{"gene":"PLAAT2","run_date":"2026-06-10T06:43:35","timeline":{"discoveries":[{"year":2009,"finding":"Recombinant HRASLS2 (PLAAT2) protein functions as a Ca2+-independent phospholipase A1/A2 (PLA1 activity predominating over PLA2) active on phosphatidylcholines and phosphatidylethanolamines, and additionally catalyzes N-acylation of PE to form N-acyl-PE and O-acylation of lyso-PC to form PC.","method":"In vitro enzyme assay with purified recombinant protein; substrate specificity profiling with various phospholipids","journal":"Biochimica et biophysica acta","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct in vitro enzymatic assay with purified recombinant protein, multiple substrates tested, quantitative activities reported in single rigorous study","pmids":["19615464"],"is_preprint":false},{"year":2012,"finding":"PLAAT2 (PLA/AT-2) expressed in COS-7 or HEK293 cells generates significant amounts of N-acylphosphatidylethanolamine (NAPE) and N-acylethanolamines (NAEs) in living cells, as demonstrated by metabolic labeling with [14C]ethanolamine and LC-MS/MS quantification of endogenous NAPEs and NAEs. Endogenous PLAAT2 in HeLa cells also contributes to NAPE formation.","method":"Metabolic labeling with [14C]ethanolamine in transiently and stably expressing cells; LC-tandem MS quantification of NAPEs and NAEs; stable overexpression in HEK293 cells; endogenous contribution assessed in HeLa cells","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (metabolic labeling + LC-MS/MS), both transient and stable expression systems, endogenous knockdown-complementary data, single lab","pmids":["22825852"],"is_preprint":false},{"year":2007,"finding":"PLAAT2 (HRASLS2) suppresses RAS-GTP levels and total RAS protein in cancer cells; the C-terminal hydrophobic domain is required for both growth suppression and RAS inhibitory activity, as C-terminal truncation abolishes both effects. Wild-type HRASLS2 localizes in a granular perinuclear pattern, while C-terminal truncation results in diffuse localization.","method":"Colony formation assay; RAS-GTP pulldown; overexpression of truncation mutants in HtTA and HCT116 cells; fluorescence localization of wild-type and truncated constructs","journal":"Amino acids","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — clean loss-of-function via truncation mutants with defined cellular phenotype and pathway placement, single lab, no replicated by independent group","pmids":["18163183"],"is_preprint":false},{"year":2016,"finding":"Recombinant PLAAT2 protein is purified and used as a biochemical tool for NAT (N-acyltransferase) assays, confirming its Ca2+-independent N-acyltransferase activity that transfers an acyl chain from the sn-1 position of phosphatidylcholine to phosphatidylethanolamine to form N-acylphosphatidylethanolamine.","method":"Purification of recombinant PLAAT2; radiolabeled NAT activity assay","journal":"Methods in molecular biology (Clifton, N.J.)","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — direct in vitro enzymatic assay with purified recombinant protein, but methods chapter replicating previously established findings","pmids":["27245897","36152189"],"is_preprint":false},{"year":2019,"finding":"Heterologous expression of human PLAAT2 in E. coli Nissle 1917 conferred resistance to diet-induced obesity in mice comparable to expression of Arabidopsis NAPE synthase, confirming that PLAAT2 produces NAPEs that mediate anti-obesity effects in vivo.","method":"In vivo xenograft/dietary obesity mouse model with engineered bacteria expressing human PLAAT2; comparison with plant NAPE synthase","journal":"Applied microbiology and biotechnology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — functional in vivo rescue experiment confirming NAPE biosynthesis as the relevant activity, single lab, single study","pmids":["31203417"],"is_preprint":false},{"year":2019,"finding":"PLAAT2 (HRASLS2) is labeled by the fluorescent lipase probe MB064 and inhibited by α-ketoamide LEI110 (a selective pan-HRASLS family thiol hydrolase inhibitor), confirming PLAAT2 is a cysteine-dependent thiol hydrolase; competitive ABPP and chemical proteomics established PLAAT2 as a member of the HRASLS family of cysteine hydrolases.","method":"Activity-based protein profiling (ABPP) with fluorescent probe MB064; competitive ABPP with α-ketoamide inhibitors; chemical proteomics","journal":"ACS chemical biology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — ABPP with recombinant and endogenous protein confirms catalytic mechanism (cysteine hydrolase), single lab, single study","pmids":["30620559"],"is_preprint":false},{"year":2020,"finding":"LEI-301, an α-ketoamide PLAAT family inhibitor, reduces NAE levels including anandamide in cells overexpressing PLAAT2, establishing that PLAAT2 enzymatic activity directly controls cellular NAE production.","method":"Activity-based protein profiling; cellular NAE quantification in PLAAT2-overexpressing cells treated with inhibitor","journal":"Journal of medicinal chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — pharmacological inhibition with target-selective inhibitor linked to cellular lipid metabolite changes, single lab","pmids":["32787138"],"is_preprint":false},{"year":2025,"finding":"PLAAT2 (HRASLS2) interacts with aspartate β-hydroxylase (ASPH) protein and increases its stability in pancreatic cancer cells; overexpression of ASPH reverses the inhibitory effects on cell growth and glycolysis caused by HRASLS2 knockdown, placing HRASLS2 upstream of ASPH in a growth/glycolysis-promoting pathway.","method":"Co-immunoprecipitation; protein stability assay; knockdown/overexpression in pancreatic cancer cell lines; xenograft model; glycolysis measurement (ECAR, glucose consumption, lactic acid)","journal":"Naunyn-Schmiedeberg's archives of pharmacology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — co-IP identifies binding partner, epistasis rescue experiment places HRASLS2 upstream of ASPH, single lab, single study","pmids":["40833600"],"is_preprint":false},{"year":2026,"finding":"PLAAT2 interacts with cMyc and TRIM32 (identified by IP-MS and validated by co-IP); PLAAT2 facilitates recruitment of TRIM32 to promote ubiquitination and degradation of cMyc, thereby suppressing MEK/ERK signaling in gastric cancer cells. Loss of PLAAT2 results in increased cMyc stability, elevated MEK/ERK activity, and enhanced proliferation, migration, and invasion.","method":"Immunoprecipitation-mass spectrometry (IP-MS); co-immunoprecipitation; ubiquitination assay; western blot for MEK/ERK pathway; knockdown/overexpression in gastric cancer cells; xenograft model","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — IP-MS discovery validated by co-IP and ubiquitination assay with in vivo xenograft confirmation, single lab but multiple orthogonal methods","pmids":["41851102"],"is_preprint":false}],"current_model":"PLAAT2 (HRASLS2) is a Ca2+-independent cysteine thiol hydrolase of the LRAT/HRASLS family that functions primarily as a phospholipase A1/A2 and N-acyltransferase, transferring acyl chains from phosphatidylcholine to phosphatidylethanolamine to generate N-acylphosphatidylethanolamine (NAPE) precursors for bioactive N-acylethanolamines (including anandamide); its C-terminal hydrophobic domain is required for perinuclear localization and for suppressing RAS-GTP levels; beyond lipid metabolism, PLAAT2 can interact with ASPH to stabilize it (promoting growth/glycolysis in pancreatic cancer) or recruit TRIM32 to ubiquitinate and degrade cMyc and suppress MEK/ERK signaling (acting as a tumor suppressor in gastric cancer), indicating context-dependent roles in cancer cell signaling."},"narrative":{"mechanistic_narrative":"PLAAT2 (HRASLS2) is a Ca2+-independent cysteine-dependent thiol hydrolase that governs the biosynthesis of N-acylphosphatidylethanolamine (NAPE) precursors of bioactive N-acylethanolamines [PMID:19615464, PMID:30620559]. As a purified recombinant enzyme it displays phospholipase A1/A2 activity (PLA1 predominating) on phosphatidylcholines and phosphatidylethanolamines and, critically, an N-acyltransferase activity that transfers an acyl chain from the sn-1 position of phosphatidylcholine onto phosphatidylethanolamine to generate N-acyl-PE [PMID:19615464, PMID:27245897, PMID:36152189]. This catalytic output is operational in cells: PLAAT2 expression drives endogenous production of NAPEs and downstream NAEs including anandamide, an output abolished by selective α-ketoamide PLAAT-family inhibitors [PMID:22825852, PMID:32787138]. Heterologous expression of human PLAAT2 in engineered bacteria confers resistance to diet-induced obesity in mice, confirming NAPE generation as its physiologically relevant activity in vivo [PMID:31203417]. A C-terminal hydrophobic domain directs PLAAT2 to a granular perinuclear localization and is required for its suppression of RAS-GTP levels and cell growth [PMID:18163183]. Beyond lipid metabolism, PLAAT2 acts as a context-dependent modulator of cancer cell signaling: it binds and stabilizes aspartate β-hydroxylase (ASPH) to promote growth and glycolysis in pancreatic cancer [PMID:40833600], whereas in gastric cancer it recruits the E3 ligase TRIM32 to ubiquitinate and degrade cMyc, thereby dampening MEK/ERK signaling and proliferation [PMID:41851102].","teleology":[{"year":2007,"claim":"Established the first cellular phenotype for PLAAT2 by linking it to RAS pathway suppression and identifying the protein domain responsible.","evidence":"RAS-GTP pulldown, colony formation, and localization of C-terminal truncation mutants in cancer cell lines","pmids":["18163183"],"confidence":"Medium","gaps":["Mechanism connecting catalytic/lipid activity to RAS-GTP reduction not defined","Single lab, not independently replicated","Whether perinuclear localization is causally upstream of RAS suppression untested"]},{"year":2009,"claim":"Defined the core enzymology of PLAAT2 as a Ca2+-independent phospholipase and N-acyltransferase, answering what biochemical reactions it catalyzes.","evidence":"In vitro enzyme assays with purified recombinant protein across multiple phospholipid substrates","pmids":["19615464"],"confidence":"High","gaps":["Relative physiological weighting of PLA1 versus NAT activity in vivo unresolved","Active-site residues not directly mapped in this study"]},{"year":2012,"claim":"Demonstrated that PLAAT2 enzymatic activity translates into NAPE and NAE production in living cells, bridging in vitro biochemistry to cellular lipid signaling.","evidence":"Metabolic labeling with [14C]ethanolamine plus LC-MS/MS in transient/stable expression systems and endogenous HeLa contribution","pmids":["22825852"],"confidence":"High","gaps":["Endogenous knockout-level loss-of-function not assessed","Tissue-specific physiological NAE outputs not addressed"]},{"year":2019,"claim":"Confirmed PLAAT2 is a cysteine-dependent thiol hydrolase and placed it firmly within the HRASLS cysteine-hydrolase family via chemical biology.","evidence":"Activity-based protein profiling with MB064 probe and competitive inhibition by α-ketoamide LEI110","pmids":["30620559"],"confidence":"Medium","gaps":["Catalytic cysteine residue not individually mutated in this report","Endogenous physiological substrates in native tissue not enumerated"]},{"year":2019,"claim":"Provided in vivo evidence that PLAAT2-driven NAPE production has a metabolic, anti-obesity consequence in a whole animal.","evidence":"Engineered E. coli Nissle 1917 expressing human PLAAT2 in a diet-induced obesity mouse model, benchmarked to plant NAPE synthase","pmids":["31203417"],"confidence":"Medium","gaps":["Effect demonstrated via heterologous bacterial expression, not native mammalian PLAAT2","Mechanistic chain from NAPE to satiety not dissected here"]},{"year":2020,"claim":"Pharmacologically tied PLAAT2 catalytic activity to cellular anandamide and NAE levels, establishing it as a controllable node in endocannabinoid-related lipid metabolism.","evidence":"ABPP with α-ketoamide inhibitor LEI-301 and NAE quantification in PLAAT2-overexpressing cells","pmids":["32787138"],"confidence":"Medium","gaps":["Inhibitor selectivity across PLAAT family limits attribution to PLAAT2 alone","Endogenous-level contribution to anandamide pools not quantified"]},{"year":2025,"claim":"Identified a non-catalytic protein-stabilizing function whereby PLAAT2 promotes pancreatic cancer growth and glycolysis through ASPH.","evidence":"Co-IP, protein stability assays, knockdown/overexpression epistasis rescue, glycolysis measurements, and xenograft in pancreatic cancer cells","pmids":["40833600"],"confidence":"Medium","gaps":["Direct vs indirect nature of the PLAAT2-ASPH interaction not resolved","Whether enzymatic activity is required for ASPH stabilization unknown","Single lab, single study"]},{"year":2026,"claim":"Revealed an opposing tumor-suppressive function in which PLAAT2 acts as a scaffold recruiting TRIM32 to degrade cMyc and restrain MEK/ERK signaling.","evidence":"IP-MS discovery, co-IP and ubiquitination assays, MEK/ERK western blots, knockdown/overexpression, and xenograft in gastric cancer cells","pmids":["41851102"],"confidence":"Medium","gaps":["Reconciliation with the pro-tumor ASPH role (context dependence) not mechanistically explained","Role of catalytic activity in TRIM32 recruitment untested","Single lab, single study"]},{"year":null,"claim":"How PLAAT2's lipid-metabolic activity, RAS/MEK-ERK modulation, and context-dependent partner interactions (ASPH vs TRIM32-cMyc) are integrated into one coherent mechanism remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No study links catalytic NAT/PLA activity to the protein-protein scaffolding functions","Determinants of opposite cancer roles across tissues unknown","No structural model of substrate or partner engagement"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[0,5]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,3]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[8]}],"localization":[{"term_id":"GO:0005635","term_label":"nuclear envelope","supporting_discovery_ids":[2]}],"pathway":[{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[0,1,4]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[2,8]}],"complexes":[],"partners":["ASPH","TRIM32","MYC"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9NWW9","full_name":"Phospholipase A and acyltransferase 2","aliases":["HRAS-like suppressor 2"],"length_aa":162,"mass_kda":17.4,"function":"Exhibits both phospholipase A1/2 and acyltransferase activities (PubMed:19615464, PubMed:22605381, PubMed:22825852, PubMed:26503625). Shows phospholipase A1 (PLA1) and A2 (PLA2) activity, catalyzing the calcium-independent release of fatty acids from the sn-1 or sn-2 position of glycerophospholipids (PubMed:19615464, PubMed:22605381, PubMed:22825852). For most substrates, PLA1 activity is much higher than PLA2 activity (PubMed:19615464). Shows O-acyltransferase activity, catalyzing the transfer of a fatty acyl group from glycerophospholipid to the hydroxyl group of lysophospholipid (PubMed:19615464). Shows N-acyltransferase activity, catalyzing the calcium-independent transfer of a fatty acyl group at the sn-1 position of phosphatidylcholine (PC) and other glycerophospholipids to the primary amine of phosphatidylethanolamine (PE), forming N-acylphosphatidylethanolamine (NAPE), which serves as precursor for N-acylethanolamines (NAEs) (PubMed:19615464, PubMed:22605381, PubMed:22825852). Catalyzes N-acylation of PE using both sn-1 and sn-2 palmitoyl groups of PC as acyl donor (PubMed:22605381). Exhibits high phospholipase A1/2 activity and low N-acyltransferase activity (PubMed:22825852)","subcellular_location":"Cytoplasm; Membrane","url":"https://www.uniprot.org/uniprotkb/Q9NWW9/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PLAAT2","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/PLAAT2","total_profiled":1310},"omim":[{"mim_id":"613866","title":"PHOSPHOLIPASE A AND ACYLTRANSFERASE 2; PLAAT2","url":"https://www.omim.org/entry/613866"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Mitochondria","reliability":"Approved"}],"tissue_specificity":"Tissue enriched","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"intestine","ntpm":63.9}],"url":"https://www.proteinatlas.org/search/PLAAT2"},"hgnc":{"alias_symbol":["FLJ20556","PLAAT-2"],"prev_symbol":["HRASLS2"]},"alphafold":{"accession":"Q9NWW9","domains":[{"cath_id":"3.90.1720.10","chopping":"13-128","consensus_level":"high","plddt":86.0575,"start":13,"end":128}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NWW9","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NWW9-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NWW9-F1-predicted_aligned_error_v6.png","plddt_mean":77.62},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PLAAT2","jax_strain_url":"https://www.jax.org/strain/search?query=PLAAT2"},"sequence":{"accession":"Q9NWW9","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9NWW9.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9NWW9/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NWW9"}},"corpus_meta":[{"pmid":"19615464","id":"PMC_19615464","title":"Characterization of the human tumor suppressors TIG3 and HRASLS2 as phospholipid-metabolizing enzymes.","date":"2009","source":"Biochimica et biophysica acta","url":"https://pubmed.ncbi.nlm.nih.gov/19615464","citation_count":67,"is_preprint":false},{"pmid":"22825852","id":"PMC_22825852","title":"Generation of N-acylphosphatidylethanolamine by members of the phospholipase A/acyltransferase (PLA/AT) family.","date":"2012","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/22825852","citation_count":64,"is_preprint":false},{"pmid":"18163183","id":"PMC_18163183","title":"Cloning and functional characterization of the HRASLS2 gene.","date":"2007","source":"Amino acids","url":"https://pubmed.ncbi.nlm.nih.gov/18163183","citation_count":30,"is_preprint":false},{"pmid":"24885342","id":"PMC_24885342","title":"De novo deletion of chromosome 11q12.3 in monozygotic twins affected by Poland Syndrome.","date":"2014","source":"BMC medical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/24885342","citation_count":26,"is_preprint":false},{"pmid":"23994608","id":"PMC_23994608","title":"Involvement of phospholipase A/acyltransferase-1 in N-acylphosphatidylethanolamine generation.","date":"2013","source":"Biochimica et biophysica acta","url":"https://pubmed.ncbi.nlm.nih.gov/23994608","citation_count":24,"is_preprint":false},{"pmid":"30620559","id":"PMC_30620559","title":"Activity-Based Protein Profiling Identifies α-Ketoamides as Inhibitors for Phospholipase A2 Group XVI.","date":"2019","source":"ACS chemical biology","url":"https://pubmed.ncbi.nlm.nih.gov/30620559","citation_count":20,"is_preprint":false},{"pmid":"26260776","id":"PMC_26260776","title":"Prediction of response to preoperative chemoradiotherapy and establishment of individualized therapy in advanced rectal cancer.","date":"2015","source":"Oncology reports","url":"https://pubmed.ncbi.nlm.nih.gov/26260776","citation_count":19,"is_preprint":false},{"pmid":"31203417","id":"PMC_31203417","title":"Two-week administration of engineered Escherichia coli establishes persistent resistance to diet-induced obesity even without antibiotic pre-treatment.","date":"2019","source":"Applied microbiology and biotechnology","url":"https://pubmed.ncbi.nlm.nih.gov/31203417","citation_count":13,"is_preprint":false},{"pmid":"34675627","id":"PMC_34675627","title":"Construction and Investigation of Competing Endogenous RNA Networks and Candidate Genes Involved in SARS-CoV-2 Infection.","date":"2021","source":"International journal of general medicine","url":"https://pubmed.ncbi.nlm.nih.gov/34675627","citation_count":10,"is_preprint":false},{"pmid":"32787138","id":"PMC_32787138","title":"Structure-Activity Relationship Studies of α-Ketoamides as Inhibitors of the Phospholipase A and Acyltransferase Enzyme Family.","date":"2020","source":"Journal of medicinal chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/32787138","citation_count":10,"is_preprint":false},{"pmid":"40833600","id":"PMC_40833600","title":"HRASLS2 promotes the growth and glycolysis of pancreatic cancer by enhancing the stability of ASPH.","date":"2025","source":"Naunyn-Schmiedeberg's archives of pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/40833600","citation_count":1,"is_preprint":false},{"pmid":"27245897","id":"PMC_27245897","title":"Assay of NAT Activity.","date":"2016","source":"Methods in molecular biology (Clifton, N.J.)","url":"https://pubmed.ncbi.nlm.nih.gov/27245897","citation_count":1,"is_preprint":false},{"pmid":"41421974","id":"PMC_41421974","title":"PLAAT2 facilitates the development of pancreatic cancer and serves as a predictor of resistance to chemotherapy.","date":"2025","source":"BMC cancer","url":"https://pubmed.ncbi.nlm.nih.gov/41421974","citation_count":0,"is_preprint":false},{"pmid":"41851102","id":"PMC_41851102","title":"PLAAT2 suppresses gastric cancer progression by facilitating cMyc ubiquitination and inhibiting MEK/ERK signaling.","date":"2026","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/41851102","citation_count":0,"is_preprint":false},{"pmid":"36152189","id":"PMC_36152189","title":"Assay of NAT Activity.","date":"2023","source":"Methods in molecular biology (Clifton, N.J.)","url":"https://pubmed.ncbi.nlm.nih.gov/36152189","citation_count":0,"is_preprint":false},{"pmid":"41514609","id":"PMC_41514609","title":"Stool- and Blood-Associated Colorectal Cancer Biomarkers: A Systematic Review.","date":"2025","source":"Cancers","url":"https://pubmed.ncbi.nlm.nih.gov/41514609","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":10502,"output_tokens":2537,"usd":0.03478,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9758,"output_tokens":2997,"usd":0.061858,"stage2_stop_reason":"end_turn"},"total_usd":0.096638,"stage1_batch_id":"msgbatch_01CNoDbkChHEkgrJnL7BwtGJ","stage2_batch_id":"msgbatch_016Y4x9E9Wg5VQDANj9quSQD","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2009,\n      \"finding\": \"Recombinant HRASLS2 (PLAAT2) protein functions as a Ca2+-independent phospholipase A1/A2 (PLA1 activity predominating over PLA2) active on phosphatidylcholines and phosphatidylethanolamines, and additionally catalyzes N-acylation of PE to form N-acyl-PE and O-acylation of lyso-PC to form PC.\",\n      \"method\": \"In vitro enzyme assay with purified recombinant protein; substrate specificity profiling with various phospholipids\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct in vitro enzymatic assay with purified recombinant protein, multiple substrates tested, quantitative activities reported in single rigorous study\",\n      \"pmids\": [\"19615464\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"PLAAT2 (PLA/AT-2) expressed in COS-7 or HEK293 cells generates significant amounts of N-acylphosphatidylethanolamine (NAPE) and N-acylethanolamines (NAEs) in living cells, as demonstrated by metabolic labeling with [14C]ethanolamine and LC-MS/MS quantification of endogenous NAPEs and NAEs. Endogenous PLAAT2 in HeLa cells also contributes to NAPE formation.\",\n      \"method\": \"Metabolic labeling with [14C]ethanolamine in transiently and stably expressing cells; LC-tandem MS quantification of NAPEs and NAEs; stable overexpression in HEK293 cells; endogenous contribution assessed in HeLa cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (metabolic labeling + LC-MS/MS), both transient and stable expression systems, endogenous knockdown-complementary data, single lab\",\n      \"pmids\": [\"22825852\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"PLAAT2 (HRASLS2) suppresses RAS-GTP levels and total RAS protein in cancer cells; the C-terminal hydrophobic domain is required for both growth suppression and RAS inhibitory activity, as C-terminal truncation abolishes both effects. Wild-type HRASLS2 localizes in a granular perinuclear pattern, while C-terminal truncation results in diffuse localization.\",\n      \"method\": \"Colony formation assay; RAS-GTP pulldown; overexpression of truncation mutants in HtTA and HCT116 cells; fluorescence localization of wild-type and truncated constructs\",\n      \"journal\": \"Amino acids\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — clean loss-of-function via truncation mutants with defined cellular phenotype and pathway placement, single lab, no replicated by independent group\",\n      \"pmids\": [\"18163183\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Recombinant PLAAT2 protein is purified and used as a biochemical tool for NAT (N-acyltransferase) assays, confirming its Ca2+-independent N-acyltransferase activity that transfers an acyl chain from the sn-1 position of phosphatidylcholine to phosphatidylethanolamine to form N-acylphosphatidylethanolamine.\",\n      \"method\": \"Purification of recombinant PLAAT2; radiolabeled NAT activity assay\",\n      \"journal\": \"Methods in molecular biology (Clifton, N.J.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — direct in vitro enzymatic assay with purified recombinant protein, but methods chapter replicating previously established findings\",\n      \"pmids\": [\"27245897\", \"36152189\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Heterologous expression of human PLAAT2 in E. coli Nissle 1917 conferred resistance to diet-induced obesity in mice comparable to expression of Arabidopsis NAPE synthase, confirming that PLAAT2 produces NAPEs that mediate anti-obesity effects in vivo.\",\n      \"method\": \"In vivo xenograft/dietary obesity mouse model with engineered bacteria expressing human PLAAT2; comparison with plant NAPE synthase\",\n      \"journal\": \"Applied microbiology and biotechnology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — functional in vivo rescue experiment confirming NAPE biosynthesis as the relevant activity, single lab, single study\",\n      \"pmids\": [\"31203417\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"PLAAT2 (HRASLS2) is labeled by the fluorescent lipase probe MB064 and inhibited by α-ketoamide LEI110 (a selective pan-HRASLS family thiol hydrolase inhibitor), confirming PLAAT2 is a cysteine-dependent thiol hydrolase; competitive ABPP and chemical proteomics established PLAAT2 as a member of the HRASLS family of cysteine hydrolases.\",\n      \"method\": \"Activity-based protein profiling (ABPP) with fluorescent probe MB064; competitive ABPP with α-ketoamide inhibitors; chemical proteomics\",\n      \"journal\": \"ACS chemical biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — ABPP with recombinant and endogenous protein confirms catalytic mechanism (cysteine hydrolase), single lab, single study\",\n      \"pmids\": [\"30620559\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"LEI-301, an α-ketoamide PLAAT family inhibitor, reduces NAE levels including anandamide in cells overexpressing PLAAT2, establishing that PLAAT2 enzymatic activity directly controls cellular NAE production.\",\n      \"method\": \"Activity-based protein profiling; cellular NAE quantification in PLAAT2-overexpressing cells treated with inhibitor\",\n      \"journal\": \"Journal of medicinal chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — pharmacological inhibition with target-selective inhibitor linked to cellular lipid metabolite changes, single lab\",\n      \"pmids\": [\"32787138\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PLAAT2 (HRASLS2) interacts with aspartate β-hydroxylase (ASPH) protein and increases its stability in pancreatic cancer cells; overexpression of ASPH reverses the inhibitory effects on cell growth and glycolysis caused by HRASLS2 knockdown, placing HRASLS2 upstream of ASPH in a growth/glycolysis-promoting pathway.\",\n      \"method\": \"Co-immunoprecipitation; protein stability assay; knockdown/overexpression in pancreatic cancer cell lines; xenograft model; glycolysis measurement (ECAR, glucose consumption, lactic acid)\",\n      \"journal\": \"Naunyn-Schmiedeberg's archives of pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — co-IP identifies binding partner, epistasis rescue experiment places HRASLS2 upstream of ASPH, single lab, single study\",\n      \"pmids\": [\"40833600\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"PLAAT2 interacts with cMyc and TRIM32 (identified by IP-MS and validated by co-IP); PLAAT2 facilitates recruitment of TRIM32 to promote ubiquitination and degradation of cMyc, thereby suppressing MEK/ERK signaling in gastric cancer cells. Loss of PLAAT2 results in increased cMyc stability, elevated MEK/ERK activity, and enhanced proliferation, migration, and invasion.\",\n      \"method\": \"Immunoprecipitation-mass spectrometry (IP-MS); co-immunoprecipitation; ubiquitination assay; western blot for MEK/ERK pathway; knockdown/overexpression in gastric cancer cells; xenograft model\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — IP-MS discovery validated by co-IP and ubiquitination assay with in vivo xenograft confirmation, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"41851102\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PLAAT2 (HRASLS2) is a Ca2+-independent cysteine thiol hydrolase of the LRAT/HRASLS family that functions primarily as a phospholipase A1/A2 and N-acyltransferase, transferring acyl chains from phosphatidylcholine to phosphatidylethanolamine to generate N-acylphosphatidylethanolamine (NAPE) precursors for bioactive N-acylethanolamines (including anandamide); its C-terminal hydrophobic domain is required for perinuclear localization and for suppressing RAS-GTP levels; beyond lipid metabolism, PLAAT2 can interact with ASPH to stabilize it (promoting growth/glycolysis in pancreatic cancer) or recruit TRIM32 to ubiquitinate and degrade cMyc and suppress MEK/ERK signaling (acting as a tumor suppressor in gastric cancer), indicating context-dependent roles in cancer cell signaling.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PLAAT2 (HRASLS2) is a Ca2+-independent cysteine-dependent thiol hydrolase that governs the biosynthesis of N-acylphosphatidylethanolamine (NAPE) precursors of bioactive N-acylethanolamines [#0, #5]. As a purified recombinant enzyme it displays phospholipase A1/A2 activity (PLA1 predominating) on phosphatidylcholines and phosphatidylethanolamines and, critically, an N-acyltransferase activity that transfers an acyl chain from the sn-1 position of phosphatidylcholine onto phosphatidylethanolamine to generate N-acyl-PE [#0, #3]. This catalytic output is operational in cells: PLAAT2 expression drives endogenous production of NAPEs and downstream NAEs including anandamide, an output abolished by selective α-ketoamide PLAAT-family inhibitors [#1, #6]. Heterologous expression of human PLAAT2 in engineered bacteria confers resistance to diet-induced obesity in mice, confirming NAPE generation as its physiologically relevant activity in vivo [#4]. A C-terminal hydrophobic domain directs PLAAT2 to a granular perinuclear localization and is required for its suppression of RAS-GTP levels and cell growth [#2]. Beyond lipid metabolism, PLAAT2 acts as a context-dependent modulator of cancer cell signaling: it binds and stabilizes aspartate β-hydroxylase (ASPH) to promote growth and glycolysis in pancreatic cancer [#7], whereas in gastric cancer it recruits the E3 ligase TRIM32 to ubiquitinate and degrade cMyc, thereby dampening MEK/ERK signaling and proliferation [#8].\",\n  \"teleology\": [\n    {\n      \"year\": 2007,\n      \"claim\": \"Established the first cellular phenotype for PLAAT2 by linking it to RAS pathway suppression and identifying the protein domain responsible.\",\n      \"evidence\": \"RAS-GTP pulldown, colony formation, and localization of C-terminal truncation mutants in cancer cell lines\",\n      \"pmids\": [\"18163183\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism connecting catalytic/lipid activity to RAS-GTP reduction not defined\", \"Single lab, not independently replicated\", \"Whether perinuclear localization is causally upstream of RAS suppression untested\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Defined the core enzymology of PLAAT2 as a Ca2+-independent phospholipase and N-acyltransferase, answering what biochemical reactions it catalyzes.\",\n      \"evidence\": \"In vitro enzyme assays with purified recombinant protein across multiple phospholipid substrates\",\n      \"pmids\": [\"19615464\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative physiological weighting of PLA1 versus NAT activity in vivo unresolved\", \"Active-site residues not directly mapped in this study\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Demonstrated that PLAAT2 enzymatic activity translates into NAPE and NAE production in living cells, bridging in vitro biochemistry to cellular lipid signaling.\",\n      \"evidence\": \"Metabolic labeling with [14C]ethanolamine plus LC-MS/MS in transient/stable expression systems and endogenous HeLa contribution\",\n      \"pmids\": [\"22825852\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Endogenous knockout-level loss-of-function not assessed\", \"Tissue-specific physiological NAE outputs not addressed\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Confirmed PLAAT2 is a cysteine-dependent thiol hydrolase and placed it firmly within the HRASLS cysteine-hydrolase family via chemical biology.\",\n      \"evidence\": \"Activity-based protein profiling with MB064 probe and competitive inhibition by α-ketoamide LEI110\",\n      \"pmids\": [\"30620559\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Catalytic cysteine residue not individually mutated in this report\", \"Endogenous physiological substrates in native tissue not enumerated\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Provided in vivo evidence that PLAAT2-driven NAPE production has a metabolic, anti-obesity consequence in a whole animal.\",\n      \"evidence\": \"Engineered E. coli Nissle 1917 expressing human PLAAT2 in a diet-induced obesity mouse model, benchmarked to plant NAPE synthase\",\n      \"pmids\": [\"31203417\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Effect demonstrated via heterologous bacterial expression, not native mammalian PLAAT2\", \"Mechanistic chain from NAPE to satiety not dissected here\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Pharmacologically tied PLAAT2 catalytic activity to cellular anandamide and NAE levels, establishing it as a controllable node in endocannabinoid-related lipid metabolism.\",\n      \"evidence\": \"ABPP with α-ketoamide inhibitor LEI-301 and NAE quantification in PLAAT2-overexpressing cells\",\n      \"pmids\": [\"32787138\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Inhibitor selectivity across PLAAT family limits attribution to PLAAT2 alone\", \"Endogenous-level contribution to anandamide pools not quantified\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identified a non-catalytic protein-stabilizing function whereby PLAAT2 promotes pancreatic cancer growth and glycolysis through ASPH.\",\n      \"evidence\": \"Co-IP, protein stability assays, knockdown/overexpression epistasis rescue, glycolysis measurements, and xenograft in pancreatic cancer cells\",\n      \"pmids\": [\"40833600\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs indirect nature of the PLAAT2-ASPH interaction not resolved\", \"Whether enzymatic activity is required for ASPH stabilization unknown\", \"Single lab, single study\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Revealed an opposing tumor-suppressive function in which PLAAT2 acts as a scaffold recruiting TRIM32 to degrade cMyc and restrain MEK/ERK signaling.\",\n      \"evidence\": \"IP-MS discovery, co-IP and ubiquitination assays, MEK/ERK western blots, knockdown/overexpression, and xenograft in gastric cancer cells\",\n      \"pmids\": [\"41851102\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Reconciliation with the pro-tumor ASPH role (context dependence) not mechanistically explained\", \"Role of catalytic activity in TRIM32 recruitment untested\", \"Single lab, single study\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How PLAAT2's lipid-metabolic activity, RAS/MEK-ERK modulation, and context-dependent partner interactions (ASPH vs TRIM32-cMyc) are integrated into one coherent mechanism remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No study links catalytic NAT/PLA activity to the protein-protein scaffolding functions\", \"Determinants of opposite cancer roles across tissues unknown\", \"No structural model of substrate or partner engagement\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [0, 5]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 3]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005635\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [0, 1, 4]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [2, 8]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"ASPH\", \"TRIM32\", \"MYC\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}