| 2000 |
NME4 (Nm23-H4) encodes a mitochondrial nucleoside diphosphate kinase. The truncated form (lacking the N-terminal mitochondrial targeting extension) possesses NDP kinase activity, while the full-length protein is inactive, indicating the N-terminal extension prevents enzyme folding/activity. X-ray crystallography showed the active form is a hexamer. The naturally occurring Ser129 (equivalent to K-pn mutation) weakens subunit interactions, and the S129P mutant is greatly stabilized. Confocal microscopy and subcellular fractionation of HEK293 cells showed mitochondrial localization with association to mitochondrial membranes, likely at contact sites between outer and inner membranes. Import into mitochondria is accompanied by cleavage of the N-terminal extension, which enables NDP kinase activity. |
X-ray crystallography, site-directed mutagenesis, GFP-fusion confocal microscopy, Western blot subcellular fractionation, in vitro enzymatic activity assay |
The Journal of biological chemistry |
High |
10799505
|
| 2008 |
NDPK-D/NME4 is a peripheral protein of the mitochondrial inner membrane, binding primarily via electrostatic interaction with cardiolipin (highest affinity among anionic phospholipids) as shown by surface plasmon resonance with recombinant protein and model liposomes. Mutation of Arg90 in the surface-exposed RRK motif strongly reduced phospholipid interaction in vitro and in vivo. Due to its symmetrical hexameric structure, NDPK-D can cross-link anionic phospholipid-containing liposomes. Respiration was significantly stimulated by NDPK substrate TDP in mitochondria expressing wild-type NDPK-D but not the R90D mutant, demonstrating functional coupling to oxidative phosphorylation that depends on membrane-bound state. |
Surface plasmon resonance, site-directed mutagenesis (R90D), stable cell expression, liposome cross-linking assay, mitochondrial respiration assay, submitochondrial fractionation |
The Journal of biological chemistry |
High |
18635542
|
| 2012 |
NME4/NDPK-D forms a complex with mitochondrial GTPase OPA1 in rat liver, suggesting direct local GTP delivery. Cardiolipin binding inhibits NDP kinase activity but enables a second function: selective intermembrane lipid transfer. Wild-type NME4, but not a membrane-binding-deficient mutant, selectively increased cardiolipin content in the outer mitochondrial membrane (analyzed by LC-MS), while other phospholipids (e.g., phosphatidylcholine) were unaffected. Cells expressing wild-type NME4 showed increased Bax accumulation in mitochondria and were sensitized to rotenone-induced apoptosis (cytochrome c release, caspase 3/7 activity, annexin V binding), demonstrating that cardiolipin transfer promotes apoptotic signaling. |
Co-immunoprecipitation (OPA1 complex), LC-MS lipid analysis, site-directed mutagenesis (membrane-binding-deficient mutant), apoptosis assays (cytochrome c release, caspase activity, annexin V), stable cell expression |
The Journal of biological chemistry |
High |
23150663
|
| 2016 |
NME4/NDPK-D facilitates cardiolipin externalization from the inner mitochondrial membrane to the outer mitochondrial membrane surface as a mitophagy signal. CCCP-induced mitophagy caused CL externalization in MLE-12 and HeLa cells; RNAi knockdown of NDPK-D decreased CL externalization and mitochondrial degradation. The R90D mutant (which does not bind CL) was inactive in promoting mitophagy. Proximity ligation assay showed that CL-transfer activity of NDPK-D is closely associated with dynamin-like GTPase OPA1, implicating fission-fusion dynamics. NDPK-D knockdown also suppressed rotenone- and 6-hydroxydopamine-triggered mitophagy in SH-SY5Y cells. |
RNAi knockdown, site-directed mutagenesis (R90D), in situ proximity ligation assay (PLA), flow cytometry/imaging of CL externalization, mitophagy assays in multiple cell lines |
Cell death and differentiation |
High |
26742431
|
| 2015 |
NDPK-D/NME4 is acetylated, and this modification is regulated by the NAD+-dependent deacetylase SIRT1, which was identified as a binding partner of NDPK-D by yeast two-hybrid screening and confirmed by co-immunoprecipitation. SIRT1 inhibition increases NDPK-D acetylation. Overexpression of NDPK-D with SIRT1, or mutation of the acetylated lysine residues, increases NDPK-D nuclear accumulation. An acetylation-mimic mutant of NDPK-D increased apoptosis in N1E-115 cells. NDPK-D knockdown induces apoptosis in neuroblastoma cells and in mouse cortex. |
Yeast two-hybrid screening, co-immunoprecipitation, site-directed mutagenesis (acetylation-mimic), confocal microscopy (co-localization), SIRT1 inhibitor treatment, RNAi knockdown, apoptosis assay |
PloS one |
Medium |
26426123
|
| 2014 |
miR-196 promotes oral cancer cell migration and invasion by inhibiting NME4 expression, which leads to activation of p-JNK, suppression of TIMP1, and augmentation of MMP1/9, placing NME4 upstream of the JNK-TIMP1-MMP signaling axis. |
RT-qPCR, Western blot, luciferase reporter assay (target validation), confocal microscopy, cell migration/invasion assays |
Molecular cancer |
Medium |
25233933
|
| 2021 |
Loss-of-function mutations in NME4 (lacking either NDP kinase activity or membrane interaction) and RNAi-mediated depletion of NME4 both promoted epithelial-mesenchymal transition, increased migratory and invasive potential, and caused mitochondrial fragmentation, loss of mitochondria, metabolic switch from respiration to glycolysis, and increased ROS generation. Immunocompromised mice developed more metastases when injected with cells expressing mutant NDPK-D versus wild-type, establishing NME4 as a metastasis suppressor acting via mitochondrial integrity. |
Loss-of-function mutagenesis, RNAi knockdown, in vivo xenograft metastasis assay, EMT marker analysis, mitochondrial morphology imaging, metabolic assays (respiration, glycolysis), ROS measurement |
BMC biology |
High |
34674701
|
| 2023 |
NME4 interacts with key enzymes in coenzyme A (CoA) metabolism and increases levels of acetyl-CoA and malonyl-CoA, leading to increased triglyceride levels and lipid accumulation in the liver. Hepatic deletion of Nme4 in mice suppressed hepatic steatosis progression, establishing NME4 as a regulator of mitochondrial lipid metabolism in NAFLD. |
Proteomics (interaction partners), metabolomics, hepatic Nme4 knockout (in vivo), high-fat diet mouse model, lipid quantification |
EMBO reports |
Medium |
38177901
|
| 2024 |
NME4 suppresses the NFκB2-CCL5 signaling axis in esophageal squamous cell carcinoma cells, thereby restricting CD8+ T cell infiltration into the tumor microenvironment. This was demonstrated using a syngeneic murine tumor model with single-cell RNA sequencing showing reduced CD8+ T cell infiltration upon NME4 expression, and quantitative proteomics/protein microarray mapping the NFκB2-CCL5 pathway as negatively regulated by NME4. |
Syngeneic murine tumor model, single-cell RNA sequencing, quantitative proteomics, protein microarray, in vivo tumor experiments |
Immunology |
Medium |
39016535
|
| 2026 |
RNF6 E3 ubiquitin ligase directly binds NME4 and facilitates its K48-linked polyubiquitination, leading to proteasomal degradation of NME4. NME4 depletion reverses the tumor-suppressive effects of RNF6 knockdown and reinstates JNK/c-JUN pathway activation, establishing an RNF6/NME4/JNK axis in ovarian cancer. |
Co-immunoprecipitation, cycloheximide (CHX) chase assay, ubiquitination assay, RNF6/NME4 co-modulation rescue experiments, nude mouse xenograft model |
Pathology, research and practice |
Medium |
41616518
|
| 2021 |
let-7f-5p directly targets the 3' UTR of Nme4 mRNA and negatively regulates Nme4 expression in mouse bone marrow-derived mesenchymal stem cells. Ectopic Nme4 expression completely reversed the inhibitory effects of let-7f-5p on osteogenic differentiation, and overexpression of Nme4 in BM-MSCs restored in vivo bone formation in an ovariectomized mouse model, placing Nme4 downstream of TNF-α/let-7f-5p in osteogenesis regulation. |
Luciferase reporter assay (3' UTR targeting), miRNA mimic/inhibitor transfection, ectopic Nme4 expression (rescue), in vivo ovariectomized animal model, ALP/alizarin red staining |
Biochemistry and cell biology |
Medium |
34297624
|
| 2024 |
Computational modeling (molecular dynamics simulations) identified conserved Arg27 in NME4 (and other group I NDPKs, NME1-4) as a key residue for hexamer assembly, mediating inter- and intra-molecular monomer interactions. Arg27 mutation decreased binding affinity and destabilized the complex. Double and triple Arg mutations in NME4, combined with a shorter C-terminal region, destabilize the hexamer into a dimer, highlighting the role of the C-terminal region in hexamer stabilization. |
Molecular dynamics simulation, computational modeling of hexameric assembly, mutation analysis (computational) |
bioRxivpreprint |
Low |
bio_10.1101_2024.09.19.613900
|