{"gene":"NME4","run_date":"2026-06-10T05:19:52","timeline":{"discoveries":[{"year":2000,"finding":"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.","method":"X-ray crystallography, site-directed mutagenesis, GFP-fusion confocal microscopy, Western blot subcellular fractionation, in vitro enzymatic activity assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure, mutagenesis, in vitro activity assays, and direct localization experiments in a single rigorous study","pmids":["10799505"],"is_preprint":false},{"year":2008,"finding":"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.","method":"Surface plasmon resonance, site-directed mutagenesis (R90D), stable cell expression, liposome cross-linking assay, mitochondrial respiration assay, submitochondrial fractionation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple orthogonal methods (SPR, mutagenesis, functional respiration assay, fractionation) in one rigorous study","pmids":["18635542"],"is_preprint":false},{"year":2012,"finding":"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.","method":"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","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — reciprocal Co-IP, quantitative lipid mass spectrometry, functional mutagenesis, and multiple apoptosis readouts in one study","pmids":["23150663"],"is_preprint":false},{"year":2016,"finding":"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.","method":"RNAi knockdown, site-directed mutagenesis (R90D), in situ proximity ligation assay (PLA), flow cytometry/imaging of CL externalization, mitophagy assays in multiple cell lines","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 2 / Strong — RNAi with specific phenotypic readout, loss-of-function mutagenesis, PLA for proximity, replicated across multiple cell lines and mitophagy triggers","pmids":["26742431"],"is_preprint":false},{"year":2015,"finding":"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.","method":"Yeast two-hybrid screening, co-immunoprecipitation, site-directed mutagenesis (acetylation-mimic), confocal microscopy (co-localization), SIRT1 inhibitor treatment, RNAi knockdown, apoptosis assay","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — yeast two-hybrid confirmed by Co-IP and mutagenesis, single lab, two orthogonal methods for interaction","pmids":["26426123"],"is_preprint":false},{"year":2014,"finding":"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.","method":"RT-qPCR, Western blot, luciferase reporter assay (target validation), confocal microscopy, cell migration/invasion assays","journal":"Molecular cancer","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — luciferase reporter confirms direct targeting, Western blot and functional assays support pathway placement, single lab","pmids":["25233933"],"is_preprint":false},{"year":2021,"finding":"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.","method":"Loss-of-function mutagenesis, RNAi knockdown, in vivo xenograft metastasis assay, EMT marker analysis, mitochondrial morphology imaging, metabolic assays (respiration, glycolysis), ROS measurement","journal":"BMC biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — complementary loss-of-function approaches (mutagenesis + RNAi), in vivo metastasis model, multiple orthogonal phenotypic readouts","pmids":["34674701"],"is_preprint":false},{"year":2023,"finding":"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.","method":"Proteomics (interaction partners), metabolomics, hepatic Nme4 knockout (in vivo), high-fat diet mouse model, lipid quantification","journal":"EMBO reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo knockout with defined metabolic phenotype, proteomics for interaction partners, single lab","pmids":["38177901"],"is_preprint":false},{"year":2024,"finding":"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.","method":"Syngeneic murine tumor model, single-cell RNA sequencing, quantitative proteomics, protein microarray, in vivo tumor experiments","journal":"Immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo syngeneic model with scRNA-seq and proteomics, single lab, novel pathway placement","pmids":["39016535"],"is_preprint":false},{"year":2026,"finding":"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.","method":"Co-immunoprecipitation, cycloheximide (CHX) chase assay, ubiquitination assay, RNF6/NME4 co-modulation rescue experiments, nude mouse xenograft model","journal":"Pathology, research and practice","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP, ubiquitination assay, CHX chase, and rescue experiments in single lab confirm K48-linked degradation mechanism","pmids":["41616518"],"is_preprint":false},{"year":2021,"finding":"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.","method":"Luciferase reporter assay (3' UTR targeting), miRNA mimic/inhibitor transfection, ectopic Nme4 expression (rescue), in vivo ovariectomized animal model, ALP/alizarin red staining","journal":"Biochemistry and cell biology","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — luciferase reporter validates direct 3' UTR targeting, rescue experiment and in vivo model support pathway placement, single lab","pmids":["34297624"],"is_preprint":false},{"year":2024,"finding":"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.","method":"Molecular dynamics simulation, computational modeling of hexameric assembly, mutation analysis (computational)","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 4 / Weak — computational prediction only, no experimental validation of the NME4-specific findings in this preprint","pmids":["bio_10.1101_2024.09.19.613900"],"is_preprint":true}],"current_model":"NME4 (NDPK-D/NM23-H4) is a mitochondrial inner membrane-associated hexameric nucleoside diphosphate kinase that, in addition to phosphotransfer activity (locally supplying GTP to OPA1 and coupling to oxidative phosphorylation via ANT), functions as a cardiolipin-binding lipid transfer protein that translocates cardiolipin from the inner to the outer mitochondrial membrane to serve as a mitophagy 'eat-me' signal (recognized by LC3) and an apoptotic signal; its membrane association depends on Arg90 in the RRK motif, its activity is regulated by SIRT1-mediated acetylation that controls nuclear-cytoplasmic shuttling, and its stability is regulated by RNF6-mediated K48-linked polyubiquitination and proteasomal degradation, while its loss promotes epithelial-mesenchymal transition, metastasis, and metabolic reprogramming via mitochondrial fragmentation and a shift to glycolysis."},"narrative":{"mechanistic_narrative":"NME4 (NDPK-D/Nm23-H4) is a mitochondrial nucleoside diphosphate kinase that doubles as a cardiolipin-binding lipid transfer protein, linking nucleotide phosphotransfer to the regulation of mitochondrial membrane lipid composition, apoptosis, mitophagy, and metabolism [PMID:10799505, PMID:23150663]. The protein is imported into mitochondria where cleavage of an N-terminal targeting extension relieves an autoinhibitory constraint to activate NDP kinase activity, and the active enzyme assembles into a hexamer that associates peripherally with the inner membrane [PMID:10799505]. Membrane binding occurs through electrostatic interaction with cardiolipin via the surface-exposed RRK motif, with Arg90 being essential; this membrane-bound state couples NDPK-D phosphotransfer to oxidative phosphorylation [PMID:18635542]. The symmetrical hexamer cross-links anionic-phospholipid membranes and, upon cardiolipin binding, switches from kinase activity to selective intermembrane transfer of cardiolipin, moving it from the inner to the outer membrane where it serves as both a pro-apoptotic and a mitophagy signal; this lipid-transfer function requires the membrane-binding state and is spatially coupled to the dynamin-like GTPase OPA1 [PMID:23150663, PMID:26742431]. Consistent with a role in mitochondrial integrity, loss of either NDP kinase activity or membrane binding drives mitochondrial fragmentation, a glycolytic metabolic shift, increased ROS, epithelial-mesenchymal transition, and metastasis, establishing NME4 as a metastasis suppressor [PMID:34674701]. NME4 abundance and localization are further controlled post-translationally: SIRT1-mediated deacetylation regulates its acetylation state and nuclear accumulation [PMID:26426123], and RNF6-mediated K48-linked polyubiquitination targets it for proteasomal degradation [PMID:41616518].","teleology":[{"year":2000,"claim":"Established that NME4 is a mitochondrial NDP kinase whose activity is gated by import-coupled processing, answering how its enzymatic function is spatially and structurally regulated.","evidence":"X-ray crystallography, GFP-fusion confocal microscopy, subcellular fractionation, and in vitro activity assays in HEK293 cells","pmids":["10799505"],"confidence":"High","gaps":["Did not define the lipid determinants of membrane association","Did not address functions beyond phosphotransfer"]},{"year":2008,"claim":"Identified cardiolipin as the principal membrane anchor of NDPK-D via the Arg90 RRK motif and showed that membrane binding couples the kinase to oxidative phosphorylation, explaining the functional basis of its inner-membrane association.","evidence":"Surface plasmon resonance with liposomes, R90D mutagenesis, mitochondrial respiration assays, and submitochondrial fractionation","pmids":["18635542"],"confidence":"High","gaps":["Did not establish a non-kinase function for the membrane-bound protein","Mechanism of respiratory coupling (partner identity) not fully resolved here"]},{"year":2012,"claim":"Revealed a moonlighting lipid-transfer function whereby cardiolipin binding switches off kinase activity and enables inner-to-outer membrane cardiolipin transfer that promotes apoptosis, redefining NME4 as a dual-function protein.","evidence":"Co-IP of the OPA1 complex, LC-MS lipid analysis, membrane-binding-deficient mutagenesis, and apoptosis assays in stable cell lines","pmids":["23150663"],"confidence":"High","gaps":["Did not establish the OPA1 interaction as direct versus indirect","Did not link the transfer activity to mitophagy"]},{"year":2016,"claim":"Demonstrated that the cardiolipin externalized by NDPK-D acts as a mitophagy 'eat-me' signal, connecting the lipid-transfer activity to organelle quality control and OPA1-dependent dynamics.","evidence":"RNAi knockdown, R90D mutagenesis, in situ proximity ligation assay, and mitophagy assays across MLE-12, HeLa, and SH-SY5Y cells with multiple triggers","pmids":["26742431"],"confidence":"High","gaps":["Did not identify the receptor recognizing externalized cardiolipin in all contexts","Did not resolve how fission-fusion and CL transfer are mechanistically coupled"]},{"year":2015,"claim":"Uncovered acetylation as a regulatory layer controlling NDPK-D localization, showing SIRT1 binds and deacetylates NDPK-D to govern its nuclear accumulation and apoptotic output.","evidence":"Yeast two-hybrid, co-IP, acetylation-mimic mutagenesis, SIRT1 inhibition, and apoptosis assays in neuroblastoma cells and mouse cortex","pmids":["26426123"],"confidence":"Medium","gaps":["Specific acetylated lysines not definitively mapped","Functional role of nuclear NDPK-D not defined","Single-lab interaction evidence"]},{"year":2021,"claim":"Established NME4 as a metastasis suppressor by showing that loss of either kinase or membrane-binding activity drives mitochondrial fragmentation, glycolytic switching, and EMT, tying its molecular functions to a cancer phenotype.","evidence":"Loss-of-function mutagenesis, RNAi, in vivo xenograft metastasis assay, and metabolic/ROS/morphology readouts","pmids":["34674701"],"confidence":"High","gaps":["Did not separate the contributions of kinase versus lipid-transfer activity to the metastasis phenotype","Upstream regulators of NME4 loss in tumors not defined here"]},{"year":2023,"claim":"Extended NME4 function into hepatic lipid metabolism, showing it interacts with CoA-metabolism enzymes and promotes lipid accumulation, with hepatic deletion protecting against steatosis.","evidence":"Proteomics, metabolomics, and hepatic Nme4 knockout in a high-fat-diet mouse model","pmids":["38177901"],"confidence":"Medium","gaps":["Direct enzymatic partners in CoA metabolism not biochemically validated","Mechanistic link to mitochondrial cardiolipin transfer not established","Single lab"]},{"year":2024,"claim":"Placed NME4 in tumor immune evasion by showing it suppresses the NFkB2-CCL5 axis to restrict CD8+ T cell infiltration, broadening its role beyond cell-intrinsic mitochondrial functions.","evidence":"Syngeneic murine tumor model with single-cell RNA sequencing, quantitative proteomics, and protein microarray in esophageal squamous cell carcinoma","pmids":["39016535"],"confidence":"Medium","gaps":["Mechanism connecting mitochondrial NME4 to NFkB2-CCL5 signaling unresolved","Direct molecular target in the pathway not identified","Single lab"]},{"year":2026,"claim":"Identified RNF6 as an E3 ligase that controls NME4 stability via K48-linked polyubiquitination, defining a degradative regulatory mechanism with downstream JNK/c-JUN consequences in ovarian cancer.","evidence":"Co-IP, CHX chase, ubiquitination assay, co-modulation rescue, and nude mouse xenograft","pmids":["41616518"],"confidence":"Medium","gaps":["Ubiquitinated lysine residues not mapped","Whether degradation is regulated under physiological signals not addressed","Single lab"]},{"year":null,"claim":"How NME4's two activities (phosphotransfer versus cardiolipin transfer) are switched in vivo, and how its post-translational regulation integrates with its metabolic and immune-modulatory roles, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model linking acetylation, ubiquitination, and lipid-transfer regulation","Structural basis of the kinase-to-transferase switch not solved","Physiological triggers governing the switch unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,1]},{"term_id":"GO:0140098","term_label":"catalytic activity, acting on RNA","supporting_discovery_ids":[0]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[1,2]},{"term_id":"GO:0140104","term_label":"molecular carrier activity","supporting_discovery_ids":[2,3]}],"localization":[{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[0,1]}],"pathway":[{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[3]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[2]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[6,7]}],"complexes":[],"partners":["OPA1","SIRT1","RNF6"],"other_free_text":[]}},"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":165,"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. 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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":14,"is_preprint":false},{"pmid":"11724361","id":"PMC_11724361","title":"Overexpression of nm23-H4 RNA in colorectal and renal tumours.","date":"2001","source":"Anticancer 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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":"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":"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":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":15212,"output_tokens":3984,"usd":0.052698,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":11758,"output_tokens":3535,"usd":0.073582,"stage2_stop_reason":"end_turn"},"total_usd":0.12628,"stage1_batch_id":"msgbatch_013a8chtv3AWyTqv4z4sEF4C","stage2_batch_id":"msgbatch_01AHsuW63vinsvuTgzmQ3n62","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 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.\",\n      \"method\": \"X-ray crystallography, site-directed mutagenesis, GFP-fusion confocal microscopy, Western blot subcellular fractionation, in vitro enzymatic activity assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure, mutagenesis, in vitro activity assays, and direct localization experiments in a single rigorous study\",\n      \"pmids\": [\"10799505\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"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.\",\n      \"method\": \"Surface plasmon resonance, site-directed mutagenesis (R90D), stable cell expression, liposome cross-linking assay, mitochondrial respiration assay, submitochondrial fractionation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple orthogonal methods (SPR, mutagenesis, functional respiration assay, fractionation) in one rigorous study\",\n      \"pmids\": [\"18635542\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"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.\",\n      \"method\": \"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\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — reciprocal Co-IP, quantitative lipid mass spectrometry, functional mutagenesis, and multiple apoptosis readouts in one study\",\n      \"pmids\": [\"23150663\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"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.\",\n      \"method\": \"RNAi knockdown, site-directed mutagenesis (R90D), in situ proximity ligation assay (PLA), flow cytometry/imaging of CL externalization, mitophagy assays in multiple cell lines\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — RNAi with specific phenotypic readout, loss-of-function mutagenesis, PLA for proximity, replicated across multiple cell lines and mitophagy triggers\",\n      \"pmids\": [\"26742431\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"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.\",\n      \"method\": \"Yeast two-hybrid screening, co-immunoprecipitation, site-directed mutagenesis (acetylation-mimic), confocal microscopy (co-localization), SIRT1 inhibitor treatment, RNAi knockdown, apoptosis assay\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — yeast two-hybrid confirmed by Co-IP and mutagenesis, single lab, two orthogonal methods for interaction\",\n      \"pmids\": [\"26426123\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"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.\",\n      \"method\": \"RT-qPCR, Western blot, luciferase reporter assay (target validation), confocal microscopy, cell migration/invasion assays\",\n      \"journal\": \"Molecular cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — luciferase reporter confirms direct targeting, Western blot and functional assays support pathway placement, single lab\",\n      \"pmids\": [\"25233933\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"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.\",\n      \"method\": \"Loss-of-function mutagenesis, RNAi knockdown, in vivo xenograft metastasis assay, EMT marker analysis, mitochondrial morphology imaging, metabolic assays (respiration, glycolysis), ROS measurement\",\n      \"journal\": \"BMC biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — complementary loss-of-function approaches (mutagenesis + RNAi), in vivo metastasis model, multiple orthogonal phenotypic 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 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.\",\n      \"method\": \"Proteomics (interaction partners), metabolomics, hepatic Nme4 knockout (in vivo), high-fat diet mouse model, lipid quantification\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo knockout with defined metabolic phenotype, proteomics for interaction partners, single lab\",\n      \"pmids\": [\"38177901\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"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.\",\n      \"method\": \"Syngeneic murine tumor model, single-cell RNA sequencing, quantitative proteomics, protein microarray, in vivo tumor experiments\",\n      \"journal\": \"Immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo syngeneic model with scRNA-seq and proteomics, single lab, novel pathway placement\",\n      \"pmids\": [\"39016535\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"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.\",\n      \"method\": \"Co-immunoprecipitation, cycloheximide (CHX) chase assay, ubiquitination assay, RNF6/NME4 co-modulation rescue experiments, nude mouse xenograft model\",\n      \"journal\": \"Pathology, research and practice\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP, ubiquitination assay, CHX chase, and rescue experiments in single lab confirm K48-linked degradation mechanism\",\n      \"pmids\": [\"41616518\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"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.\",\n      \"method\": \"Luciferase reporter assay (3' UTR targeting), miRNA mimic/inhibitor transfection, ectopic Nme4 expression (rescue), in vivo ovariectomized animal model, ALP/alizarin red staining\",\n      \"journal\": \"Biochemistry and cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — luciferase reporter validates direct 3' UTR targeting, rescue experiment and in vivo model support pathway placement, single lab\",\n      \"pmids\": [\"34297624\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"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.\",\n      \"method\": \"Molecular dynamics simulation, computational modeling of hexameric assembly, mutation analysis (computational)\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 / Weak — computational prediction only, no experimental validation of the NME4-specific findings in this preprint\",\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 inner membrane-associated hexameric nucleoside diphosphate kinase that, in addition to phosphotransfer activity (locally supplying GTP to OPA1 and coupling to oxidative phosphorylation via ANT), functions as a cardiolipin-binding lipid transfer protein that translocates cardiolipin from the inner to the outer mitochondrial membrane to serve as a mitophagy 'eat-me' signal (recognized by LC3) and an apoptotic signal; its membrane association depends on Arg90 in the RRK motif, its activity is regulated by SIRT1-mediated acetylation that controls nuclear-cytoplasmic shuttling, and its stability is regulated by RNF6-mediated K48-linked polyubiquitination and proteasomal degradation, while its loss promotes epithelial-mesenchymal transition, metastasis, and metabolic reprogramming via mitochondrial fragmentation and a shift to glycolysis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"NME4 (NDPK-D/Nm23-H4) is a mitochondrial nucleoside diphosphate kinase that doubles as a cardiolipin-binding lipid transfer protein, linking nucleotide phosphotransfer to the regulation of mitochondrial membrane lipid composition, apoptosis, mitophagy, and metabolism [#0, #2]. The protein is imported into mitochondria where cleavage of an N-terminal targeting extension relieves an autoinhibitory constraint to activate NDP kinase activity, and the active enzyme assembles into a hexamer that associates peripherally with the inner membrane [#0]. Membrane binding occurs through electrostatic interaction with cardiolipin via the surface-exposed RRK motif, with Arg90 being essential; this membrane-bound state couples NDPK-D phosphotransfer to oxidative phosphorylation [#1]. The symmetrical hexamer cross-links anionic-phospholipid membranes and, upon cardiolipin binding, switches from kinase activity to selective intermembrane transfer of cardiolipin, moving it from the inner to the outer membrane where it serves as both a pro-apoptotic and a mitophagy signal; this lipid-transfer function requires the membrane-binding state and is spatially coupled to the dynamin-like GTPase OPA1 [#2, #3]. Consistent with a role in mitochondrial integrity, loss of either NDP kinase activity or membrane binding drives mitochondrial fragmentation, a glycolytic metabolic shift, increased ROS, epithelial-mesenchymal transition, and metastasis, establishing NME4 as a metastasis suppressor [#6]. NME4 abundance and localization are further controlled post-translationally: SIRT1-mediated deacetylation regulates its acetylation state and nuclear accumulation [#4], and RNF6-mediated K48-linked polyubiquitination targets it for proteasomal degradation [#9].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Established that NME4 is a mitochondrial NDP kinase whose activity is gated by import-coupled processing, answering how its enzymatic function is spatially and structurally regulated.\",\n      \"evidence\": \"X-ray crystallography, GFP-fusion confocal microscopy, subcellular fractionation, and in vitro activity assays in HEK293 cells\",\n      \"pmids\": [\"10799505\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the lipid determinants of membrane association\", \"Did not address functions beyond phosphotransfer\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Identified cardiolipin as the principal membrane anchor of NDPK-D via the Arg90 RRK motif and showed that membrane binding couples the kinase to oxidative phosphorylation, explaining the functional basis of its inner-membrane association.\",\n      \"evidence\": \"Surface plasmon resonance with liposomes, R90D mutagenesis, mitochondrial respiration assays, and submitochondrial fractionation\",\n      \"pmids\": [\"18635542\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish a non-kinase function for the membrane-bound protein\", \"Mechanism of respiratory coupling (partner identity) not fully resolved here\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Revealed a moonlighting lipid-transfer function whereby cardiolipin binding switches off kinase activity and enables inner-to-outer membrane cardiolipin transfer that promotes apoptosis, redefining NME4 as a dual-function protein.\",\n      \"evidence\": \"Co-IP of the OPA1 complex, LC-MS lipid analysis, membrane-binding-deficient mutagenesis, and apoptosis assays in stable cell lines\",\n      \"pmids\": [\"23150663\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish the OPA1 interaction as direct versus indirect\", \"Did not link the transfer activity to mitophagy\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Demonstrated that the cardiolipin externalized by NDPK-D acts as a mitophagy 'eat-me' signal, connecting the lipid-transfer activity to organelle quality control and OPA1-dependent dynamics.\",\n      \"evidence\": \"RNAi knockdown, R90D mutagenesis, in situ proximity ligation assay, and mitophagy assays across MLE-12, HeLa, and SH-SY5Y cells with multiple triggers\",\n      \"pmids\": [\"26742431\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the receptor recognizing externalized cardiolipin in all contexts\", \"Did not resolve how fission-fusion and CL transfer are mechanistically coupled\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Uncovered acetylation as a regulatory layer controlling NDPK-D localization, showing SIRT1 binds and deacetylates NDPK-D to govern its nuclear accumulation and apoptotic output.\",\n      \"evidence\": \"Yeast two-hybrid, co-IP, acetylation-mimic mutagenesis, SIRT1 inhibition, and apoptosis assays in neuroblastoma cells and mouse cortex\",\n      \"pmids\": [\"26426123\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific acetylated lysines not definitively mapped\", \"Functional role of nuclear NDPK-D not defined\", \"Single-lab interaction evidence\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Established NME4 as a metastasis suppressor by showing that loss of either kinase or membrane-binding activity drives mitochondrial fragmentation, glycolytic switching, and EMT, tying its molecular functions to a cancer phenotype.\",\n      \"evidence\": \"Loss-of-function mutagenesis, RNAi, in vivo xenograft metastasis assay, and metabolic/ROS/morphology readouts\",\n      \"pmids\": [\"34674701\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not separate the contributions of kinase versus lipid-transfer activity to the metastasis phenotype\", \"Upstream regulators of NME4 loss in tumors not defined here\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Extended NME4 function into hepatic lipid metabolism, showing it interacts with CoA-metabolism enzymes and promotes lipid accumulation, with hepatic deletion protecting against steatosis.\",\n      \"evidence\": \"Proteomics, metabolomics, and hepatic Nme4 knockout in a high-fat-diet mouse model\",\n      \"pmids\": [\"38177901\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct enzymatic partners in CoA metabolism not biochemically validated\", \"Mechanistic link to mitochondrial cardiolipin transfer not established\", \"Single lab\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Placed NME4 in tumor immune evasion by showing it suppresses the NFkB2-CCL5 axis to restrict CD8+ T cell infiltration, broadening its role beyond cell-intrinsic mitochondrial functions.\",\n      \"evidence\": \"Syngeneic murine tumor model with single-cell RNA sequencing, quantitative proteomics, and protein microarray in esophageal squamous cell carcinoma\",\n      \"pmids\": [\"39016535\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism connecting mitochondrial NME4 to NFkB2-CCL5 signaling unresolved\", \"Direct molecular target in the pathway not identified\", \"Single lab\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Identified RNF6 as an E3 ligase that controls NME4 stability via K48-linked polyubiquitination, defining a degradative regulatory mechanism with downstream JNK/c-JUN consequences in ovarian cancer.\",\n      \"evidence\": \"Co-IP, CHX chase, ubiquitination assay, co-modulation rescue, and nude mouse xenograft\",\n      \"pmids\": [\"41616518\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Ubiquitinated lysine residues not mapped\", \"Whether degradation is regulated under physiological signals not addressed\", \"Single lab\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How NME4's two activities (phosphotransfer versus cardiolipin transfer) are switched in vivo, and how its post-translational regulation integrates with its metabolic and immune-modulatory roles, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model linking acetylation, ubiquitination, and lipid-transfer regulation\", \"Structural basis of the kinase-to-transferase switch not solved\", \"Physiological triggers governing the switch unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [1, 2]},\n      {\"term_id\": \"GO:0140104\", \"supporting_discovery_ids\": [2, 3]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0005743\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [3]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [2]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [6, 7]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"OPA1\", \"SIRT1\", \"RNF6\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"tie","faith_supported":5,"faith_total":6,"faith_pct":83.33333333333333}}