{"gene":"NME4","run_date":"2026-04-29T11:37:57","timeline":{"discoveries":[{"year":2000,"finding":"NME4 (Nm23-H4) encodes a mitochondrial nucleoside diphosphate kinase; the full-length protein is inactive due to its N-terminal mitochondrial targeting extension, while the truncated form lacking the extension possesses NDP kinase activity. Import into mitochondria is accompanied by cleavage of the N-terminal extension, restoring activity. X-ray crystallography confirmed the protein forms a hexamer, and submito-chondrial fractionation showed it is associated with mitochondrial membranes, possibly at contact sites between outer and inner membranes.","method":"Recombinant protein expression in E. coli, NDP kinase activity assay, X-ray crystallography, site-directed mutagenesis (S129P), GFP-fusion confocal microscopy, Western blot subcellular fractionation in HEK293 cells","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — reconstitution in vitro, crystal structure, mutagenesis, and direct localization with functional consequence in single rigorous study","pmids":["10799505"],"is_preprint":false},{"year":2008,"finding":"NME4/NDPK-D binds the inner mitochondrial membrane primarily through electrostatic interaction with cardiolipin (the anionic phospholipid most enriched in the inner membrane), mediated by a surface-exposed basic RRK motif (Arg-90). Mutation R90D strongly reduces phospholipid binding in vitro and in vivo. The membrane-bound state of NME4 is required for functional coupling with oxidative phosphorylation (respiration stimulated by TDP only in mitochondria expressing wild-type, not R90D, NME4). NME4's symmetrical hexameric structure allows it to cross-link anionic phospholipid-containing liposomes, suggesting a role in promoting intermembrane contacts.","method":"Surface plasmon resonance with recombinant protein and model liposomes, site-directed mutagenesis (R90D), stable expression in HeLa cells, respiration assays, latency assays with isolated mitochondria, antibody binding to mitoplasts","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution, mutagenesis, and functional coupling assay; replicated in cellular and liposome systems","pmids":["18635542"],"is_preprint":false},{"year":2012,"finding":"NME4 has a dual function acting as a mitochondrial switch: (1) phosphotransfer/NDP kinase activity supplying GTP locally, and (2) selective intermembrane cardiolipin transfer from inner to outer mitochondrial membrane. Cardiolipin binding inhibits NDP kinase activity but is required for lipid transfer. Wild-type NME4 (but not a membrane-binding-deficient mutant) selectively increased cardiolipin content in the outer mitochondrial membrane. NME4 forms a complex with the mitochondrial GTPase OPA1 in rat liver, suggesting direct local GTP delivery. Wild-type NME4-expressing HeLa cells showed increased Bax accumulation in mitochondria and were sensitized to rotenone-induced apoptosis (cytochrome c release, caspase 3/7 activation, annexin V binding).","method":"Co-immunoprecipitation (NME4-OPA1 complex), LC-MS lipid analysis of HeLa cells expressing wild-type vs. membrane-binding-deficient mutant NME4, apoptosis assays (cytochrome c release, caspase 3/7, annexin V), molecular modeling","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — multiple orthogonal methods (Co-IP, lipidomics, functional apoptosis assays, mutagenesis) in single study with strong internal controls","pmids":["23150663"],"is_preprint":false},{"year":2016,"finding":"NME4/NDPK-D facilitates translocation of cardiolipin from the inner mitochondrial membrane to the outer mitochondrial membrane surface upon mitophagy induction (CCCP treatment), enabling cardiolipin to serve as an 'eat-me' signal recognized by LC3. RNAi knockdown of NME4 decreased CCCP-induced CL externalization and mitochondrial degradation. The CL-binding deficient mutant R90D was inactive in promoting mitophagy. Proximity ligation assay showed NME4's CL-transfer activity is closely associated with the dynamin-like GTPase OPA1, implicating fission-fusion dynamics. NME4 knockdown also suppressed rotenone- and 6-hydroxydopamine-triggered mitophagy in SH-SY5Y cells.","method":"RNAi knockdown, CCCP/rotenone/6-OHDA-induced mitophagy, CL externalization assay, R90D mutant functional analysis, in situ proximity ligation assay (PLA) for NME4-OPA1 association, mitochondrial degradation assays in MLE-12, HeLa, and SH-SY5Y cells","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 2 — multiple cell lines, RNAi plus mutagenesis, multiple mitophagy inducers, replicated across labs","pmids":["26742431"],"is_preprint":false},{"year":2015,"finding":"NME4/NDPK-D is acetylated, and its acetylation state regulates its subcellular localization between nucleus and cytoplasm, as well as cell survival. SIRT1 was identified as a binding partner of NME4 by yeast two-hybrid screening, confirmed by co-immunoprecipitation. SIRT1 inhibition increases NME4 acetylation. Overexpression of NME4 with SIRT1, or mutation of acetylated lysine residues in NME4, increases nuclear accumulation. Acetylation-mimic mutant NME4 increased apoptosis in N1E-115 cells. NME4 knockdown induced apoptosis in neuroblastoma cells and mouse cortex.","method":"Yeast two-hybrid screening, co-immunoprecipitation, site-directed mutagenesis (acetylation-mimic), SIRT1 inhibitor treatment, confocal microscopy, apoptosis assays, in vivo knockdown in mouse cortex","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2–3 — yeast two-hybrid + Co-IP + mutagenesis + functional apoptosis readout, single lab","pmids":["26426123"],"is_preprint":false},{"year":2014,"finding":"NME4 suppresses cell migration and invasion in oral cancer through the NME4-JNK-TIMP1-MMP signaling pathway; miR-196 inhibits NME4 expression, thereby activating p-JNK, suppressing TIMP1, and augmenting MMP1/9, promoting invasive phenotype.","method":"miR-196 overexpression/inhibition, RT-qPCR, Western blot, luciferase reporter assay for miR-196 targeting of NME4 3'UTR, cell migration and invasion assays, confocal microscopy","journal":"Molecular cancer","confidence":"Medium","confidence_rationale":"Tier 2–3 — luciferase reporter validates direct targeting, pathway placement by downstream marker analysis, single lab","pmids":["25233933"],"is_preprint":false},{"year":2021,"finding":"NME4 acts as a metastasis suppressor in cancer cells. Loss-of-function mutations (lacking either NDP kinase activity or membrane interaction) or RNAi depletion of NME4 promoted epithelial-mesenchymal transition, increased migratory and invasive potential, and increased metastasis formation in immunocompromised mice. Mechanistically, NME4 loss caused mitochondrial fragmentation and loss, metabolic switch from respiration to glycolysis, and increased ROS generation, triggering pro-metastatic signaling cascades.","method":"Loss-of-function mutants (kinase-dead and membrane interaction-deficient), RNAi knockdown, in vitro migration/invasion assays, EMT marker analysis, in vivo xenograft metastasis model in immunocompromised mice, metabolic profiling","journal":"BMC biology","confidence":"High","confidence_rationale":"Tier 2 — complementary genetic approaches (mutants + RNAi), in vitro and in vivo validation, multiple mechanistic readouts","pmids":["34674701"],"is_preprint":false},{"year":2023,"finding":"NME4 interacts with key enzymes in coenzyme A (CoA) metabolism and increases the levels of acetyl-CoA and malonyl-CoA in the liver, promoting triglyceride accumulation and NAFLD progression. Hepatic deletion of Nme4 in mice suppressed hepatic steatosis progression.","method":"Hepatic-specific Nme4 knockout in mice (high-fat diet model), proteomics, metabolomics, CoA metabolite measurements","journal":"EMBO reports","confidence":"Medium","confidence_rationale":"Tier 2 — in vivo KO with metabolomics and proteomics, single lab","pmids":["38177901"],"is_preprint":false},{"year":2024,"finding":"NME4 negatively regulates the NFκB2-CCL5 signaling axis in esophageal squamous cell carcinoma, preventing CD8+ T cell infiltration into the tumor microenvironment. Mechanistically, NME4 suppresses NFκB2 activity, which controls CCL5 chemokine expression.","method":"Syngeneic tumor model in C57BL/6 mice, single-cell RNA sequencing, quantitative proteomics, protein microarray screening, NME4 modulation in murine ESCC cell line AKR","journal":"Immunology","confidence":"Medium","confidence_rationale":"Tier 2 — in vivo syngeneic model with scRNA-seq and proteomics pathway mapping, single lab","pmids":["39016535"],"is_preprint":false},{"year":2021,"finding":"Let-7f-5p miRNA directly targets the 3' UTR of Nme4 mRNA and negatively regulates Nme4 expression in mouse bone marrow-derived mesenchymal stem cells (BM-MSCs). TNF-α upregulates let-7f-5p (via NF-κB), reducing Nme4 and impairing osteogenic differentiation. Ectopic Nme4 expression reversed the inhibitory effects of let-7f-5p on osteogenesis in vitro and restored bone formation in ovariectomized mice in vivo.","method":"miRNA mimic/inhibitor transfection, luciferase reporter assay (let-7f-5p targeting Nme4 3'UTR), NME4 overexpression rescue, in vivo ovariectomized mouse model, osteogenic differentiation assays (ALP, Alizarin Red staining)","journal":"Biochemistry and cell biology","confidence":"Medium","confidence_rationale":"Tier 2–3 — luciferase reporter validates targeting, rescue experiments in vitro and in vivo, single lab","pmids":["34297624"],"is_preprint":false},{"year":2026,"finding":"E3 ubiquitin ligase RNF6 directly binds NME4 and promotes its K48-linked polyubiquitination, leading to proteasomal degradation of NME4. NME4 degradation by RNF6 activates the JNK/c-JUN signaling pathway, promoting ovarian cancer malignancy.","method":"Co-immunoprecipitation (RNF6-NME4 interaction), cycloheximide chase assay, ubiquitination assay (K48-linked), RNF6/NME4 co-modulation rescue experiments, in vivo nude mouse xenograft model","journal":"Pathology, research and practice","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP, ubiquitination assay, rescue experiments, and in vivo validation; single lab","pmids":["41616518"],"is_preprint":false},{"year":2024,"finding":"Conserved Arg27 across group I NDPKs (NME1-4) is a key residue for hexamer assembly; Arg27 mutation leads to decreased binding affinity, altered dynamics, and complex destabilization. For NME4 specifically, double and triple Arg mutations destabilize the hexamer into a dimer, partly due to its shorter C-terminal region.","method":"Molecular dynamics simulations, structural modeling, binding affinity calculations with mutant NME4 constructs","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 4 — computational modeling only, no experimental validation reported","pmids":["bio_10.1101_2024.09.19.613900"],"is_preprint":true}],"current_model":"NME4 (NDPK-D/NM23-H4) is a mitochondrial intermembrane space hexameric NDP kinase that binds the inner mitochondrial membrane via cardiolipin interaction through its RRK motif (Arg-90); it acts as a bifunctional molecular switch—in its membrane-bound, cardiolipin-crosslinking state it transfers cardiolipin from the inner to the outer mitochondrial membrane to serve as a mitophagy/apoptosis 'eat-me' signal (recognized by LC3), while in a soluble or loosely membrane-associated state it provides phosphotransfer activity to locally regenerate NTPs (including GTP for OPA1); its acetylation by/deacetylation via SIRT1 controls its nuclear-cytoplasmic shuttling, and RNF6-mediated K48-linked ubiquitination targets it for proteasomal degradation, activating JNK/c-JUN signaling, collectively establishing NME4 as a metastasis suppressor that maintains mitochondrial integrity and suppresses EMT."},"narrative":{"teleology":[{"year":2000,"claim":"Establishing that NME4 encodes a mitochondrial NDP kinase resolved its enzymatic identity and organellar context: the full-length precursor is inactive, and import-coupled cleavage of the N-terminal extension activates the hexameric enzyme at mitochondrial membranes.","evidence":"Recombinant expression, X-ray crystallography of hexamer, NDP kinase assays, GFP-fusion confocal microscopy, and subcellular fractionation in HEK293 cells","pmids":["10799505"],"confidence":"High","gaps":["Precise sub-mitochondrial topology (inner vs. outer membrane contact sites) not resolved","No identification of the protease responsible for N-terminal cleavage","Physiological substrates in vivo not determined"]},{"year":2008,"claim":"Demonstrating that Arg-90-dependent cardiolipin binding tethers NME4 to the inner membrane and is required for functional coupling to oxidative phosphorylation answered how the kinase integrates with the respiratory chain.","evidence":"Surface plasmon resonance with model liposomes, R90D mutagenesis, respiration assays and latency measurements in HeLa mitochondria","pmids":["18635542"],"confidence":"High","gaps":["Stoichiometry of NME4–cardiolipin interaction not quantified","Whether NME4 contacts other lipid species at physiological ratios unknown","Structural basis of hexamer-mediated membrane cross-linking not resolved at atomic level"]},{"year":2012,"claim":"Revealing that NME4 selectively transfers cardiolipin from the inner to the outer mitochondrial membrane—while simultaneously forming a complex with the GTPase OPA1—established its dual role as both a lipid transfer protein and a local GTP supplier.","evidence":"LC-MS lipidomics of outer membrane fractions, Co-IP of NME4–OPA1 in rat liver, apoptosis assays (cytochrome c release, caspase 3/7) in HeLa cells with wild-type versus membrane-binding-deficient mutant","pmids":["23150663"],"confidence":"High","gaps":["Whether NME4 directly channels GTP to OPA1 or acts indirectly not distinguished","Cardiolipin transfer mechanism (tunnel, flip-flop, or facilitated diffusion) undetermined","Reciprocal OPA1-to-NME4 regulation not tested"]},{"year":2014,"claim":"Placing NME4 upstream of JNK–TIMP1–MMP signaling in oral cancer provided the first pathway-level explanation for its invasion-suppressive function, with miR-196 identified as a direct negative regulator of NME4 mRNA.","evidence":"Luciferase reporter assay confirming miR-196 targeting of NME4 3′ UTR, migration/invasion assays, downstream marker analysis in oral cancer cells","pmids":["25233933"],"confidence":"Medium","gaps":["Whether JNK activation by NME4 loss is direct or secondary to mitochondrial dysfunction not clarified","Single cancer type tested","No in vivo metastasis data"]},{"year":2015,"claim":"Discovering that SIRT1 deacetylates NME4 and that acetylation status controls its nuclear versus cytoplasmic distribution revealed a post-translational regulatory axis beyond mitochondrial function.","evidence":"Yeast two-hybrid identification of SIRT1, Co-IP confirmation, acetylation-mimic mutagenesis, confocal localization, apoptosis assays in N1E-115 cells and mouse cortex knockdown","pmids":["26426123"],"confidence":"Medium","gaps":["Identity of the acetyltransferase is unknown","Specific acetylated lysine residues on NME4 not fully mapped by mass spectrometry","Nuclear function of NME4 remains uncharacterized"]},{"year":2016,"claim":"Showing that NME4-mediated cardiolipin externalization serves as an LC3-recognized 'eat-me' signal for mitophagy unified the lipid-transfer and quality-control roles, with NME4 required across multiple mitophagy inducers and cell types.","evidence":"RNAi knockdown and R90D mutant analysis, CCCP/rotenone/6-OHDA-induced mitophagy assays, proximity ligation assay for NME4–OPA1, in MLE-12, HeLa, and SH-SY5Y cells","pmids":["26742431"],"confidence":"High","gaps":["Whether NME4 cooperates with PINK1/Parkin pathway or acts in parallel not determined","Degree of cardiolipin externalization required for LC3 recognition not quantified","In vivo relevance in neurodegeneration models not established"]},{"year":2021,"claim":"Comprehensive loss-of-function analysis established NME4 as a bona fide metastasis suppressor: loss of either kinase or membrane-binding activity caused mitochondrial fragmentation, metabolic reprogramming to glycolysis, EMT, and metastasis in xenograft models.","evidence":"Kinase-dead and membrane-binding-deficient mutants, RNAi knockdown, in vitro migration/invasion, EMT markers, in vivo xenograft metastasis in immunocompromised mice, metabolic profiling","pmids":["34674701"],"confidence":"High","gaps":["Whether the two functions (kinase and lipid transfer) contribute equally to suppression is unresolved","Relevance to immunocompetent tumor microenvironment not addressed in this study","Downstream effectors linking metabolic shift to EMT not fully mapped"]},{"year":2023,"claim":"Hepatic Nme4 knockout revealed an unexpected metabolic role: NME4 interacts with CoA-metabolizing enzymes and its loss suppresses acetyl-CoA/malonyl-CoA accumulation and steatosis, expanding its function beyond cancer biology.","evidence":"Liver-specific Nme4 knockout mice on high-fat diet, proteomics, metabolomics, CoA metabolite quantification","pmids":["38177901"],"confidence":"Medium","gaps":["Whether NME4 directly phosphorylates CoA pathway enzymes or acts indirectly is unclear","Relevance to human NAFLD not yet demonstrated","Interaction with CoA enzymes not validated by reciprocal pull-down"]},{"year":2024,"claim":"In esophageal squamous cell carcinoma, NME4 suppresses NFκB2-CCL5 signaling and thereby limits CD8+ T cell infiltration, connecting its tumor-suppressive function to immune modulation in the tumor microenvironment.","evidence":"Syngeneic C57BL/6 tumor model, single-cell RNA sequencing, quantitative proteomics, protein microarray in murine ESCC cells","pmids":["39016535"],"confidence":"Medium","gaps":["Mechanism by which NME4 suppresses NFκB2 activity not defined","Whether immune modulation is direct or secondary to mitochondrial dysfunction unclear","Single tumor type in syngeneic setting"]},{"year":2026,"claim":"Identification of RNF6 as the E3 ligase that K48-ubiquitinates NME4 for proteasomal degradation, activating JNK/c-JUN, defined the major degradation pathway controlling NME4 protein levels in ovarian cancer.","evidence":"Co-IP of RNF6–NME4, K48-linked ubiquitination assay, cycloheximide chase, rescue experiments, nude mouse xenograft","pmids":["41616518"],"confidence":"Medium","gaps":["Specific ubiquitinated lysine residues on NME4 not mapped","Whether RNF6-NME4 axis operates in non-cancer contexts unknown","Deubiquitinase that opposes RNF6 not identified"]},{"year":null,"claim":"How NME4's two biochemical activities—NDP kinase and cardiolipin transfer—are coordinately regulated in vivo, and whether its nuclear pool performs distinct functions, remain major open questions.","evidence":"","pmids":[],"confidence":"Low","gaps":["No structural model of NME4 bound to cardiolipin at atomic resolution","Nuclear function of NME4 entirely uncharacterized","Relationship between NME4-mediated mitophagy and PINK1/Parkin pathway not resolved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,1,2]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[1,2,3]},{"term_id":"GO:0140104","term_label":"molecular carrier activity","supporting_discovery_ids":[2,3]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[5,6,8]}],"localization":[{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[0,1,2,3]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[4]}],"pathway":[{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[3,6]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[2,4]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[0,1,7]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[5,6,8,10]}],"complexes":["NDPK-D hexamer"],"partners":["OPA1","SIRT1","RNF6"],"other_free_text":[]},"mechanistic_narrative":"NME4 (NDPK-D/NM23-H4) is a mitochondrial nucleoside diphosphate kinase that functions as a bifunctional molecular switch governing mitochondrial integrity, lipid signaling, and metastasis suppression. The processed hexameric enzyme resides in the mitochondrial intermembrane space, where it binds the inner membrane via electrostatic interaction between its RRK motif (Arg-90) and cardiolipin; in this membrane-bound state it cross-links membranes and selectively transfers cardiolipin to the outer membrane surface, generating an 'eat-me' signal recognized by LC3 to initiate mitophagy, while also coupling with OPA1-dependent GTP supply to support oxidative phosphorylation [PMID:10799505, PMID:18635542, PMID:23150663, PMID:26742431]. Loss of NME4 kinase or membrane-binding function causes mitochondrial fragmentation, a metabolic shift to glycolysis, epithelial–mesenchymal transition, and enhanced metastasis in vivo, establishing NME4 as a metastasis suppressor that operates through JNK-TIMP1-MMP and NFκB2-CCL5 signaling axes [PMID:34674701, PMID:25233933, PMID:39016535]. NME4 protein turnover is controlled by RNF6-mediated K48-linked ubiquitination targeting it for proteasomal degradation, while SIRT1-dependent deacetylation regulates its nuclear-cytoplasmic partitioning [PMID:41616518, PMID:26426123]."},"prefetch_data":{"uniprot":{"accession":"O00746","full_name":"Nucleoside diphosphate kinase D, mitochondrial","aliases":["Nucleoside diphosphate kinase 4","NDK4","nm23-H4"],"length_aa":187,"mass_kda":20.7,"function":"Mitochondria-specific nucleoside diphosphate kinase that catalyzes the transfer of a gamma-phosphoryl group from ATP to a nucleoside diphosphate via a ping-pong mechanism involving a phosphohistidine intermediate, participating in nucleoside triphosphate homeostasis (PubMed:10799505, PubMed:16313181, PubMed:18635542, PubMed:23150663). In vitro, purine nucleoside triphosphates are much more effective as donors and acceptors than pyrimidine nucleoside triphosphates, and ribonucleosides derivatives are superior to their deoxyribonucleosides counterparts (By similarity). Associates with cardiolipin-containing mitochondrial inner membrane and locally produces ADP in the mitochondrial intermembrane space, which is directly taken up via the ADP/ATP translocase (ANT) into the matrix to stimulate respiratory ATP regeneration (PubMed:18635542, PubMed:24970086). Also directly provides GTP for mitochondrial GTPases, such as the dynamin-related GTPase OPA1 (PubMed:24970086). Additionally, catalyzes the anionic phospholipid transfer, namely cardiolipin, from the mitochondrial inner membrane (IM) to the outer membrane (OM) through the formation of contact sites between IM and OM, in a nucleoside diphosphate kinase-independent manner (PubMed:17028143, PubMed:23150663). The switch between the nucleoside diphosphate kinase and the phospholipid transfer activity may depend on the availability and accessibility of cardiolipin and other anionic phospholipids in the IM outer leaflet and OM inner leaflet (PubMed:23150663)","subcellular_location":"Mitochondrion intermembrane space; Mitochondrion matrix; Mitochondrion outer membrane","url":"https://www.uniprot.org/uniprotkb/O00746/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/NME4","classification":"Not Classified","n_dependent_lines":3,"n_total_lines":1208,"dependency_fraction":0.0024834437086092716},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/NME4","total_profiled":1310},"omim":[{"mim_id":"611787","title":"CYTIDINE MONOPHOSPHATE (UMP-CMP) KINASE 2, MITOCHONDRIAL; CMPK2","url":"https://www.omim.org/entry/611787"},{"mim_id":"601818","title":"NME/NM23 NUCLEOSIDE DIPHOSPHATE KINASE 4; NME4","url":"https://www.omim.org/entry/601818"},{"mim_id":"300032","title":"ATRX CHROMATIN REMODELER; ATRX","url":"https://www.omim.org/entry/300032"},{"mim_id":"156491","title":"NME/NM23 NUCLEOSIDE DIPHOSPHATE KINASE 2; NME2","url":"https://www.omim.org/entry/156491"},{"mim_id":"156490","title":"NME/NM23 NUCLEOSIDE DIPHOSPHATE KINASE 1; NME1","url":"https://www.omim.org/entry/156490"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Mitochondria","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/NME4"},"hgnc":{"alias_symbol":["nm23-H4","NM23H4","NDPKD"],"prev_symbol":[]},"alphafold":{"accession":"O00746","domains":[{"cath_id":"3.30.70.141","chopping":"40-75_107-172","consensus_level":"high","plddt":97.5397,"start":40,"end":172}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O00746","model_url":"https://alphafold.ebi.ac.uk/files/AF-O00746-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O00746-F1-predicted_aligned_error_v6.png","plddt_mean":85.0},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=NME4","jax_strain_url":"https://www.jax.org/strain/search?query=NME4"},"sequence":{"accession":"O00746","fasta_url":"https://rest.uniprot.org/uniprotkb/O00746.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O00746/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O00746"}},"corpus_meta":[{"pmid":"26742431","id":"PMC_26742431","title":"NDPK-D (NM23-H4)-mediated externalization of cardiolipin enables elimination of depolarized mitochondria by mitophagy.","date":"2016","source":"Cell death and differentiation","url":"https://pubmed.ncbi.nlm.nih.gov/26742431","citation_count":164,"is_preprint":false},{"pmid":"16834293","id":"PMC_16834293","title":"Cation [M = H+, Li+, Na+, K+, Ca2+, Mg2+, NH4+, and NMe4+] interactions with the aromatic motifs of naturally occurring amino acids: a theoretical study.","date":"2005","source":"The journal of physical chemistry. A","url":"https://pubmed.ncbi.nlm.nih.gov/16834293","citation_count":152,"is_preprint":false},{"pmid":"10799505","id":"PMC_10799505","title":"The human nm23-H4 gene product is a mitochondrial nucleoside diphosphate kinase.","date":"2000","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/10799505","citation_count":126,"is_preprint":false},{"pmid":"9099850","id":"PMC_9099850","title":"nm23-H4, a new member of the family of human nm23/nucleoside diphosphate kinase genes localised on chromosome 16p13.","date":"1997","source":"Human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/9099850","citation_count":115,"is_preprint":false},{"pmid":"23150663","id":"PMC_23150663","title":"Dual function of mitochondrial Nm23-H4 protein in phosphotransfer and intermembrane lipid transfer: a cardiolipin-dependent switch.","date":"2012","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/23150663","citation_count":95,"is_preprint":false},{"pmid":"25233933","id":"PMC_25233933","title":"OncomiR-196 promotes an invasive phenotype in oral cancer through the NME4-JNK-TIMP1-MMP signaling pathway.","date":"2014","source":"Molecular cancer","url":"https://pubmed.ncbi.nlm.nih.gov/25233933","citation_count":84,"is_preprint":false},{"pmid":"18635542","id":"PMC_18635542","title":"The nucleoside diphosphate kinase D (NM23-H4) binds the inner mitochondrial membrane with high affinity to cardiolipin and couples nucleotide transfer with respiration.","date":"2008","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/18635542","citation_count":83,"is_preprint":false},{"pmid":"19254751","id":"PMC_19254751","title":"Interaction of NDPK-D with cardiolipin-containing membranes: Structural basis and implications for mitochondrial physiology.","date":"2009","source":"Biochimie","url":"https://pubmed.ncbi.nlm.nih.gov/19254751","citation_count":36,"is_preprint":false},{"pmid":"29035377","id":"PMC_29035377","title":"NME4/nucleoside diphosphate kinase D in cardiolipin signaling and mitophagy.","date":"2017","source":"Laboratory investigation; a journal of technical methods and pathology","url":"https://pubmed.ncbi.nlm.nih.gov/29035377","citation_count":30,"is_preprint":false},{"pmid":"29491425","id":"PMC_29491425","title":"The mitochondrial nucleoside diphosphate kinase (NDPK-D/NME4), a moonlighting protein for cell homeostasis.","date":"2018","source":"Laboratory investigation; a journal of technical methods and pathology","url":"https://pubmed.ncbi.nlm.nih.gov/29491425","citation_count":27,"is_preprint":false},{"pmid":"34674701","id":"PMC_34674701","title":"The mitochondrially-localized nucleoside diphosphate kinase D (NME4) is a novel metastasis suppressor.","date":"2021","source":"BMC biology","url":"https://pubmed.ncbi.nlm.nih.gov/34674701","citation_count":26,"is_preprint":false},{"pmid":"15726650","id":"PMC_15726650","title":"Expression of the nm23 homologues nm23-H4, nm23-H6, and nm23-H7 in human gastric and colon cancer.","date":"2005","source":"The Journal of pathology","url":"https://pubmed.ncbi.nlm.nih.gov/15726650","citation_count":25,"is_preprint":false},{"pmid":"26426123","id":"PMC_26426123","title":"Acetylation of NDPK-D Regulates Its Subcellular Localization and Cell Survival.","date":"2015","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/26426123","citation_count":20,"is_preprint":false},{"pmid":"22052250","id":"PMC_22052250","title":"Probing Lewis acidity of Y(BH4)3 via its reactions with MBH4 (M = Li, Na, K, NMe4).","date":"2011","source":"Dalton transactions (Cambridge, England : 2003)","url":"https://pubmed.ncbi.nlm.nih.gov/22052250","citation_count":19,"is_preprint":false},{"pmid":"31257488","id":"PMC_31257488","title":"NME4 may enhance non‑small cell lung cancer progression by overcoming cell cycle arrest and promoting cellular proliferation.","date":"2019","source":"Molecular medicine reports","url":"https://pubmed.ncbi.nlm.nih.gov/31257488","citation_count":17,"is_preprint":false},{"pmid":"25231795","id":"PMC_25231795","title":"Mitochondrial NM23-H4/NDPK-D: a bifunctional nanoswitch for bioenergetics and lipid signaling.","date":"2014","source":"Naunyn-Schmiedeberg's archives of pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/25231795","citation_count":15,"is_preprint":false},{"pmid":"39016535","id":"PMC_39016535","title":"NME4 suppresses NFκB2-CCL5 axis, restricting CD8+ T cell tumour infiltration in oesophageal squamous cell carcinoma.","date":"2024","source":"Immunology","url":"https://pubmed.ncbi.nlm.nih.gov/39016535","citation_count":12,"is_preprint":false},{"pmid":"11724361","id":"PMC_11724361","title":"Overexpression of nm23-H4 RNA in colorectal and renal tumours.","date":"2001","source":"Anticancer research","url":"https://pubmed.ncbi.nlm.nih.gov/11724361","citation_count":12,"is_preprint":false},{"pmid":"32192776","id":"PMC_32192776","title":"NME4 modulates PD-L1 expression via the STAT3 signaling pathway in squamous cell carcinoma.","date":"2020","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/32192776","citation_count":11,"is_preprint":false},{"pmid":"38177901","id":"PMC_38177901","title":"NME4 mediates metabolic reprogramming and promotes nonalcoholic fatty liver disease progression.","date":"2023","source":"EMBO reports","url":"https://pubmed.ncbi.nlm.nih.gov/38177901","citation_count":8,"is_preprint":false},{"pmid":"32504364","id":"PMC_32504364","title":"Widely targeted metabolomic analyses unveil the metabolic variations after stable knock-down of NME4 in esophageal squamous cell carcinoma cells.","date":"2020","source":"Molecular and cellular biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/32504364","citation_count":6,"is_preprint":false},{"pmid":"37096311","id":"PMC_37096311","title":"Differential Expression of NME4 in Trophoblast Stem-Like Cells and Peripheral Blood Mononuclear Cells of Normal Pregnancy and Preeclampsia.","date":"2023","source":"Journal of Korean medical science","url":"https://pubmed.ncbi.nlm.nih.gov/37096311","citation_count":3,"is_preprint":false},{"pmid":"34297624","id":"PMC_34297624","title":"The let-7f-5p-Nme4 pathway mediates tumor necrosis factor α-induced impairment in osteogenesis of bone marrow-derived mesenchymal stem cells.","date":"2021","source":"Biochemistry and cell biology = Biochimie et biologie cellulaire","url":"https://pubmed.ncbi.nlm.nih.gov/34297624","citation_count":2,"is_preprint":false},{"pmid":"41616518","id":"PMC_41616518","title":"RNF6 activates JNK/c-JUN pathway in ovarian cancer by promoting K48-linked NME4 ubiquitination.","date":"2026","source":"Pathology, research and practice","url":"https://pubmed.ncbi.nlm.nih.gov/41616518","citation_count":0,"is_preprint":false},{"pmid":"38502357","id":"PMC_38502357","title":"Creatine kinase elevation in chronic hepatitis B patients with telbivudine therapy: influence of telbivudine plasma concentration and single nucleotide polymorphisms of TK2, RRM2B, and NME4.","date":"2024","source":"European journal of clinical pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/38502357","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2024.09.19.613900","title":"Modelling dynamics of human NDPK hexamer structure, stability and interactions","date":"2024-09-23","source":"bioRxiv","url":"https://doi.org/10.1101/2024.09.19.613900","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":14440,"output_tokens":3884,"usd":0.05079},"stage2":{"model":"claude-opus-4-6","input_tokens":7254,"output_tokens":3159,"usd":0.172868},"total_usd":0.223658,"stage1_batch_id":"msgbatch_012ZiiCUBfB62NCTQd4Bk1Jo","stage2_batch_id":"msgbatch_01Xuqq9aUHoQouS7UVGZNStZ","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2000,\n      \"finding\": \"NME4 (Nm23-H4) encodes a mitochondrial nucleoside diphosphate kinase; the full-length protein is inactive due to its N-terminal mitochondrial targeting extension, while the truncated form lacking the extension possesses NDP kinase activity. Import into mitochondria is accompanied by cleavage of the N-terminal extension, restoring activity. X-ray crystallography confirmed the protein forms a hexamer, and submito-chondrial fractionation showed it is associated with mitochondrial membranes, possibly at contact sites between outer and inner membranes.\",\n      \"method\": \"Recombinant protein expression in E. coli, NDP kinase activity assay, X-ray crystallography, site-directed mutagenesis (S129P), GFP-fusion confocal microscopy, Western blot subcellular fractionation in HEK293 cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstitution in vitro, crystal structure, mutagenesis, and direct localization with functional consequence in single rigorous study\",\n      \"pmids\": [\"10799505\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"NME4/NDPK-D binds the inner mitochondrial membrane primarily through electrostatic interaction with cardiolipin (the anionic phospholipid most enriched in the inner membrane), mediated by a surface-exposed basic RRK motif (Arg-90). Mutation R90D strongly reduces phospholipid binding in vitro and in vivo. The membrane-bound state of NME4 is required for functional coupling with oxidative phosphorylation (respiration stimulated by TDP only in mitochondria expressing wild-type, not R90D, NME4). NME4's symmetrical hexameric structure allows it to cross-link anionic phospholipid-containing liposomes, suggesting a role in promoting intermembrane contacts.\",\n      \"method\": \"Surface plasmon resonance with recombinant protein and model liposomes, site-directed mutagenesis (R90D), stable expression in HeLa cells, respiration assays, latency assays with isolated mitochondria, antibody binding to mitoplasts\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution, mutagenesis, and functional coupling assay; replicated in cellular and liposome systems\",\n      \"pmids\": [\"18635542\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"NME4 has a dual function acting as a mitochondrial switch: (1) phosphotransfer/NDP kinase activity supplying GTP locally, and (2) selective intermembrane cardiolipin transfer from inner to outer mitochondrial membrane. Cardiolipin binding inhibits NDP kinase activity but is required for lipid transfer. Wild-type NME4 (but not a membrane-binding-deficient mutant) selectively increased cardiolipin content in the outer mitochondrial membrane. NME4 forms a complex with the mitochondrial GTPase OPA1 in rat liver, suggesting direct local GTP delivery. Wild-type NME4-expressing HeLa cells showed increased Bax accumulation in mitochondria and were sensitized to rotenone-induced apoptosis (cytochrome c release, caspase 3/7 activation, annexin V binding).\",\n      \"method\": \"Co-immunoprecipitation (NME4-OPA1 complex), LC-MS lipid analysis of HeLa cells expressing wild-type vs. membrane-binding-deficient mutant NME4, apoptosis assays (cytochrome c release, caspase 3/7, annexin V), molecular modeling\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — multiple orthogonal methods (Co-IP, lipidomics, functional apoptosis assays, mutagenesis) in single study with strong internal controls\",\n      \"pmids\": [\"23150663\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"NME4/NDPK-D facilitates translocation of cardiolipin from the inner mitochondrial membrane to the outer mitochondrial membrane surface upon mitophagy induction (CCCP treatment), enabling cardiolipin to serve as an 'eat-me' signal recognized by LC3. RNAi knockdown of NME4 decreased CCCP-induced CL externalization and mitochondrial degradation. The CL-binding deficient mutant R90D was inactive in promoting mitophagy. Proximity ligation assay showed NME4's CL-transfer activity is closely associated with the dynamin-like GTPase OPA1, implicating fission-fusion dynamics. NME4 knockdown also suppressed rotenone- and 6-hydroxydopamine-triggered mitophagy in SH-SY5Y cells.\",\n      \"method\": \"RNAi knockdown, CCCP/rotenone/6-OHDA-induced mitophagy, CL externalization assay, R90D mutant functional analysis, in situ proximity ligation assay (PLA) for NME4-OPA1 association, mitochondrial degradation assays in MLE-12, HeLa, and SH-SY5Y cells\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple cell lines, RNAi plus mutagenesis, multiple mitophagy inducers, replicated across labs\",\n      \"pmids\": [\"26742431\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"NME4/NDPK-D is acetylated, and its acetylation state regulates its subcellular localization between nucleus and cytoplasm, as well as cell survival. SIRT1 was identified as a binding partner of NME4 by yeast two-hybrid screening, confirmed by co-immunoprecipitation. SIRT1 inhibition increases NME4 acetylation. Overexpression of NME4 with SIRT1, or mutation of acetylated lysine residues in NME4, increases nuclear accumulation. Acetylation-mimic mutant NME4 increased apoptosis in N1E-115 cells. NME4 knockdown induced apoptosis in neuroblastoma cells and mouse cortex.\",\n      \"method\": \"Yeast two-hybrid screening, co-immunoprecipitation, site-directed mutagenesis (acetylation-mimic), SIRT1 inhibitor treatment, confocal microscopy, apoptosis assays, in vivo knockdown in mouse cortex\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — yeast two-hybrid + Co-IP + mutagenesis + functional apoptosis readout, single lab\",\n      \"pmids\": [\"26426123\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"NME4 suppresses cell migration and invasion in oral cancer through the NME4-JNK-TIMP1-MMP signaling pathway; miR-196 inhibits NME4 expression, thereby activating p-JNK, suppressing TIMP1, and augmenting MMP1/9, promoting invasive phenotype.\",\n      \"method\": \"miR-196 overexpression/inhibition, RT-qPCR, Western blot, luciferase reporter assay for miR-196 targeting of NME4 3'UTR, cell migration and invasion assays, confocal microscopy\",\n      \"journal\": \"Molecular cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — luciferase reporter validates direct targeting, pathway placement by downstream marker analysis, single lab\",\n      \"pmids\": [\"25233933\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"NME4 acts as a metastasis suppressor in cancer cells. Loss-of-function mutations (lacking either NDP kinase activity or membrane interaction) or RNAi depletion of NME4 promoted epithelial-mesenchymal transition, increased migratory and invasive potential, and increased metastasis formation in immunocompromised mice. Mechanistically, NME4 loss caused mitochondrial fragmentation and loss, metabolic switch from respiration to glycolysis, and increased ROS generation, triggering pro-metastatic signaling cascades.\",\n      \"method\": \"Loss-of-function mutants (kinase-dead and membrane interaction-deficient), RNAi knockdown, in vitro migration/invasion assays, EMT marker analysis, in vivo xenograft metastasis model in immunocompromised mice, metabolic profiling\",\n      \"journal\": \"BMC biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — complementary genetic approaches (mutants + RNAi), in vitro and in vivo validation, multiple mechanistic readouts\",\n      \"pmids\": [\"34674701\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"NME4 interacts with key enzymes in coenzyme A (CoA) metabolism and increases the levels of acetyl-CoA and malonyl-CoA in the liver, promoting triglyceride accumulation and NAFLD progression. Hepatic deletion of Nme4 in mice suppressed hepatic steatosis progression.\",\n      \"method\": \"Hepatic-specific Nme4 knockout in mice (high-fat diet model), proteomics, metabolomics, CoA metabolite measurements\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo KO with metabolomics and proteomics, single lab\",\n      \"pmids\": [\"38177901\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"NME4 negatively regulates the NFκB2-CCL5 signaling axis in esophageal squamous cell carcinoma, preventing CD8+ T cell infiltration into the tumor microenvironment. Mechanistically, NME4 suppresses NFκB2 activity, which controls CCL5 chemokine expression.\",\n      \"method\": \"Syngeneic tumor model in C57BL/6 mice, single-cell RNA sequencing, quantitative proteomics, protein microarray screening, NME4 modulation in murine ESCC cell line AKR\",\n      \"journal\": \"Immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo syngeneic model with scRNA-seq and proteomics pathway mapping, single lab\",\n      \"pmids\": [\"39016535\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Let-7f-5p miRNA directly targets the 3' UTR of Nme4 mRNA and negatively regulates Nme4 expression in mouse bone marrow-derived mesenchymal stem cells (BM-MSCs). TNF-α upregulates let-7f-5p (via NF-κB), reducing Nme4 and impairing osteogenic differentiation. Ectopic Nme4 expression reversed the inhibitory effects of let-7f-5p on osteogenesis in vitro and restored bone formation in ovariectomized mice in vivo.\",\n      \"method\": \"miRNA mimic/inhibitor transfection, luciferase reporter assay (let-7f-5p targeting Nme4 3'UTR), NME4 overexpression rescue, in vivo ovariectomized mouse model, osteogenic differentiation assays (ALP, Alizarin Red staining)\",\n      \"journal\": \"Biochemistry and cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — luciferase reporter validates targeting, rescue experiments in vitro and in vivo, single lab\",\n      \"pmids\": [\"34297624\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"E3 ubiquitin ligase RNF6 directly binds NME4 and promotes its K48-linked polyubiquitination, leading to proteasomal degradation of NME4. NME4 degradation by RNF6 activates the JNK/c-JUN signaling pathway, promoting ovarian cancer malignancy.\",\n      \"method\": \"Co-immunoprecipitation (RNF6-NME4 interaction), cycloheximide chase assay, ubiquitination assay (K48-linked), RNF6/NME4 co-modulation rescue experiments, in vivo nude mouse xenograft model\",\n      \"journal\": \"Pathology, research and practice\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP, ubiquitination assay, rescue experiments, and in vivo validation; single lab\",\n      \"pmids\": [\"41616518\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Conserved Arg27 across group I NDPKs (NME1-4) is a key residue for hexamer assembly; Arg27 mutation leads to decreased binding affinity, altered dynamics, and complex destabilization. For NME4 specifically, double and triple Arg mutations destabilize the hexamer into a dimer, partly due to its shorter C-terminal region.\",\n      \"method\": \"Molecular dynamics simulations, structural modeling, binding affinity calculations with mutant NME4 constructs\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 — computational modeling only, no experimental validation reported\",\n      \"pmids\": [\"bio_10.1101_2024.09.19.613900\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"NME4 (NDPK-D/NM23-H4) is a mitochondrial intermembrane space hexameric NDP kinase that binds the inner mitochondrial membrane via cardiolipin interaction through its RRK motif (Arg-90); it acts as a bifunctional molecular switch—in its membrane-bound, cardiolipin-crosslinking state it transfers cardiolipin from the inner to the outer mitochondrial membrane to serve as a mitophagy/apoptosis 'eat-me' signal (recognized by LC3), while in a soluble or loosely membrane-associated state it provides phosphotransfer activity to locally regenerate NTPs (including GTP for OPA1); its acetylation by/deacetylation via SIRT1 controls its nuclear-cytoplasmic shuttling, and RNF6-mediated K48-linked ubiquitination targets it for proteasomal degradation, activating JNK/c-JUN signaling, collectively establishing NME4 as a metastasis suppressor that maintains mitochondrial integrity and suppresses EMT.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"NME4 (NDPK-D/NM23-H4) is a mitochondrial nucleoside diphosphate kinase that functions as a bifunctional molecular switch governing mitochondrial integrity, lipid signaling, and metastasis suppression. The processed hexameric enzyme resides in the mitochondrial intermembrane space, where it binds the inner membrane via electrostatic interaction between its RRK motif (Arg-90) and cardiolipin; in this membrane-bound state it cross-links membranes and selectively transfers cardiolipin to the outer membrane surface, generating an 'eat-me' signal recognized by LC3 to initiate mitophagy, while also coupling with OPA1-dependent GTP supply to support oxidative phosphorylation [PMID:10799505, PMID:18635542, PMID:23150663, PMID:26742431]. Loss of NME4 kinase or membrane-binding function causes mitochondrial fragmentation, a metabolic shift to glycolysis, epithelial–mesenchymal transition, and enhanced metastasis in vivo, establishing NME4 as a metastasis suppressor that operates through JNK-TIMP1-MMP and NFκB2-CCL5 signaling axes [PMID:34674701, PMID:25233933, PMID:39016535]. NME4 protein turnover is controlled by RNF6-mediated K48-linked ubiquitination targeting it for proteasomal degradation, while SIRT1-dependent deacetylation regulates its nuclear-cytoplasmic partitioning [PMID:41616518, PMID:26426123].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Establishing that NME4 encodes a mitochondrial NDP kinase resolved its enzymatic identity and organellar context: the full-length precursor is inactive, and import-coupled cleavage of the N-terminal extension activates the hexameric enzyme at mitochondrial membranes.\",\n      \"evidence\": \"Recombinant expression, X-ray crystallography of hexamer, NDP kinase assays, GFP-fusion confocal microscopy, and subcellular fractionation in HEK293 cells\",\n      \"pmids\": [\"10799505\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Precise sub-mitochondrial topology (inner vs. outer membrane contact sites) not resolved\", \"No identification of the protease responsible for N-terminal cleavage\", \"Physiological substrates in vivo not determined\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Demonstrating that Arg-90-dependent cardiolipin binding tethers NME4 to the inner membrane and is required for functional coupling to oxidative phosphorylation answered how the kinase integrates with the respiratory chain.\",\n      \"evidence\": \"Surface plasmon resonance with model liposomes, R90D mutagenesis, respiration assays and latency measurements in HeLa mitochondria\",\n      \"pmids\": [\"18635542\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry of NME4–cardiolipin interaction not quantified\", \"Whether NME4 contacts other lipid species at physiological ratios unknown\", \"Structural basis of hexamer-mediated membrane cross-linking not resolved at atomic level\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Revealing that NME4 selectively transfers cardiolipin from the inner to the outer mitochondrial membrane—while simultaneously forming a complex with the GTPase OPA1—established its dual role as both a lipid transfer protein and a local GTP supplier.\",\n      \"evidence\": \"LC-MS lipidomics of outer membrane fractions, Co-IP of NME4–OPA1 in rat liver, apoptosis assays (cytochrome c release, caspase 3/7) in HeLa cells with wild-type versus membrane-binding-deficient mutant\",\n      \"pmids\": [\"23150663\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether NME4 directly channels GTP to OPA1 or acts indirectly not distinguished\", \"Cardiolipin transfer mechanism (tunnel, flip-flop, or facilitated diffusion) undetermined\", \"Reciprocal OPA1-to-NME4 regulation not tested\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Placing NME4 upstream of JNK–TIMP1–MMP signaling in oral cancer provided the first pathway-level explanation for its invasion-suppressive function, with miR-196 identified as a direct negative regulator of NME4 mRNA.\",\n      \"evidence\": \"Luciferase reporter assay confirming miR-196 targeting of NME4 3′ UTR, migration/invasion assays, downstream marker analysis in oral cancer cells\",\n      \"pmids\": [\"25233933\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether JNK activation by NME4 loss is direct or secondary to mitochondrial dysfunction not clarified\", \"Single cancer type tested\", \"No in vivo metastasis data\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Discovering that SIRT1 deacetylates NME4 and that acetylation status controls its nuclear versus cytoplasmic distribution revealed a post-translational regulatory axis beyond mitochondrial function.\",\n      \"evidence\": \"Yeast two-hybrid identification of SIRT1, Co-IP confirmation, acetylation-mimic mutagenesis, confocal localization, apoptosis assays in N1E-115 cells and mouse cortex knockdown\",\n      \"pmids\": [\"26426123\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Identity of the acetyltransferase is unknown\", \"Specific acetylated lysine residues on NME4 not fully mapped by mass spectrometry\", \"Nuclear function of NME4 remains uncharacterized\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Showing that NME4-mediated cardiolipin externalization serves as an LC3-recognized 'eat-me' signal for mitophagy unified the lipid-transfer and quality-control roles, with NME4 required across multiple mitophagy inducers and cell types.\",\n      \"evidence\": \"RNAi knockdown and R90D mutant analysis, CCCP/rotenone/6-OHDA-induced mitophagy assays, proximity ligation assay for NME4–OPA1, in MLE-12, HeLa, and SH-SY5Y cells\",\n      \"pmids\": [\"26742431\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether NME4 cooperates with PINK1/Parkin pathway or acts in parallel not determined\", \"Degree of cardiolipin externalization required for LC3 recognition not quantified\", \"In vivo relevance in neurodegeneration models not established\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Comprehensive loss-of-function analysis established NME4 as a bona fide metastasis suppressor: loss of either kinase or membrane-binding activity caused mitochondrial fragmentation, metabolic reprogramming to glycolysis, EMT, and metastasis in xenograft models.\",\n      \"evidence\": \"Kinase-dead and membrane-binding-deficient mutants, RNAi knockdown, in vitro migration/invasion, EMT markers, in vivo xenograft metastasis in immunocompromised mice, metabolic profiling\",\n      \"pmids\": [\"34674701\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the two functions (kinase and lipid transfer) contribute equally to suppression is unresolved\", \"Relevance to immunocompetent tumor microenvironment not addressed in this study\", \"Downstream effectors linking metabolic shift to EMT not fully mapped\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Hepatic Nme4 knockout revealed an unexpected metabolic role: NME4 interacts with CoA-metabolizing enzymes and its loss suppresses acetyl-CoA/malonyl-CoA accumulation and steatosis, expanding its function beyond cancer biology.\",\n      \"evidence\": \"Liver-specific Nme4 knockout mice on high-fat diet, proteomics, metabolomics, CoA metabolite quantification\",\n      \"pmids\": [\"38177901\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether NME4 directly phosphorylates CoA pathway enzymes or acts indirectly is unclear\", \"Relevance to human NAFLD not yet demonstrated\", \"Interaction with CoA enzymes not validated by reciprocal pull-down\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"In esophageal squamous cell carcinoma, NME4 suppresses NFκB2-CCL5 signaling and thereby limits CD8+ T cell infiltration, connecting its tumor-suppressive function to immune modulation in the tumor microenvironment.\",\n      \"evidence\": \"Syngeneic C57BL/6 tumor model, single-cell RNA sequencing, quantitative proteomics, protein microarray in murine ESCC cells\",\n      \"pmids\": [\"39016535\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which NME4 suppresses NFκB2 activity not defined\", \"Whether immune modulation is direct or secondary to mitochondrial dysfunction unclear\", \"Single tumor type in syngeneic setting\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Identification of RNF6 as the E3 ligase that K48-ubiquitinates NME4 for proteasomal degradation, activating JNK/c-JUN, defined the major degradation pathway controlling NME4 protein levels in ovarian cancer.\",\n      \"evidence\": \"Co-IP of RNF6–NME4, K48-linked ubiquitination assay, cycloheximide chase, rescue experiments, nude mouse xenograft\",\n      \"pmids\": [\"41616518\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific ubiquitinated lysine residues on NME4 not mapped\", \"Whether RNF6-NME4 axis operates in non-cancer contexts unknown\", \"Deubiquitinase that opposes RNF6 not identified\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How NME4's two biochemical activities—NDP kinase and cardiolipin transfer—are coordinately regulated in vivo, and whether its nuclear pool performs distinct functions, remain major open questions.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structural model of NME4 bound to cardiolipin at atomic resolution\", \"Nuclear function of NME4 entirely uncharacterized\", \"Relationship between NME4-mediated mitophagy and PINK1/Parkin pathway not resolved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 1, 2]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [1, 2, 3]},\n      {\"term_id\": \"GO:0140104\", \"supporting_discovery_ids\": [2, 3]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [5, 6, 8]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [0, 1, 2, 3]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [3, 6]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [2, 4]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [0, 1, 7]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [5, 6, 8, 10]}\n    ],\n    \"complexes\": [\n      \"NDPK-D hexamer\"\n    ],\n    \"partners\": [\n      \"OPA1\",\n      \"SIRT1\",\n      \"RNF6\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}