{"gene":"DDHD2","run_date":"2026-06-09T23:54:41","timeline":{"discoveries":[{"year":2009,"finding":"iPLA1γ/DDHD2 localizes to the cytosol, cis-Golgi, and ERGIC, and mediates a COPI- and Rab6-independent retrograde membrane transport pathway from the Golgi complex to the ER; RNAi knockdown delayed BFA-induced Golgi-to-ER transfer and cholera toxin B retrograde transport without affecting anterograde VSVGts045 transport or ERGIC-53/Shiga toxin recycling.","method":"RNAi knockdown, time-lapse microscopy, GFP-fusion localization, BFA assay, cholera toxin B retrograde transport assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal functional knockdown with multiple orthogonal transport assays, live imaging, single lab","pmids":["19632984"],"is_preprint":false},{"year":2012,"finding":"Mutations in DDHD2 that affect the DDHD domain (required for phospholipase activity) cause SPG54; knockdown of the Drosophila ortholog reduced active zones at synaptic terminals, implicating DDHD2 in synaptic function.","method":"Drosophila Ddhd RNAi knockdown with synapse morphology analysis; human genetics (mutation identification)","journal":"American journal of human genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — in vivo model organism knockdown with defined morphological readout, single lab, no biochemical reconstitution","pmids":["23176823"],"is_preprint":false},{"year":2014,"finding":"DDHD2 is the principal triglyceride (triacylglycerol/TAG) hydrolase in the brain; DDHD2−/− mice accumulate TAGs selectively in the CNS (not peripheral tissues), with lipid droplets localizing to neuronal intracellular compartments; recombinant DDHD2 displays TAG hydrolase activity in vitro; a selective in vivo DDHD2 inhibitor also causes brain TAG accumulation in wild-type mice.","method":"DDHD2−/− mouse generation, mass spectrometry-based lipidomics, in vitro TAG hydrolase assay with recombinant enzyme, selective inhibitor pharmacology, electron microscopy","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — genetic KO + recombinant enzyme assay + selective inhibitor, replicated across multiple approaches in single rigorous study","pmids":["25267624"],"is_preprint":false},{"year":2015,"finding":"A truncating mutation (p.R287X) in DDHD2 removes the SAM and DDHD domains, which are crucial for phosphoinositide binding and phospholipase activity, confirming these domains are essential for DDHD2 function.","method":"Exome sequencing, homozygosity mapping, domain analysis of truncated protein","journal":"BMC research notes","confidence":"Low","confidence_rationale":"Tier 3 / Weak — domain inference from truncation mutation, no direct biochemical assay of truncated protein","pmids":["26113134"],"is_preprint":false},{"year":2014,"finding":"A DDHD2 missense mutation (identified in late-onset spastic ataxia patients) caused a marked reduction in phospholipase A1 activity, and patients showed abnormal lipid peak on MRS consistent with lipid accumulation.","method":"Phospholipase A1 activity assay of mutant DDHD2, patient MR spectroscopy","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — direct enzymatic assay of disease-associated mutant, single lab, single method","pmids":["25417924"],"is_preprint":false},{"year":2016,"finding":"Recombinant rat DDHD2 functions preferentially as a diacylglycerol (DG) lipase; kcat/Km for DG(18:0/20:4) was much higher than for TAG or phosphatidic acid; DDHD2 shows preference for DG substrates with polyunsaturated fatty acids at the sn-2 position; DDHD2-expressing CHO cells showed elevated 2-arachidonoylglycerol (2-AG) levels, supporting a DG lipase role in vivo.","method":"Enzymatic assay with purified recombinant protein, LC-MS substrate specificity profiling, GC-MS/MS quantification of 2-AG in transfected CHO cells","journal":"Journal of biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with purified enzyme, multiple substrates tested by LC-MS, and cellular validation with GC-MS/MS","pmids":["27198176"],"is_preprint":false},{"year":2017,"finding":"HSP-related mutations in DDHD2 disrupt its triglyceride hydrolase activity in vitro and impair DDHD2's capacity to protect cells from lipid droplet accumulation upon free fatty acid exposure; lipid droplets in DDHD2−/− brains contain both known LD-associated proteins and CNS-enriched proteins including several with links to neurological disease.","method":"In vitro TAG hydrolase assay of HSP mutants, cell-based LD accumulation assay with free fatty acid treatment, DDHD2 inhibitor, LD proteomics from KO brain tissue","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro enzyme assay of multiple disease mutants plus cell-based functional assay plus LD proteomics, single lab, multiple orthogonal methods","pmids":["29278326"],"is_preprint":false},{"year":2018,"finding":"DDHD2 ablation causes age-dependent apoptosis of motor neurons in mouse spinal cord; DDHD2 KO cells show decreased cardiolipin content and increased reactive oxygen species (ROS); ROS increase was reversed by wild-type DDHD2 re-expression but not by catalytically inactive DDHD2, HSP-associated DDHD2 mutants, or DDHD1, establishing that DDHD2 lipase activity is required for mitochondrial protection.","method":"DDHD2−/− mice, ROS measurement (chemical and probe-based), cardiolipin quantification, rescue by wild-type vs. active-site or HSP mutant DDHD2 re-expression, apoptosis assays","journal":"Cell death & disease","confidence":"High","confidence_rationale":"Tier 2 / Moderate — KO with defined phenotype, active-site mutagenesis rescue, multiple orthogonal assays, single lab","pmids":["30038238"],"is_preprint":false},{"year":2020,"finding":"DDHD2 depletion prevents neurite elongation in SH-SY5Y and PC12 cells (opposite to DDHD1 depletion, which promotes elongation), indicating a distinct and non-redundant role for DDHD2 in neurite outgrowth regulation.","method":"siRNA depletion, neurite length measurement in SH-SY5Y and PC12 cells","journal":"Frontiers in cell and developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — loss-of-function with defined cellular phenotype, single lab, single method per condition","pmids":["32850804"],"is_preprint":false},{"year":2023,"finding":"In primary cortical neurons, DDHD2 functions as a dual TAG/DAG hydrolase and complements ATGL-dependent TAG hydrolysis; in neuroblastoma cells, DDHD2 acts exclusively downstream of ATGL on DAG isomers but is dispensable for TAG hydrolysis, revealing cell-type-specific lipolysome configurations.","method":"In vitro acylglycerol hydrolase assays, neutral lipid hydrolase activity measurements in neuroblastoma cells and brain tissue, primary cortical neuron studies with DDHD2 and ATGL modulation","journal":"Journal of lipid research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro enzyme assays plus cell-based studies in two cell types, single lab","pmids":["37832604"],"is_preprint":false},{"year":2024,"finding":"DDHD2 interacts with multiple ATG8-family proteins (LC3s and GABARAPs) via two authentic LIR motifs; this interaction promotes lipophagy, as DDHD2 enhances colocalization of LC3B with lipid droplets; LC3/GABARAP-binding capacity and canonical autophagy both contribute to DDHD2's LD-eliminating activity.","method":"Affinity purification-mass spectrometry (AP-MS), mutational analysis of LIR motifs, LC3B/LD colocalization assays, LD quantification upon DDHD2 overexpression/deficiency, LD·ATTEC compound rescue","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 2 / Moderate — AP-MS identification of interactors, LIR mutational validation, multiple functional assays in single rigorous study","pmids":["38332048"],"is_preprint":false},{"year":2024,"finding":"DDHD2 binds the synaptic protein STXBP1 (identified by pulldown-mass spectrometry); STXBP1 controls targeting of DDHD2 to the plasma membrane and generation of saturated free fatty acids in the brain; genetic ablation of DDHD2 dramatically reduces saturated FFA responses to memory acquisition and impairs reward-based and spatial memory performance.","method":"Pulldown-mass spectrometry, STXBP1/2 KO neurosecretory cells, haploinsufficient STXBP1+/− mouse model, DDHD2 KO mice, lipidomics, behavioral memory assays","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Moderate — pulldown-MS binding identification plus genetic models (KO and haploinsufficient) plus lipidomics and behavioral phenotyping, multiple orthogonal approaches in single study","pmids":["38316990"],"is_preprint":false},{"year":2024,"finding":"DDHD2 knockdown in human artery endothelial cells reduces GPX4 and Nrf2 protein levels and increases lipid peroxidation; co-immunoprecipitation indicates a physical interaction between DDHD2 and Nrf2, suggesting DDHD2 regulates the Nrf2/GPX4 ferroptosis-protective pathway.","method":"DDHD2 siRNA knockdown, co-immunoprecipitation, western blot for GPX4/Nrf2, lipid peroxidation assay","journal":"Biomolecules","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP plus KD phenotype, single lab, no reciprocal IP or direct mechanistic follow-up","pmids":["39062593"],"is_preprint":false},{"year":2025,"finding":"Recombinant human DDHD2 preferentially hydrolyzes DAG over phospholipids and shows slight preference for DAG over TAG; DDHD2 also exhibits transacylase activity, transferring acyl chains from TAGs to DAGs and monoacylglycerols to remodel TAG acyl chains; a predicted hydrophobic amphipathic helix is essential for lipid droplet binding in vitro and in cells and is required for full enzymatic activity and TAG acyl-chain remodeling.","method":"In vitro enzyme assays with recombinant human DDHD2, substrate specificity profiling, transacylase activity assay, amphipathic helix deletion mutagenesis, in vitro LD-binding assay, cell-based LD localization","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Moderate — reconstitution with purified recombinant enzyme, multiple substrate assays, mutagenesis of structural element, in vitro and cell-based validation","pmids":["41264248"],"is_preprint":false},{"year":2025,"finding":"DDHD2-dependent release of long-chain saturated free fatty acids (myristic, palmitic, stearic acids) in an activity-dependent manner supports mitochondrial β-oxidation and ATP synthesis in neurons; genetic ablation of Ddhd2 impairs mitochondrial respiration and ATP production despite increased glycolysis; saturated fatty acyl-CoA supplementation rescues mitochondrial energy production, membrane trafficking, synaptic function, and protein homeostasis defects in Ddhd2 KO neurons.","method":"Ddhd2 KO neurons, Seahorse mitochondrial respiration assay, ATP measurement, glycolysis assay, mitochondrial fatty acid import inhibition, saturated fatty acyl-CoA rescue experiments","journal":"Nature metabolism","confidence":"High","confidence_rationale":"Tier 2 / Moderate — KO with mechanistic rescue, multiple orthogonal assays (respiration, ATP, glycolysis, inhibitor experiments), single lab","pmids":["41028912"],"is_preprint":false},{"year":2025,"finding":"Acute DDHD2 inhibition (by compound KLH45) causing lipid droplet accumulation in primary rat cortical neurons did not affect total α-synuclein levels, phosphoserine-129 status, or solubility, and no colocalization between LDs and α-synuclein was detected.","method":"DDHD2 inhibitor KLH45 in primary rat cortical neurons, western blot for α-synuclein and pSer129, detergent solubility fractionation, immunofluorescence colocalization","journal":"Metabolic brain disease","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — pharmacological inhibition with defined negative outcome, multiple readouts, single lab — this is a mechanistically informative negative result","pmids":["39853540"],"is_preprint":false}],"current_model":"DDHD2 is a serine lipase/phospholipase A1 that functions as the principal brain triglyceride and diacylglycerol hydrolase, releasing long-chain saturated free fatty acids to fuel neuronal mitochondrial β-oxidation and ATP synthesis; it also exhibits transacylase activity to remodel TAG acyl chains, promotes lipid droplet catabolism via direct interaction with ATG8-family proteins (LC3/GABARAPs) through LIR motifs to enhance lipophagy, mediates COPI/Rab6-independent Golgi-to-ER retrograde membrane transport, and interacts with STXBP1 to enable plasma membrane targeting and saturated FFA generation required for synaptic plasticity and memory; loss-of-function causes lipid droplet accumulation in neurons, mitochondrial dysfunction, motor neuron apoptosis, and the SPG54 form of hereditary spastic paraplegia."},"narrative":{"mechanistic_narrative":"DDHD2 is a brain-enriched serine lipase that serves as the principal neuronal triacylglycerol and diacylglycerol hydrolase, coupling lipid droplet catabolism to neuronal energy metabolism and synaptic function [PMID:25267624, PMID:27198176, PMID:41028912]. Genetic ablation in mice causes CNS-selective accumulation of triglycerides and neuronal lipid droplets, and recombinant enzyme directly hydrolyzes TAG; a selective inhibitor reproduces the brain lipid phenotype in wild-type animals [PMID:25267624]. Beyond TAG, DDHD2 preferentially acts on diacylglycerol with polyunsaturated sn-2 chains, generating 2-arachidonoylglycerol, and exhibits transacylase activity that remodels TAG acyl chains, an activity dependent on an amphipathic helix required for lipid droplet binding [PMID:27198176, PMID:41264248]. The activity-dependent release of long-chain saturated free fatty acids by DDHD2 fuels mitochondrial β-oxidation and ATP synthesis, and saturated fatty acyl-CoA supplementation rescues the respiratory, trafficking, and synaptic defects of DDHD2-null neurons [PMID:41028912]. DDHD2 promotes lipophagy through two LIR motifs that bind LC3 and GABARAP ATG8-family proteins, enhancing colocalization of LC3B with lipid droplets [PMID:38332048], and its binding to STXBP1 directs plasma-membrane targeting and saturated FFA generation required for memory [PMID:38316990]. Loss of lipase activity decreases cardiolipin, raises reactive oxygen species, and triggers age-dependent motor neuron apoptosis, with catalytically dead and disease mutants failing to rescue [PMID:30038238]. DDHD2 additionally mediates a COPI- and Rab6-independent Golgi-to-ER retrograde transport pathway [PMID:19632984], and disease-associated mutations that disrupt its DDHD/SAM domains and phospholipase activity cause the SPG54 form of hereditary spastic paraplegia [PMID:23176823, PMID:25417924, PMID:29278326].","teleology":[{"year":2009,"claim":"Established the first cellular function for DDHD2 by showing it operates in a defined membrane-trafficking route, before any lipase role was known.","evidence":"RNAi knockdown with live imaging and orthogonal retrograde/anterograde transport assays in cultured cells","pmids":["19632984"],"confidence":"High","gaps":["Did not connect transport function to lipase activity","Mechanism of how DDHD2 drives retrograde transport unresolved","No substrate identified at this stage"]},{"year":2012,"claim":"Linked DDHD2 to human disease and synaptic function by tying DDHD-domain mutations to SPG54 and showing ortholog loss reduces synaptic active zones.","evidence":"Human mutation identification plus Drosophila ortholog RNAi with synapse morphology analysis","pmids":["23176823"],"confidence":"Medium","gaps":["No biochemical reconstitution of mutant enzyme activity","Connection between synaptic phenotype and lipid metabolism unestablished"]},{"year":2014,"claim":"Defined DDHD2's core enzymatic identity as the principal brain TAG hydrolase, explaining the CNS-selective lipid accumulation in disease.","evidence":"DDHD2 KO mice, lipidomics, recombinant TAG hydrolase assay, and selective inhibitor pharmacology","pmids":["25267624","25417924"],"confidence":"High","gaps":["Did not resolve preferred physiological substrate (TAG vs DAG)","Downstream metabolic consequence of accumulation not yet defined"]},{"year":2016,"claim":"Refined substrate specificity, showing DDHD2 acts preferentially as a diacylglycerol lipase generating the endocannabinoid 2-AG, not solely a TAG hydrolase.","evidence":"Purified recombinant enzyme kinetics, LC-MS substrate profiling, and 2-AG quantification in transfected cells","pmids":["27198176"],"confidence":"High","gaps":["Reconciliation with TAG-hydrolase role in vivo incomplete","Physiological relevance of 2-AG production in neurons untested here"]},{"year":2017,"claim":"Connected disease mutations mechanistically to enzyme dysfunction and lipid droplet protection, and catalogued the protein content of pathological LDs.","evidence":"In vitro TAG hydrolase assays of HSP mutants, cell-based LD accumulation assays, and KO brain LD proteomics","pmids":["29278326"],"confidence":"High","gaps":["Functional role of CNS-enriched LD-associated proteins unexplored","Mechanism of LD targeting not defined"]},{"year":2018,"claim":"Demonstrated that DDHD2 lipase activity protects mitochondria and motor neurons, linking lipid hydrolysis to redox/cardiolipin homeostasis and cell survival.","evidence":"KO mice with ROS and cardiolipin measurements and rescue by wild-type versus catalytically dead/HSP-mutant DDHD2","pmids":["30038238"],"confidence":"High","gaps":["Mechanistic link from lipase activity to cardiolipin content unresolved","Whether ROS rise is cause or consequence of LD accumulation unclear"]},{"year":2020,"claim":"Revealed a non-redundant, paralog-distinct role for DDHD2 in neurite outgrowth, separating it functionally from DDHD1.","evidence":"siRNA depletion with neurite length measurement in SH-SY5Y and PC12 cells","pmids":["32850804"],"confidence":"Medium","gaps":["Molecular basis of neurite phenotype not defined","Single readout per condition"]},{"year":2023,"claim":"Showed DDHD2's lipolytic role is cell-type-specific, acting as a dual TAG/DAG hydrolase in neurons but only downstream of ATGL on DAG in neuroblastoma cells.","evidence":"In vitro acylglycerol hydrolase assays and ATGL/DDHD2 modulation in primary neurons and neuroblastoma cells","pmids":["37832604"],"confidence":"Medium","gaps":["Determinants of lipolysome configuration unknown","In vivo relevance of cell-type differences untested"]},{"year":2024,"claim":"Identified the lipophagy arm of DDHD2 function through direct LIR-mediated binding to ATG8 proteins, complementing its direct hydrolase activity in LD elimination.","evidence":"AP-MS, LIR motif mutagenesis, and LC3B/LD colocalization with LD quantification","pmids":["38332048"],"confidence":"High","gaps":["Relative contribution of direct hydrolysis versus lipophagy in vivo unquantified","Regulation of LIR-mediated recruitment unknown"]},{"year":2024,"claim":"Established a synaptic-protein partner (STXBP1) that controls DDHD2 plasma-membrane targeting and links its saturated FFA output to memory.","evidence":"Pulldown-MS, STXBP1 KO and haploinsufficient models, DDHD2 KO mice, lipidomics, and behavioral memory assays","pmids":["38316990"],"confidence":"High","gaps":["Structural basis of DDHD2-STXBP1 interaction undefined","How plasma-membrane targeting alters FFA release mechanism unclear"]},{"year":2024,"claim":"Tested whether DDHD2-driven LD accumulation engages alpha-synuclein pathology, returning an informative negative result.","evidence":"Pharmacological DDHD2 inhibition in primary rat cortical neurons with alpha-synuclein blots, solubility, and colocalization","pmids":["39853540"],"confidence":"Medium","gaps":["Negative result limited to acute inhibition","Does not exclude chronic or in vivo synuclein effects"]},{"year":2024,"claim":"Proposed a link between DDHD2 and the Nrf2/GPX4 ferroptosis-protective pathway in endothelial cells.","evidence":"siRNA knockdown, single co-immunoprecipitation, GPX4/Nrf2 western blots, and lipid peroxidation assay","pmids":["39062593"],"confidence":"Low","gaps":["Single Co-IP without reciprocal validation","No demonstration of direct binding or mechanism","Relevance outside endothelial cells untested"]},{"year":2025,"claim":"Mechanistically completed the metabolic picture: DDHD2 releases long-chain saturated FFAs to fuel mitochondrial beta-oxidation and ATP synthesis, with acyl-CoA rescue restoring multiple neuronal defects.","evidence":"KO neurons with Seahorse respiration, ATP and glycolysis assays, mitochondrial import inhibition, and saturated fatty acyl-CoA rescue","pmids":["41028912"],"confidence":"High","gaps":["How activity-dependent FFA release is triggered remains unresolved","Coupling between LD pool and mitochondrial import undefined"]},{"year":2025,"claim":"Resolved substrate preference and added a transacylase activity for human DDHD2, and identified an amphipathic helix required for LD binding and full activity.","evidence":"Purified recombinant human enzyme assays, substrate profiling, transacylase assays, and amphipathic-helix deletion mutagenesis","pmids":["41264248"],"confidence":"High","gaps":["Structure of the LD-bound enzyme unsolved","Physiological role of transacylase remodeling in vivo untested"]},{"year":null,"claim":"How DDHD2's distinct activities — Golgi-to-ER transport, direct acylglycerol hydrolysis, transacylation, lipophagy, and STXBP1-dependent membrane targeting — are integrated and regulated within a single neuron remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of the full-length enzyme on membranes","Regulatory triggers for activity-dependent FFA release unknown","Mechanistic relationship between transport and lipase functions undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[2,5,13]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[13]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[13]}],"localization":[{"term_id":"GO:0005811","term_label":"lipid droplet","supporting_discovery_ids":[2,10,13]},{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[0]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[11]}],"pathway":[{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[2,5,14]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[10]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[0]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[1,6]}],"complexes":[],"partners":["STXBP1","MAP1LC3B","GABARAP"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O94830","full_name":"Triacylglycerol hydrolase DDHD2","aliases":["DDHD domain-containing protein 2","KIAA0725p","Phospholipase DDHD2","SAM, WWE and DDHD domain-containing protein 1","Triglyceride hydrolase DDHD2","Triglyceride lipase"],"length_aa":711,"mass_kda":81.0,"function":"Diacylglycerol (DAG) and triacylglycerol (TAG) lipase required for proper lipid homeostasis in the central nervous system (PubMed:29278326, PubMed:37832604). It cooperates with PNPLA2/ATGL in neuronal TAG catabolism and hydrolyzes sn-1,3 DAG downstream of PNPLA2/ATGL (By similarity). In vitro, it also acts as a phospholipase that hydrolyzes preferentially phosphatidic acids, including 1,2-dioleoyl-sn-phosphatidic acid, phosphatidylcholine and phosphatidylethanolamine. Specifically binds to phosphatidylinositol 3-phosphate (PI(3)P), phosphatidylinositol 4-phosphate (PI(4)P), phosphatidylinositol 5-phosphate (PI(5)P) and possibly phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). May be involved in the maintenance of the endoplasmic reticulum and/or Golgi structures. May regulate the transport between Golgi apparatus and plasma membrane","subcellular_location":"Cytoplasm, cytosol; Endoplasmic reticulum-Golgi intermediate compartment; Golgi apparatus, cis-Golgi network","url":"https://www.uniprot.org/uniprotkb/O94830/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/DDHD2","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/DDHD2","total_profiled":1310},"omim":[{"mim_id":"615033","title":"SPASTIC PARAPLEGIA 54, AUTOSOMAL RECESSIVE; SPG54","url":"https://www.omim.org/entry/615033"},{"mim_id":"615003","title":"DDHD DOMAIN-CONTAINING PROTEIN 2; DDHD2","url":"https://www.omim.org/entry/615003"},{"mim_id":"611225","title":"SPASTIC PARAPLEGIA 18B, AUTOSOMAL RECESSIVE; SPG18B","url":"https://www.omim.org/entry/611225"},{"mim_id":"270800","title":"SPASTIC PARAPLEGIA 5A, AUTOSOMAL RECESSIVE; SPG5A","url":"https://www.omim.org/entry/270800"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Cytosol","reliability":"Supported"},{"location":"Centriolar satellite","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/DDHD2"},"hgnc":{"alias_symbol":["KIAA0725","SPG54","p125B","iPLA1gamma","iPLA1γ"],"prev_symbol":["SAMWD1"]},"alphafold":{"accession":"O94830","domains":[{"cath_id":"-","chopping":"44-176_189-197","consensus_level":"high","plddt":88.8673,"start":44,"end":197},{"cath_id":"3.40.50.1820","chopping":"214-365_493-567_650-703","consensus_level":"high","plddt":86.2789,"start":214,"end":703},{"cath_id":"1.10.150.50","chopping":"391-448","consensus_level":"high","plddt":83.2398,"start":391,"end":448}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O94830","model_url":"https://alphafold.ebi.ac.uk/files/AF-O94830-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O94830-F1-predicted_aligned_error_v6.png","plddt_mean":73.69},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=DDHD2","jax_strain_url":"https://www.jax.org/strain/search?query=DDHD2"},"sequence":{"accession":"O94830","fasta_url":"https://rest.uniprot.org/uniprotkb/O94830.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O94830/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O94830"}},"corpus_meta":[{"pmid":"23176823","id":"PMC_23176823","title":"Mutations in DDHD2, encoding an intracellular phospholipase A(1), cause a recessive form of complex hereditary spastic paraplegia.","date":"2012","source":"American journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/23176823","citation_count":150,"is_preprint":false},{"pmid":"25267624","id":"PMC_25267624","title":"The hereditary spastic paraplegia-related enzyme DDHD2 is a principal brain triglyceride lipase.","date":"2014","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/25267624","citation_count":146,"is_preprint":false},{"pmid":"32747698","id":"PMC_32747698","title":"Common genetic risk variants identified in the SPARK cohort support DDHD2 as a candidate risk gene for autism.","date":"2020","source":"Translational psychiatry","url":"https://pubmed.ncbi.nlm.nih.gov/32747698","citation_count":73,"is_preprint":false},{"pmid":"23486545","id":"PMC_23486545","title":"Mutations in phospholipase DDHD2 cause autosomal recessive hereditary spastic paraplegia (SPG54).","date":"2013","source":"European journal of human genetics : EJHG","url":"https://pubmed.ncbi.nlm.nih.gov/23486545","citation_count":62,"is_preprint":false},{"pmid":"24337409","id":"PMC_24337409","title":"Mutations in CYP2U1, DDHD2 and GBA2 genes are rare causes of complicated forms of hereditary spastic paraparesis.","date":"2013","source":"Journal of neurology","url":"https://pubmed.ncbi.nlm.nih.gov/24337409","citation_count":61,"is_preprint":false},{"pmid":"29278326","id":"PMC_29278326","title":"Functional Contribution of the Spastic Paraplegia-Related Triglyceride Hydrolase DDHD2 to the Formation and Content of Lipid Droplets.","date":"2017","source":"Biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/29278326","citation_count":47,"is_preprint":false},{"pmid":"19632984","id":"PMC_19632984","title":"Intracellular phospholipase A1gamma (iPLA1gamma) is a novel factor involved in coat protein complex I- and Rab6-independent retrograde transport between the endoplasmic reticulum and the Golgi complex.","date":"2009","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/19632984","citation_count":42,"is_preprint":false},{"pmid":"33479208","id":"PMC_33479208","title":"Circular RNA circRUNX1 promotes papillary thyroid cancer progression and metastasis by sponging MiR-296-3p and regulating DDHD2 expression.","date":"2021","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/33479208","citation_count":39,"is_preprint":false},{"pmid":"30038238","id":"PMC_30038238","title":"Loss of DDHD2, whose mutation causes spastic paraplegia, promotes reactive oxygen species generation and apoptosis.","date":"2018","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/30038238","citation_count":31,"is_preprint":false},{"pmid":"25417924","id":"PMC_25417924","title":"Late-onset spastic ataxia phenotype in a patient with a homozygous DDHD2 mutation.","date":"2014","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/25417924","citation_count":28,"is_preprint":false},{"pmid":"38316990","id":"PMC_38316990","title":"The DDHD2-STXBP1 interaction mediates long-term memory via generation of saturated free fatty acids.","date":"2024","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/38316990","citation_count":22,"is_preprint":false},{"pmid":"25653011","id":"PMC_25653011","title":"miR-503 represses human cell proliferation and directly targets the oncogene DDHD2 by non-canonical target pairing.","date":"2015","source":"BMC genomics","url":"https://pubmed.ncbi.nlm.nih.gov/25653011","citation_count":20,"is_preprint":false},{"pmid":"26113134","id":"PMC_26113134","title":"Truncating mutation in intracellular phospholipase A₁ gene (DDHD2) in hereditary spastic paraplegia with intellectual disability (SPG54).","date":"2015","source":"BMC research notes","url":"https://pubmed.ncbi.nlm.nih.gov/26113134","citation_count":19,"is_preprint":false},{"pmid":"41028912","id":"PMC_41028912","title":"DDHD2 provides a flux of saturated fatty acids for neuronal energy and function.","date":"2025","source":"Nature metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/41028912","citation_count":18,"is_preprint":false},{"pmid":"38332048","id":"PMC_38332048","title":"DDHD2, whose mutations cause spastic paraplegia type 54, enhances lipophagy via engaging ATG8 family proteins.","date":"2024","source":"Cell death and differentiation","url":"https://pubmed.ncbi.nlm.nih.gov/38332048","citation_count":16,"is_preprint":false},{"pmid":"27198176","id":"PMC_27198176","title":"Enzymatic characterization of recombinant rat DDHD2: a soluble diacylglycerol lipase.","date":"2016","source":"Journal of biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/27198176","citation_count":16,"is_preprint":false},{"pmid":"37832604","id":"PMC_37832604","title":"Cooperative lipolytic control of neuronal triacylglycerol by spastic paraplegia-associated enzyme DDHD2 and ATGL.","date":"2023","source":"Journal of lipid research","url":"https://pubmed.ncbi.nlm.nih.gov/37832604","citation_count":13,"is_preprint":false},{"pmid":"39062593","id":"PMC_39062593","title":"Radiation-Induced Endothelial Ferroptosis Accelerates Atherosclerosis via the DDHD2-Mediated Nrf2/GPX4 Pathway.","date":"2024","source":"Biomolecules","url":"https://pubmed.ncbi.nlm.nih.gov/39062593","citation_count":10,"is_preprint":false},{"pmid":"37420318","id":"PMC_37420318","title":"Biallelic DDHD2 mutations in patients with adult-onset complex hereditary spastic paraplegia.","date":"2023","source":"Annals of clinical and translational neurology","url":"https://pubmed.ncbi.nlm.nih.gov/37420318","citation_count":6,"is_preprint":false},{"pmid":"32850804","id":"PMC_32850804","title":"DDHD1, but Not DDHD2, Suppresses Neurite Outgrowth in SH-SY5Y and PC12 Cells by Regulating Protein Transport From Recycling Endosomes.","date":"2020","source":"Frontiers in cell and developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/32850804","citation_count":5,"is_preprint":false},{"pmid":"38909316","id":"PMC_38909316","title":"DDHD2 promotes lipid droplet catabolism by acting as a TAG lipase and a cargo receptor for lipophagy.","date":"2024","source":"Autophagy","url":"https://pubmed.ncbi.nlm.nih.gov/38909316","citation_count":4,"is_preprint":false},{"pmid":"41264248","id":"PMC_41264248","title":"DDHD2 possesses both lipase and transacylase capacities that remodel triglyceride acyl chains.","date":"2025","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/41264248","citation_count":3,"is_preprint":false},{"pmid":"36090575","id":"PMC_36090575","title":"Case report: Novel compound heterozygous missense mutations in the DDHD2 gene in a Chinese patient associated with spastic paraplegia type 54.","date":"2022","source":"Frontiers in pediatrics","url":"https://pubmed.ncbi.nlm.nih.gov/36090575","citation_count":3,"is_preprint":false},{"pmid":"33246910","id":"PMC_33246910","title":"Coinheritance of novel mutations in NAGLU causing mucopolysaccharidosis type IIIB and in DDHD2 causing spastic paraplegia54 in a Turkish family.","date":"2020","source":"Journal of clinical neuroscience : official journal of the Neurosurgical Society of Australasia","url":"https://pubmed.ncbi.nlm.nih.gov/33246910","citation_count":2,"is_preprint":false},{"pmid":"41040251","id":"PMC_41040251","title":"DDHD2 possesses both lipase and transacylase capacities that remodel triglyceride acyl chains.","date":"2025","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/41040251","citation_count":0,"is_preprint":false},{"pmid":"39853540","id":"PMC_39853540","title":"Acute lipid droplet accumulation induced by the inhibition of the phospholipase DDHD2 does not affect the level, solubility, or phosphoserine-129 status of α-synuclein.","date":"2025","source":"Metabolic brain disease","url":"https://pubmed.ncbi.nlm.nih.gov/39853540","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":14449,"output_tokens":4267,"usd":0.053676,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":12191,"output_tokens":4421,"usd":0.08574,"stage2_stop_reason":"end_turn"},"total_usd":0.139416,"stage1_batch_id":"msgbatch_01AsR7g8dGTEiEw9U83bJeWn","stage2_batch_id":"msgbatch_01YV5ZyxtFNUZEjxRcpd9jzb","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2009,\n      \"finding\": \"iPLA1γ/DDHD2 localizes to the cytosol, cis-Golgi, and ERGIC, and mediates a COPI- and Rab6-independent retrograde membrane transport pathway from the Golgi complex to the ER; RNAi knockdown delayed BFA-induced Golgi-to-ER transfer and cholera toxin B retrograde transport without affecting anterograde VSVGts045 transport or ERGIC-53/Shiga toxin recycling.\",\n      \"method\": \"RNAi knockdown, time-lapse microscopy, GFP-fusion localization, BFA assay, cholera toxin B retrograde transport assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal functional knockdown with multiple orthogonal transport assays, live imaging, single lab\",\n      \"pmids\": [\"19632984\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Mutations in DDHD2 that affect the DDHD domain (required for phospholipase activity) cause SPG54; knockdown of the Drosophila ortholog reduced active zones at synaptic terminals, implicating DDHD2 in synaptic function.\",\n      \"method\": \"Drosophila Ddhd RNAi knockdown with synapse morphology analysis; human genetics (mutation identification)\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — in vivo model organism knockdown with defined morphological readout, single lab, no biochemical reconstitution\",\n      \"pmids\": [\"23176823\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"DDHD2 is the principal triglyceride (triacylglycerol/TAG) hydrolase in the brain; DDHD2−/− mice accumulate TAGs selectively in the CNS (not peripheral tissues), with lipid droplets localizing to neuronal intracellular compartments; recombinant DDHD2 displays TAG hydrolase activity in vitro; a selective in vivo DDHD2 inhibitor also causes brain TAG accumulation in wild-type mice.\",\n      \"method\": \"DDHD2−/− mouse generation, mass spectrometry-based lipidomics, in vitro TAG hydrolase assay with recombinant enzyme, selective inhibitor pharmacology, electron microscopy\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — genetic KO + recombinant enzyme assay + selective inhibitor, replicated across multiple approaches in single rigorous study\",\n      \"pmids\": [\"25267624\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"A truncating mutation (p.R287X) in DDHD2 removes the SAM and DDHD domains, which are crucial for phosphoinositide binding and phospholipase activity, confirming these domains are essential for DDHD2 function.\",\n      \"method\": \"Exome sequencing, homozygosity mapping, domain analysis of truncated protein\",\n      \"journal\": \"BMC research notes\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — domain inference from truncation mutation, no direct biochemical assay of truncated protein\",\n      \"pmids\": [\"26113134\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"A DDHD2 missense mutation (identified in late-onset spastic ataxia patients) caused a marked reduction in phospholipase A1 activity, and patients showed abnormal lipid peak on MRS consistent with lipid accumulation.\",\n      \"method\": \"Phospholipase A1 activity assay of mutant DDHD2, patient MR spectroscopy\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — direct enzymatic assay of disease-associated mutant, single lab, single method\",\n      \"pmids\": [\"25417924\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Recombinant rat DDHD2 functions preferentially as a diacylglycerol (DG) lipase; kcat/Km for DG(18:0/20:4) was much higher than for TAG or phosphatidic acid; DDHD2 shows preference for DG substrates with polyunsaturated fatty acids at the sn-2 position; DDHD2-expressing CHO cells showed elevated 2-arachidonoylglycerol (2-AG) levels, supporting a DG lipase role in vivo.\",\n      \"method\": \"Enzymatic assay with purified recombinant protein, LC-MS substrate specificity profiling, GC-MS/MS quantification of 2-AG in transfected CHO cells\",\n      \"journal\": \"Journal of biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with purified enzyme, multiple substrates tested by LC-MS, and cellular validation with GC-MS/MS\",\n      \"pmids\": [\"27198176\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"HSP-related mutations in DDHD2 disrupt its triglyceride hydrolase activity in vitro and impair DDHD2's capacity to protect cells from lipid droplet accumulation upon free fatty acid exposure; lipid droplets in DDHD2−/− brains contain both known LD-associated proteins and CNS-enriched proteins including several with links to neurological disease.\",\n      \"method\": \"In vitro TAG hydrolase assay of HSP mutants, cell-based LD accumulation assay with free fatty acid treatment, DDHD2 inhibitor, LD proteomics from KO brain tissue\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro enzyme assay of multiple disease mutants plus cell-based functional assay plus LD proteomics, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"29278326\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"DDHD2 ablation causes age-dependent apoptosis of motor neurons in mouse spinal cord; DDHD2 KO cells show decreased cardiolipin content and increased reactive oxygen species (ROS); ROS increase was reversed by wild-type DDHD2 re-expression but not by catalytically inactive DDHD2, HSP-associated DDHD2 mutants, or DDHD1, establishing that DDHD2 lipase activity is required for mitochondrial protection.\",\n      \"method\": \"DDHD2−/− mice, ROS measurement (chemical and probe-based), cardiolipin quantification, rescue by wild-type vs. active-site or HSP mutant DDHD2 re-expression, apoptosis assays\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO with defined phenotype, active-site mutagenesis rescue, multiple orthogonal assays, single lab\",\n      \"pmids\": [\"30038238\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"DDHD2 depletion prevents neurite elongation in SH-SY5Y and PC12 cells (opposite to DDHD1 depletion, which promotes elongation), indicating a distinct and non-redundant role for DDHD2 in neurite outgrowth regulation.\",\n      \"method\": \"siRNA depletion, neurite length measurement in SH-SY5Y and PC12 cells\",\n      \"journal\": \"Frontiers in cell and developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — loss-of-function with defined cellular phenotype, single lab, single method per condition\",\n      \"pmids\": [\"32850804\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"In primary cortical neurons, DDHD2 functions as a dual TAG/DAG hydrolase and complements ATGL-dependent TAG hydrolysis; in neuroblastoma cells, DDHD2 acts exclusively downstream of ATGL on DAG isomers but is dispensable for TAG hydrolysis, revealing cell-type-specific lipolysome configurations.\",\n      \"method\": \"In vitro acylglycerol hydrolase assays, neutral lipid hydrolase activity measurements in neuroblastoma cells and brain tissue, primary cortical neuron studies with DDHD2 and ATGL modulation\",\n      \"journal\": \"Journal of lipid research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro enzyme assays plus cell-based studies in two cell types, single lab\",\n      \"pmids\": [\"37832604\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"DDHD2 interacts with multiple ATG8-family proteins (LC3s and GABARAPs) via two authentic LIR motifs; this interaction promotes lipophagy, as DDHD2 enhances colocalization of LC3B with lipid droplets; LC3/GABARAP-binding capacity and canonical autophagy both contribute to DDHD2's LD-eliminating activity.\",\n      \"method\": \"Affinity purification-mass spectrometry (AP-MS), mutational analysis of LIR motifs, LC3B/LD colocalization assays, LD quantification upon DDHD2 overexpression/deficiency, LD·ATTEC compound rescue\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — AP-MS identification of interactors, LIR mutational validation, multiple functional assays in single rigorous study\",\n      \"pmids\": [\"38332048\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"DDHD2 binds the synaptic protein STXBP1 (identified by pulldown-mass spectrometry); STXBP1 controls targeting of DDHD2 to the plasma membrane and generation of saturated free fatty acids in the brain; genetic ablation of DDHD2 dramatically reduces saturated FFA responses to memory acquisition and impairs reward-based and spatial memory performance.\",\n      \"method\": \"Pulldown-mass spectrometry, STXBP1/2 KO neurosecretory cells, haploinsufficient STXBP1+/− mouse model, DDHD2 KO mice, lipidomics, behavioral memory assays\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pulldown-MS binding identification plus genetic models (KO and haploinsufficient) plus lipidomics and behavioral phenotyping, multiple orthogonal approaches in single study\",\n      \"pmids\": [\"38316990\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"DDHD2 knockdown in human artery endothelial cells reduces GPX4 and Nrf2 protein levels and increases lipid peroxidation; co-immunoprecipitation indicates a physical interaction between DDHD2 and Nrf2, suggesting DDHD2 regulates the Nrf2/GPX4 ferroptosis-protective pathway.\",\n      \"method\": \"DDHD2 siRNA knockdown, co-immunoprecipitation, western blot for GPX4/Nrf2, lipid peroxidation assay\",\n      \"journal\": \"Biomolecules\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP plus KD phenotype, single lab, no reciprocal IP or direct mechanistic follow-up\",\n      \"pmids\": [\"39062593\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Recombinant human DDHD2 preferentially hydrolyzes DAG over phospholipids and shows slight preference for DAG over TAG; DDHD2 also exhibits transacylase activity, transferring acyl chains from TAGs to DAGs and monoacylglycerols to remodel TAG acyl chains; a predicted hydrophobic amphipathic helix is essential for lipid droplet binding in vitro and in cells and is required for full enzymatic activity and TAG acyl-chain remodeling.\",\n      \"method\": \"In vitro enzyme assays with recombinant human DDHD2, substrate specificity profiling, transacylase activity assay, amphipathic helix deletion mutagenesis, in vitro LD-binding assay, cell-based LD localization\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstitution with purified recombinant enzyme, multiple substrate assays, mutagenesis of structural element, in vitro and cell-based validation\",\n      \"pmids\": [\"41264248\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"DDHD2-dependent release of long-chain saturated free fatty acids (myristic, palmitic, stearic acids) in an activity-dependent manner supports mitochondrial β-oxidation and ATP synthesis in neurons; genetic ablation of Ddhd2 impairs mitochondrial respiration and ATP production despite increased glycolysis; saturated fatty acyl-CoA supplementation rescues mitochondrial energy production, membrane trafficking, synaptic function, and protein homeostasis defects in Ddhd2 KO neurons.\",\n      \"method\": \"Ddhd2 KO neurons, Seahorse mitochondrial respiration assay, ATP measurement, glycolysis assay, mitochondrial fatty acid import inhibition, saturated fatty acyl-CoA rescue experiments\",\n      \"journal\": \"Nature metabolism\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO with mechanistic rescue, multiple orthogonal assays (respiration, ATP, glycolysis, inhibitor experiments), single lab\",\n      \"pmids\": [\"41028912\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Acute DDHD2 inhibition (by compound KLH45) causing lipid droplet accumulation in primary rat cortical neurons did not affect total α-synuclein levels, phosphoserine-129 status, or solubility, and no colocalization between LDs and α-synuclein was detected.\",\n      \"method\": \"DDHD2 inhibitor KLH45 in primary rat cortical neurons, western blot for α-synuclein and pSer129, detergent solubility fractionation, immunofluorescence colocalization\",\n      \"journal\": \"Metabolic brain disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — pharmacological inhibition with defined negative outcome, multiple readouts, single lab — this is a mechanistically informative negative result\",\n      \"pmids\": [\"39853540\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"DDHD2 is a serine lipase/phospholipase A1 that functions as the principal brain triglyceride and diacylglycerol hydrolase, releasing long-chain saturated free fatty acids to fuel neuronal mitochondrial β-oxidation and ATP synthesis; it also exhibits transacylase activity to remodel TAG acyl chains, promotes lipid droplet catabolism via direct interaction with ATG8-family proteins (LC3/GABARAPs) through LIR motifs to enhance lipophagy, mediates COPI/Rab6-independent Golgi-to-ER retrograde membrane transport, and interacts with STXBP1 to enable plasma membrane targeting and saturated FFA generation required for synaptic plasticity and memory; loss-of-function causes lipid droplet accumulation in neurons, mitochondrial dysfunction, motor neuron apoptosis, and the SPG54 form of hereditary spastic paraplegia.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"DDHD2 is a brain-enriched serine lipase that serves as the principal neuronal triacylglycerol and diacylglycerol hydrolase, coupling lipid droplet catabolism to neuronal energy metabolism and synaptic function [#2, #5, #14]. Genetic ablation in mice causes CNS-selective accumulation of triglycerides and neuronal lipid droplets, and recombinant enzyme directly hydrolyzes TAG; a selective inhibitor reproduces the brain lipid phenotype in wild-type animals [#2]. Beyond TAG, DDHD2 preferentially acts on diacylglycerol with polyunsaturated sn-2 chains, generating 2-arachidonoylglycerol, and exhibits transacylase activity that remodels TAG acyl chains, an activity dependent on an amphipathic helix required for lipid droplet binding [#5, #13]. The activity-dependent release of long-chain saturated free fatty acids by DDHD2 fuels mitochondrial \\u03b2-oxidation and ATP synthesis, and saturated fatty acyl-CoA supplementation rescues the respiratory, trafficking, and synaptic defects of DDHD2-null neurons [#14]. DDHD2 promotes lipophagy through two LIR motifs that bind LC3 and GABARAP ATG8-family proteins, enhancing colocalization of LC3B with lipid droplets [#10], and its binding to STXBP1 directs plasma-membrane targeting and saturated FFA generation required for memory [#11]. Loss of lipase activity decreases cardiolipin, raises reactive oxygen species, and triggers age-dependent motor neuron apoptosis, with catalytically dead and disease mutants failing to rescue [#7]. DDHD2 additionally mediates a COPI- and Rab6-independent Golgi-to-ER retrograde transport pathway [#0], and disease-associated mutations that disrupt its DDHD/SAM domains and phospholipase activity cause the SPG54 form of hereditary spastic paraplegia [#1, #4, #6].\",\n  \"teleology\": [\n    {\n      \"year\": 2009,\n      \"claim\": \"Established the first cellular function for DDHD2 by showing it operates in a defined membrane-trafficking route, before any lipase role was known.\",\n      \"evidence\": \"RNAi knockdown with live imaging and orthogonal retrograde/anterograde transport assays in cultured cells\",\n      \"pmids\": [\"19632984\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not connect transport function to lipase activity\", \"Mechanism of how DDHD2 drives retrograde transport unresolved\", \"No substrate identified at this stage\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Linked DDHD2 to human disease and synaptic function by tying DDHD-domain mutations to SPG54 and showing ortholog loss reduces synaptic active zones.\",\n      \"evidence\": \"Human mutation identification plus Drosophila ortholog RNAi with synapse morphology analysis\",\n      \"pmids\": [\"23176823\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No biochemical reconstitution of mutant enzyme activity\", \"Connection between synaptic phenotype and lipid metabolism unestablished\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Defined DDHD2's core enzymatic identity as the principal brain TAG hydrolase, explaining the CNS-selective lipid accumulation in disease.\",\n      \"evidence\": \"DDHD2 KO mice, lipidomics, recombinant TAG hydrolase assay, and selective inhibitor pharmacology\",\n      \"pmids\": [\"25267624\", \"25417924\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve preferred physiological substrate (TAG vs DAG)\", \"Downstream metabolic consequence of accumulation not yet defined\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Refined substrate specificity, showing DDHD2 acts preferentially as a diacylglycerol lipase generating the endocannabinoid 2-AG, not solely a TAG hydrolase.\",\n      \"evidence\": \"Purified recombinant enzyme kinetics, LC-MS substrate profiling, and 2-AG quantification in transfected cells\",\n      \"pmids\": [\"27198176\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Reconciliation with TAG-hydrolase role in vivo incomplete\", \"Physiological relevance of 2-AG production in neurons untested here\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Connected disease mutations mechanistically to enzyme dysfunction and lipid droplet protection, and catalogued the protein content of pathological LDs.\",\n      \"evidence\": \"In vitro TAG hydrolase assays of HSP mutants, cell-based LD accumulation assays, and KO brain LD proteomics\",\n      \"pmids\": [\"29278326\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional role of CNS-enriched LD-associated proteins unexplored\", \"Mechanism of LD targeting not defined\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Demonstrated that DDHD2 lipase activity protects mitochondria and motor neurons, linking lipid hydrolysis to redox/cardiolipin homeostasis and cell survival.\",\n      \"evidence\": \"KO mice with ROS and cardiolipin measurements and rescue by wild-type versus catalytically dead/HSP-mutant DDHD2\",\n      \"pmids\": [\"30038238\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanistic link from lipase activity to cardiolipin content unresolved\", \"Whether ROS rise is cause or consequence of LD accumulation unclear\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Revealed a non-redundant, paralog-distinct role for DDHD2 in neurite outgrowth, separating it functionally from DDHD1.\",\n      \"evidence\": \"siRNA depletion with neurite length measurement in SH-SY5Y and PC12 cells\",\n      \"pmids\": [\"32850804\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular basis of neurite phenotype not defined\", \"Single readout per condition\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Showed DDHD2's lipolytic role is cell-type-specific, acting as a dual TAG/DAG hydrolase in neurons but only downstream of ATGL on DAG in neuroblastoma cells.\",\n      \"evidence\": \"In vitro acylglycerol hydrolase assays and ATGL/DDHD2 modulation in primary neurons and neuroblastoma cells\",\n      \"pmids\": [\"37832604\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Determinants of lipolysome configuration unknown\", \"In vivo relevance of cell-type differences untested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identified the lipophagy arm of DDHD2 function through direct LIR-mediated binding to ATG8 proteins, complementing its direct hydrolase activity in LD elimination.\",\n      \"evidence\": \"AP-MS, LIR motif mutagenesis, and LC3B/LD colocalization with LD quantification\",\n      \"pmids\": [\"38332048\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contribution of direct hydrolysis versus lipophagy in vivo unquantified\", \"Regulation of LIR-mediated recruitment unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Established a synaptic-protein partner (STXBP1) that controls DDHD2 plasma-membrane targeting and links its saturated FFA output to memory.\",\n      \"evidence\": \"Pulldown-MS, STXBP1 KO and haploinsufficient models, DDHD2 KO mice, lipidomics, and behavioral memory assays\",\n      \"pmids\": [\"38316990\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of DDHD2-STXBP1 interaction undefined\", \"How plasma-membrane targeting alters FFA release mechanism unclear\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Tested whether DDHD2-driven LD accumulation engages alpha-synuclein pathology, returning an informative negative result.\",\n      \"evidence\": \"Pharmacological DDHD2 inhibition in primary rat cortical neurons with alpha-synuclein blots, solubility, and colocalization\",\n      \"pmids\": [\"39853540\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Negative result limited to acute inhibition\", \"Does not exclude chronic or in vivo synuclein effects\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Proposed a link between DDHD2 and the Nrf2/GPX4 ferroptosis-protective pathway in endothelial cells.\",\n      \"evidence\": \"siRNA knockdown, single co-immunoprecipitation, GPX4/Nrf2 western blots, and lipid peroxidation assay\",\n      \"pmids\": [\"39062593\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single Co-IP without reciprocal validation\", \"No demonstration of direct binding or mechanism\", \"Relevance outside endothelial cells untested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Mechanistically completed the metabolic picture: DDHD2 releases long-chain saturated FFAs to fuel mitochondrial beta-oxidation and ATP synthesis, with acyl-CoA rescue restoring multiple neuronal defects.\",\n      \"evidence\": \"KO neurons with Seahorse respiration, ATP and glycolysis assays, mitochondrial import inhibition, and saturated fatty acyl-CoA rescue\",\n      \"pmids\": [\"41028912\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How activity-dependent FFA release is triggered remains unresolved\", \"Coupling between LD pool and mitochondrial import undefined\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Resolved substrate preference and added a transacylase activity for human DDHD2, and identified an amphipathic helix required for LD binding and full activity.\",\n      \"evidence\": \"Purified recombinant human enzyme assays, substrate profiling, transacylase assays, and amphipathic-helix deletion mutagenesis\",\n      \"pmids\": [\"41264248\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure of the LD-bound enzyme unsolved\", \"Physiological role of transacylase remodeling in vivo untested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How DDHD2's distinct activities — Golgi-to-ER transport, direct acylglycerol hydrolysis, transacylation, lipophagy, and STXBP1-dependent membrane targeting — are integrated and regulated within a single neuron remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of the full-length enzyme on membranes\", \"Regulatory triggers for activity-dependent FFA release unknown\", \"Mechanistic relationship between transport and lipase functions undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [2, 5, 13]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [13]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [13]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005811\", \"supporting_discovery_ids\": [2, 10, 13]},\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [11]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [2, 5, 14]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [10]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [1, 6]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"STXBP1\", \"MAP1LC3B\", \"GABARAP\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":7,"faith_pct":85.71428571428571}}