrolasesABSTRACT
Development of hepatocellular carcinoma (HCC) is a complex and progressive disease that involves cycles of liver cell death, inflammation, and tissue regeneration/remodelling. Chemokines and chemokine receptors play numerous and integral roles in the disease progression of HCC. Here we investigated the novel chemokine receptor CXCR7/RDC1 in HCC progression, its two known ligands CXCL12 and CXCL11, as well as the other CXCL12 receptor, CXCR4. Our results show that in a cohort of 408 human HCCs, CXCR7 and CXCL11 were significantly higher in tumours compared to normal liver controls (5- and 10-fold, respectively). Immunohistochemical (IHC) staining on human HCC sections confirmed that both CXCL11 and CXCR7 were much higher in cancer tissues. Furthermore, IHC staining revealed that CXCR7 protein was only expressed in endothelial cells whereas CXCL11 exhibited a much broader tissue expression. At the cellular level we observed that in vitro, human microvascular endothelial cells (HMEC-1) up-regulated CXCR7 under hypoxic and acidic pH conditions, which are well known characteristics of the HCC tumour micro-environment. As for its ligand, we observed that IFNγ robustly induced CXCL11 in hepatic stellate cells, hepatocytes, and HMEC-1s. In addition, in the mouse Diethylnitrosamine model of hepatocarcinogenesis we observed a very strong induction of CXCR7 and CXCL11 transcripts, confirming that CXCR7/CXCL11 up-regulation is conserved between human and mice liver cancer. Altogether, our results strongly support the hypothesis that the CXCL11/CXCR7 pathway is involved HCC progression.
Subject(s)
Carcinoma, Hepatocellular/genetics , Endothelial Cells/metabolism , Liver Neoplasms/genetics , Receptors, CXCR/genetics , Aged , Animals , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Disease Progression , Endothelial Cells/pathology , Female , Gene Expression Regulation, Neoplastic , Humans , Jurkat Cells , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Male , Mice , Mice, Inbred C57BL , Middle Aged , Organ Specificity/genetics , Receptors, CXCR/metabolism , Tumor Cells, CulturedABSTRACT
Mitotic kinesin-like protein 2 (MKlp2), a microtubule-associated motor, is required during mitosis exit for the final step of cytokinesis. It also contributes to retrograde vesicular trafficking from the Golgi apparatus to the endoplasmic reticulum in interphase. The KIF20A gene encoding MKlp2 is controlled by the E2F-retinoblastoma protein-p16 pathway, and its widely expressed mRNA is found in fetal and proliferating adult tissues. The expression pattern and function of MKlp2 in the adult liver, however, have not been investigated. We report herein that MKlp2 transiently accumulates in vivo during mouse liver regeneration after partial hepatectomy and is strongly overexpressed in preneoplastic and neoplastic mouse liver. In vitro in mitogen-stimulated primary hepatocytes, MKlp2 accumulated in the nucleus during the G2 phase of the cell cycle coincident with the mitotic kinase Aurora B. Human hepatoma cell lines exhibited high levels of MKlp2; however, it was undetectable in normal human hepatocytes. RNAi-mediated MKlp2 knockdown in hepatoma cells induced polyploidization consistent with its essential function in promoting cytokinesis and inhibited cell proliferation without inducing apoptosis. KIF20A mRNA was strongly accumulated in a large series of human hepatocellular carcinomas, with the highest expression observed in tumors with genomic instability. Accumulation of MKlp2 in normal proliferating, preneoplastic, and transformed hepatocytes suggests that MKlp2 contributes to both normal and pathologic hepatocyte proliferation and is linked to tumor aggressiveness in human hepatocellular carcinomas.
Subject(s)
Carcinoma, Hepatocellular/metabolism , Cell Transformation, Neoplastic/metabolism , Kinesins/metabolism , Liver Neoplasms/metabolism , Liver Regeneration/physiology , Precancerous Conditions/metabolism , Aged , Animals , Carcinoma, Hepatocellular/pathology , Cell Cycle/physiology , Cell Line, Tumor , Female , Hepatocytes/metabolism , Hepatocytes/pathology , Humans , Ki-67 Antigen/metabolism , Liver Neoplasms/pathology , Male , Mice , Mice, Inbred C57BL , Middle Aged , Mitogens/pharmacology , Precancerous Conditions/pathology , Tumor Cells, CulturedABSTRACT
The human NME gene family (also known as NM23) comprises ten genes that are involved in diverse physiological and pathological processes including proliferation, differentiation, development, ciliary functions, and metastasis. For the moment, only the NME1, NME2, and NME7 genes have been inactivated in transgenic knockout mice, as well as a double NME1-NME2 gene knockout. Mice lacking NME1 or NME2 grow to adulthood without health problems, although NME1 (-/-) mice have modest growth retardation. Double knockout NME1 (-/-)-NME2 (-/-) mice, by contrast, are highly hypotrophic and die at birth from profound anemia due to impaired erythroblast development. Evidence for a metastasis suppressor function of NME1 in vivo comes from crossing NME1 (-/-) mice with mice prone to develop hepatocellular carcinoma; the double transgenic mice present a higher incidence of lung metastases. Silencing of NME1 by siRNA interference has confirmed this function by conferring a "metastatic phenotype" on non-invasive human epithelial cancer cell lines. This function is specific to NME1 and is not observed when the NME2 is silenced. The data indicate that NME1 loss is causally involved at the early stages of the metastatic cascade. NME2 (-/-) mice and NME2 silencing experiments reveal a specific role of NME2 in activation of heterotrimeric G proteins and of KCa3.1 channel in T cells, pointing to a role of NME2 as a histidine phosphotransferase. Regarding NME7, consistent with its expression in axonemal structures, NME7 (-/-) mice present lesions similar to primary ciliary dyskinesia. This review summarizes the recent data obtained by knockout and silencing of NME/NM23 genes that provide mechanistic insights into their respective roles in physiology and pathology.
Subject(s)
Gene Silencing , Models, Genetic , NM23 Nucleoside Diphosphate Kinases/genetics , NM23 Nucleoside Diphosphate Kinases/physiology , Animals , Cell Line, Tumor , Humans , Mice , Mice, Knockout , Neoplasm Metastasis , Neoplasms/enzymology , Neoplasms/genetics , Neoplasms/pathology , Protein SubunitsABSTRACT
NME/NDPK family proteins are involved in the control of intracellular nucleotide homeostasis as well as in both physiological and pathological cellular processes, such as proliferation, differentiation, development, apoptosis, and metastasis dissemination, through mechanisms still largely unknown. One family member, NME1/NDPK-A, is a metastasis suppressor, yet the primary physiological functions of this protein are still missing. The purpose of this study was to identify new NME1/NDPK-A-dependent biological functions and pathways regulated by this gene in the liver. We analyzed the proteomes of wild-type and transgenic NME1-null mouse livers by combining two-dimensional gel electrophoresis and mass spectrometry (matrix-assisted laser desorption/ionization time of flight and liquid chromatography-tandem mass spectrometry). We found that the levels of three proteins, namely, phenylalanine hydroxylase, annexin IV, and elongation factor 1 Bα (EF-1Bα), were strongly reduced in the cytosolic fraction of NME1(-/-) mouse livers when compared to the wild type. This was confirmed by immunoblotting analysis. No concomitant reduction in the corresponding messenger RNAs or of total protein level was observed, however, suggesting that NME1 controls annexin IV and EF-1Bα amounts by post-translational mechanisms. NME1 deletion induced a change in the subcellular location of annexin IV in hepatocytes resulting in enrichment of this protein at the plasma membrane. We also observed a redistribution of EF-1Bα in NME1(-/-) hepatocytes to an intracytoplasmic compartment that colocalized with a marker of the reticulum endoplasmic. Finally, we found reduced expression of annexin IV coincident with decreased NME1 expression in a panel of different carcinoma cell lines. Taken together, our data suggest for the first time that NME1 might regulate the subcellular trafficking of annexin IV and EF-1Bα. The potential role of these proteins in metastatic dissemination is discussed.
Subject(s)
Annexin A4/metabolism , Liver/enzymology , NM23 Nucleoside Diphosphate Kinases/physiology , Peptide Elongation Factor 1/genetics , Peptide Elongation Factor 1/metabolism , Protein Processing, Post-Translational , Animals , Annexin A4/genetics , Blotting, Western , Cell Line, Tumor , Cytosol/enzymology , Cytosol/metabolism , Electrophoresis, Gel, Two-Dimensional , Endoplasmic Reticulum/enzymology , Endoplasmic Reticulum/metabolism , Humans , Liver/metabolism , Male , Mice , Mice, Knockout , NM23 Nucleoside Diphosphate Kinases/genetics , Protein Transport , Proteomics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Tandem Mass SpectrometryABSTRACT
The human nucleoside diphosphate (NDP) kinase A, product of the NME1 gene also named NM23-H1, is known as a metastasis suppressor protein. A naturally occurring variant, S120G, identified in neuroblastomas, possesses native three-dimensional structure and enzymatic activity but displays reduced conformational stability and a folding defect with the accumulation of a "molten globule" folding intermediate during refolding in vitro. As such intermediate has been postulated to be involved in amyloid formation, NDP kinase A may serve as a model protein for studying the relationship between folding intermediates and amyloid fibrils. The NDP kinase A S120G was heated in phosphate buffer (pH 7.0). The protein precipitated as amyloid fibrils, as demonstrated by electron microscopy, Congo red, and thioflavin T binding and FTIR spectroscopy. The NDP kinase A S120G, at neutral pH and at moderate temperature experiences a transition towards amyloid fibrils. The aggregation process was faster if seeded by preformed fibrils. The fibrils presented a large proteinase K-resistant core not including residue Gly 120, as shown by mass spectrometry. This suggests that the aggregation process is triggered by the reduced stability of the S120G variant and not by a specific increase in the kinase domain intrinsic aggregation propensity at the place of mutation. This constitutes one of the few reports on a protein involved in cancer biology able to aggregate into amyloid structures under mild conditions.
Subject(s)
Amyloid/chemistry , Amyloid/genetics , Mutation , NM23 Nucleoside Diphosphate Kinases/chemistry , NM23 Nucleoside Diphosphate Kinases/genetics , Neuroblastoma/genetics , Circular Dichroism , Electrophoresis, Polyacrylamide Gel , Humans , Microscopy, Electron, Transmission , Protein Conformation , Protein Folding , Protein Stability , Spectroscopy, Fourier Transform Infrared/methods , TemperatureABSTRACT
Loss of NM23-H1 expression correlates with the degree of metastasis and with unfavorable clinical prognosis in several types of human carcinoma. However, the mechanistic basis for the metastasis suppressor function of NM23-H1 is obscure. We silenced NM23-H1 expression in human hepatoma and colon carcinoma cells and methodologically investigated effects on cell-cell adhesion, migration, invasion, and signaling linked to cancer progression. NM23-H1 silencing disrupted cell-cell adhesion mediated by E-cadherin, resulting in ß-catenin nuclear translocation and T-cell factor/lymphoid-enhancing factor-1 transactivation. Further, NM23-H1 silencing promoted cellular scattering, motility, and extracellular matrix invasion by promoting invadopodia formation and upregulating several matrix metalloproteinases (MMP), including membrane type 1 MMP. In contrast, silencing the related NM23-H2 gene was ineffective at promoting invasion. NM23-H1 silencing activated proinvasive signaling pathways involving Rac1, mitogen-activated protein kinases, phosphatidylinositol 3-kinase (PI3K)/Akt, and src kinase. Conversely, NM23-H1 was dispensable for cancer cell proliferation in vitro and liver regeneration in NM23-M1 null mice, instead inducing cellular resistance to chemotherapeutic drugs in vitro. Analysis of NM23-H1 expression in clinical specimens revealed high expression in premalignant lesions (liver cirrhosis and colon adenoma) and the central body of primary liver or colon tumors, but downregulation at the invasive front of tumors. Our findings reveal that NM23-H1 is critical for control of cell-cell adhesion and cell migration at early stages of the invasive program in epithelial cancers, orchestrating a barrier against conversion of in situ carcinoma into invasive malignancy.
Subject(s)
Adherens Junctions/genetics , NM23 Nucleoside Diphosphate Kinases/genetics , Neoplasms/genetics , Actins/metabolism , Adenocarcinoma/genetics , Adenocarcinoma/metabolism , Adenocarcinoma/pathology , Adherens Junctions/metabolism , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Cell Movement/genetics , Colonic Neoplasms/genetics , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Cytoskeleton/metabolism , Cytoskeleton/pathology , Gene Silencing , Humans , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Matrix Metalloproteinase 14/metabolism , NM23 Nucleoside Diphosphate Kinases/biosynthesis , Neoplasm Invasiveness , Neoplasms/metabolism , Neoplasms/pathology , Wnt Proteins/metabolismABSTRACT
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.
Subject(s)
Cardiolipins/metabolism , Creatine Kinase, Mitochondrial Form/metabolism , Mitochondria/metabolism , Nucleoside-Diphosphate Kinase/metabolism , Animals , HumansABSTRACT
Nucleoside diphosphate kinases (NDPK) are encoded by the NME genes, also called NM23. They catalyze the transfer of gamma-phosphate from nucleoside triphosphates to nucleoside diphosphates by a ping-pong mechanism involving the formation of a high energy phospho-histidine intermediate [1, 2]. Besides their known functions in the control of intracellular nucleotide homeostasis, they are involved in multiple physiological and pathological cellular processes such as differentiation, development, metastastic dissemination or cilia functions. Over the past 15 years, ten human genes have been discovered encoding partial, full length, and/or tandemly repeated Nm23/NDPK domains, with or without N-or C-terminal extensions and/or additional domains. These genes encode proteins exhibiting different functions at various tissular and subcellular localizations. Most of these genes appear late in evolution with the emergence of the vertebrate lineage. This review summarizes the present knowledge on these multitalented proteins.
Subject(s)
Cilia/metabolism , NM23 Nucleoside Diphosphate Kinases/metabolism , Neoplasms/pathology , Nucleoside-Diphosphate Kinase/metabolism , Animals , Cilia/genetics , Humans , Isoenzymes/metabolism , Mammals/genetics , Mammals/metabolism , Mitochondria/enzymology , NM23 Nucleoside Diphosphate Kinases/genetics , Neoplasm Metastasis/genetics , Nucleoside-Diphosphate Kinase/genetics , Phylogeny , Protein Binding , Signal TransductionABSTRACT
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.