{"gene":"PLAAT4","run_date":"2026-06-10T06:43:35","timeline":{"discoveries":[{"year":2009,"finding":"Purified recombinant TIG3 (PLAAT4) functions as a Ca2+-independent phospholipase A1/2 with maximal activity of 0.53 µmol/min/mg, active on phosphatidylcholines and phosphatidylethanolamines, with PLA1 activity predominating over PLA2. TIG3 also catalyzes N-acylation of PE and O-acylation of lyso-PC at relatively low rates.","method":"In vitro enzymatic assay with purified recombinant protein; substrate specificity profiling","journal":"Biochimica et biophysica acta","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct in vitro reconstitution with purified recombinant protein, quantitative activity measurements, replicated across multiple substrates","pmids":["19615464"],"is_preprint":false},{"year":2010,"finding":"The N-terminal hydrophilic region of TIG3 (residues 1–134) is sufficient for Ca2+-independent phospholipase A2 enzymatic activity, while the C-terminal hydrophobic region is important for cellular localization rather than catalysis.","method":"Expression and purification of truncated N-terminal domain; in vitro phospholipase activity assay; limited proteolysis mapping structural domain boundaries","journal":"Protein expression and purification","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro enzymatic assay with domain-truncation constructs, limited proteolysis structural mapping, single lab","pmids":["20100577"],"is_preprint":false},{"year":2000,"finding":"The C-terminal hydrophobic domain of TIG3 is required for perinuclear/membrane localization and for full growth-suppressive activity. Truncated TIG3 lacking this domain (TIG3 1–134) redistributes to the cytoplasm and shows partial loss of colony-suppression activity.","method":"Vector-mediated expression of full-length vs. C-terminal truncation mutants; GFP-fusion localization by fluorescence microscopy; colony formation assay in CHO, T47D and HaCaT cells","journal":"International journal of oncology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — direct localization experiment with functional consequence, multiple cell lines, domain-truncation mutagenesis, two orthogonal readouts (localization + colony formation)","pmids":["11078805"],"is_preprint":false},{"year":2007,"finding":"TIG3 interacts with type I transglutaminase (TG1) through a domain spanning amino acids 112–164. The N-terminal conserved region of TIG3 is required for keratinocyte differentiation; its removal converts TIG3 into a proapoptotic protein characterized by cell rounding, membrane blebbing, cytochrome c release, and caspase-3/PARP cleavage. Loss of the N-terminal region also shifts TIG3 to increased membrane association.","method":"Co-precipitation of TG1 with TIG3 truncation mutants; apoptosis assays (cytochrome c release, caspase-3/PARP cleavage, p53/p21 levels); fluorescence localization of mutant series","journal":"The Journal of investigative dermatology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-precipitation, multiple truncation mutants, multiple orthogonal apoptosis readouts, functional differentiation assay; replicated in context of prior TG1-interaction work","pmids":["17762858"],"is_preprint":false},{"year":2008,"finding":"TIG3 interacts with and activates type I transglutaminase (TG1) to promote cornified envelope formation during keratinocyte terminal differentiation. TIG3 expression in the suprabasal epidermis is associated with TG1 activation.","method":"Co-immunoprecipitation; transglutaminase activity assay; immunofluorescence localization in epidermis","journal":"Amino acids","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP and activity assay from single lab; consistent with companion papers on the same interaction","pmids":["18612777"],"is_preprint":false},{"year":2011,"finding":"TIG3 localizes near the centrosome in squamous cell carcinoma cells, and pericentrosomal accumulation of TIG3 alters microtubule and microfilament organization, drives pericentrosomal organelle clustering (a hallmark of apoptosis), reduces cyclin D1/E/A, increases p21, elevates Bax, reduces Bcl-XL, and promotes cleavage of procaspase-3/-9 and PARP.","method":"Fluorescence microscopy of GFP-TIG3; organelle distribution assays; Western blot for cell-cycle and apoptosis markers in SCC-13 cells expressing TIG3","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization with functional consequence, multiple downstream markers, single lab","pmids":["21858038"],"is_preprint":false},{"year":2012,"finding":"TIG3 colocalizes with γ-tubulin and pericentrin at the centrosome, alters microtubule nucleation and anterograde growth, increases α-tubulin acetylation and detyrosination, increases insoluble tubulin, drives formation of a peripheral microtubule ring, suppresses centrosome separation (but not duplication), and reduces cell proliferation.","method":"Immunofluorescence co-localization with centrosome markers; microtubule dynamics assays; tubulin modification Western blots; centrosome separation counting; proliferation assays","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization with functional consequences, multiple orthogonal readouts, single lab","pmids":["22427689"],"is_preprint":false},{"year":2013,"finding":"The C-terminal hydrophobic domain of TIG3 targets intact TIG3 to the plasma membrane but, when isolated independently, localizes to mitochondria. A segment within the N-terminal hydrophilic region (amino acids 1–135) is necessary and sufficient for centrosomal targeting, indicating dual localization signals for membrane vs. centrosome functions.","method":"GFP-fusion constructs of isolated domains; fluorescence microscopy in keratinocytes; subcellular fractionation","journal":"The Journal of investigative dermatology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization experiment with domain dissection, functional consequence inferred from prior work; single lab","pmids":["24401997"],"is_preprint":false},{"year":2014,"finding":"TIG3 distributes to the cell membrane (where it activates TG1 for terminal differentiation) and to the centrosome (where it inhibits centrosome separation during mitosis and alters microtubule function), establishing two spatially distinct mechanisms for controlling keratinocyte proliferation and survival.","method":"Immunofluorescence localization; TG1 activity assay; centrosome separation assay; cell proliferation assay (review/synthesis of prior experimental work)","journal":"The Journal of investigative dermatology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — review synthesizing direct experimental findings from the same lab across multiple papers; each individual claim is from prior Tier-2 work","pmids":["24599174"],"is_preprint":false},{"year":2014,"finding":"RARRES3 phospholipase A1/A2 enzymatic activity contributes to tumor cell differentiation; loss of this activity promotes lung metastasis of breast cancer cells. RARRES3 downregulation also facilitates adhesion of tumor cells to the lung parenchyma.","method":"Loss-of-function (shRNA knockdown) and re-expression of wild-type vs. catalytically inactive RARRES3 mutants; in vitro adhesion assays; in vivo lung metastasis mouse models","journal":"EMBO molecular medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — active-site mutant rescue experiment linking enzymatic activity to biological phenotype, combined with in vivo metastasis model; multiple orthogonal methods","pmids":["24867881"],"is_preprint":false},{"year":2014,"finding":"RARRES3 acts as an acyl protein thioesterase that binds Wnt proteins and LRP6, modulates their acylation status, and thereby suppresses Wnt/β-catenin signaling, epithelial-mesenchymal transition, and cancer stem cell properties. Mutation of conserved active-site residues abolishes this deacylation activity. p53 induces RARRES3 expression, linking p53 to Wnt pathway regulation through protein deacylation.","method":"Co-immunoprecipitation of RARRES3 with Wnt proteins and LRP6; active-site mutagenesis; acylation status assays; EMT and cancer stem cell phenotype assays; p53 modulation experiments in breast cancer cells","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — active-site mutagenesis combined with co-IP and functional pathway assays; novel enzymatic mechanism (thioesterase) with multiple orthogonal readouts; single lab","pmids":["25361079"],"is_preprint":false},{"year":2015,"finding":"RARRES3 interacts with MTDH (metadherin/AEG-1) as determined by co-immunoprecipitation, and their interaction is inversely correlated; RARRES3 suppresses EMT and metastasis of colorectal cancer cells in vitro and in vivo through this suppression of MTDH.","method":"Co-immunoprecipitation; knockdown and re-expression in CRC cell lines; transwell/wound healing migration assays; tail-vein xenograft metastasis model","journal":"American journal of cancer research","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — co-IP binding partner identification combined with functional in vivo assay; single lab, limited mechanistic depth","pmids":["26269758"],"is_preprint":false},{"year":2015,"finding":"The NMR solution structure of the TIG3 N-terminal domain (NTD) is similar in overall fold to H-REV107 NTD, but the CTD-binding regions on the NTD differ between TIG3 and H-REV107. The TIG3 NTD enhances cell death induced by the CTD, while the H-REV107 NTD is inhibitory; the flexible main loop of H-REV107, but not TIG3, is critical for this NTD-CTD modulatory function.","method":"NMR solution structure determination; cell death assays with domain constructs; domain-interaction studies in HeLa cells","journal":"FEBS letters","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR structure with functional validation (cell death assay), domain-swap and deletion analysis; single lab","pmids":["25871522"],"is_preprint":false},{"year":2016,"finding":"Overexpression of TIG3 in HCC Hep3B cells suppresses tumor growth in vitro and in vivo via inhibition of ERK1/2 signaling, promoting apoptosis and inhibiting proliferation and migration.","method":"TIG3 cDNA overexpression; Western blot for pERK1/2; apoptosis assays; nude mouse xenograft model","journal":"Tumour biology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single overexpression experiment with Western blot for pathway marker; no direct mechanistic link between TIG3 and ERK demonstrated; single lab, single method","pmids":["26951515"],"is_preprint":false},{"year":2017,"finding":"G9a histone methyltransferase epigenetically silences RARRES3 through H3K9 di-methylation at the RARRES3 locus, and this silencing is a key downstream mechanism by which G9a promotes HCC progression. Inactivation of G9a (RNAi, CRISPR, or pharmacological) restores RARRES3 expression and suppresses HCC cell proliferation and metastasis.","method":"ChIP assay (H3K9me2 at RARRES3 promoter); RNA-seq; G9a shRNA/CRISPR KO; pharmacological inhibition; in vivo nude mouse model","journal":"Journal of hepatology","confidence":"High","confidence_rationale":"Tier 2 / Strong — ChIP directly linking G9a to H3K9me2 at RARRES3 locus, confirmed by multiple independent genetic and pharmacological loss-of-function approaches, in vitro and in vivo","pmids":["28532996"],"is_preprint":false},{"year":2017,"finding":"RARRES3 knockdown increases transcript and protein levels of immunoproteasome subunits (but not constitutive proteasome subunits) in mammary epithelial and breast cancer cell lines, identifying RARRES3 as an endogenous inhibitor of immunoproteasome expression. RARRES3 expression is regulated by IRF1 and is sensitive to RORA depletion.","method":"RARRES3 siRNA knockdown; Western blot and RT-qPCR for immunoproteasome subunits; RORA depletion; IRF1 functional analysis","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — knockdown with protein/mRNA readout in two cell line types; no direct binding or mechanistic reconstitution; single lab","pmids":["28051153"],"is_preprint":false},{"year":2019,"finding":"PLAAT4 physically interacts with the ribosomal protein RPLP0, as identified by yeast two-hybrid screening and confirmed by co-immunoprecipitation and co-localization. PLAAT4 expression suppresses RPLP0 protein levels; cells expressing PLAAT4 or with RPLP0 silenced show similar patterns of decreased cell viability/proliferation, increased cell death, and reduced levels of cell-cycle-associated and anti-apoptotic proteins, indicating that RPLP0 downregulation mediates PLAAT4-induced cell cycle arrest and apoptosis.","method":"Yeast two-hybrid screening; co-immunoprecipitation; co-localization by fluorescence microscopy; RPLP0 siRNA knockdown; cell viability/death assays; Western blot for cell-cycle and apoptosis proteins","journal":"Cell biochemistry and biophysics","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — interaction confirmed by three methods (Y2H, Co-IP, co-localization); functional phenocopy of PLAAT4 OE by RPLP0 KD; single lab","pmids":["31131438"],"is_preprint":false},{"year":2021,"finding":"RARRES3 restricts Toxoplasma gondii infection in human cells by inducing premature egress of the parasite. RARRES3 is an IFNγ-stimulated gene whose individual expression is sufficient to restrict parasite growth across multiple human cell lines.","method":"Overexpression screen of 414 IFNγ-induced ISGs; T. gondii infection assays; parasite egress assays in multiple human cell lines","journal":"eLife","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional overexpression screen with validation in multiple cell lines; defined cellular mechanism (premature egress); single lab","pmids":["34871166"],"is_preprint":false},{"year":2022,"finding":"KDM2A (H3K36me2 demethylase) suppresses RARRES3 expression via demethylation of H3K36me2 at the RARRES3 promoter. RARRES3 knockdown attenuates the inhibitory effects of KDM2A depletion on bladder cancer cell malignant phenotypes, placing RARRES3 downstream of KDM2A in an epigenetic regulatory axis.","method":"ChIP for H3K36me2 at RARRES3 promoter; KDM2A knockdown; RARRES3 knockdown rescue epistasis; xenograft mouse model; KDM2A inhibitor + ATRA combination treatment","journal":"Cell death & disease","confidence":"High","confidence_rationale":"Tier 2 / Moderate — direct ChIP showing histone mark at RARRES3 promoter, genetic epistasis (RARRES3 KD rescues KDM2A KD phenotype), in vivo validation; single lab","pmids":["35697678"],"is_preprint":false},{"year":2025,"finding":"CRABP2 physically binds PLAAT4 and decreases its protein stability; inhibition of PLAAT4 reverses the suppression of NSCLC cell malignant phenotypes and lipid droplet formation caused by CRABP2 knockdown, defining a CRABP2/PLAAT4-mediated lipid metabolic axis in lung cancer progression.","method":"Co-immunoprecipitation of CRABP2 and PLAAT4; protein stability assay; PLAAT4 knockdown rescue experiment; lipid droplet quantification; xenograft mouse model","journal":"Journal of Cancer","confidence":"Medium","confidence_rationale":"Tier 2–3 / Weak — Co-IP binding confirmed, genetic epistasis via PLAAT4 KD rescue, functional lipid droplet and xenograft readouts; single lab, single paper","pmids":["40657374"],"is_preprint":false},{"year":2025,"finding":"A peptide derived from TIG3 binds near the Switch II domain of KRAS G12V with moderate affinity, induces conformational changes in KRAS G12V as determined by X-ray crystallography, and reduces viability of cancer cell lines harboring KRAS G12V mutation.","method":"X-ray crystallography of TIG3 peptide–KRAS G12V complex; binding affinity measurement; cell viability assay","journal":"Biochimica et biophysica acta. Proteins and proteomics","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — crystal structure provides direct structural evidence of interaction; functional cell viability data; single lab, single paper, peptide-based interaction not full-length protein","pmids":["40752582"],"is_preprint":false},{"year":2025,"finding":"BCL6 transcriptionally represses PLAAT4 expression in high-grade serous ovarian cancer (HGSOC), and BCL6-mediated downregulation of PLAAT4 activates the PI3K/AKT signaling pathway to promote tumor cell proliferation, invasion, and migration in vitro and in vivo.","method":"CUT&Tag + RNA-seq to identify BCL6 target genes; PLAAT4 knockdown and overexpression; Western blot for PI3K/AKT and EMT markers; xenograft and abdominal metastasis mouse models","journal":"Frontiers in pharmacology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CUT&Tag identifies direct BCL6 binding to PLAAT4 locus; genetic epistasis with functional rescue; in vivo model; single lab","pmids":["40777995"],"is_preprint":false},{"year":2003,"finding":"TIG3 mRNA induction by ATRA in head and neck and lung carcinoma cells is blocked by pan-RAR antagonist AGN193109 and RARα antagonist Ro 41-5253, demonstrating that TIG3 transcription is regulated through retinoid receptors (RAR-dependent). Induction of TIG3 by ATRA is associated with suppression of anchorage-independent colony formation.","method":"Pharmacological RAR antagonist treatment; RT-PCR; anchorage-independent colony formation assay in HNSCC and NSCLC cell lines","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — receptor antagonist experiments defining upstream regulatory pathway, multiple cell lines, functional colony suppression readout; single lab","pmids":["12879006"],"is_preprint":false},{"year":2005,"finding":"TIG3 expression in ovarian carcinoma cells is negatively regulated by an activated MEK-ERK signaling pathway; specific MEK inhibition restores TIG3 mRNA and is correlated with growth inhibition. In a subset of ovarian carcinoma cells, TIG3 suppression is MEK-ERK-independent but can be partially reversed by IFNγ, indicating multiple upstream regulatory mechanisms.","method":"MEK-ERK pathway inhibition with small-molecule MEK inhibitors; IFNγ treatment; RT-PCR; cell growth assay; in situ hybridization","journal":"International journal of cancer","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — pharmacological epistasis with MEK inhibitors in multiple cell lines; mechanistic pathway placement; single lab","pmids":["15856468"],"is_preprint":false}],"current_model":"PLAAT4/TIG3/RARRES3 is a Ca2+-independent phospholipase A1/A2 and acyl protein thioesterase (LRAT-family) whose N-terminal domain harbors catalytic activity while its C-terminal hydrophobic domain drives plasma membrane localization; at the membrane it activates type I transglutaminase to promote keratinocyte terminal differentiation, and at the centrosome (directed by an N-terminal motif) it inhibits centrosome separation and microtubule dynamics to suppress cell proliferation; its thioesterase activity deacylates Wnt proteins and LRP6 to suppress Wnt/β-catenin signaling and EMT; its expression is induced by retinoids through RAR-dependent transcription and suppressed epigenetically by G9a (H3K9me2) and KDM2A (H3K36me2 demethylation), as well as transcriptionally by BCL6; it interacts with RPLP0 to mediate cell cycle arrest and apoptosis, and with MTDH to suppress metastasis; and it restricts Toxoplasma gondii infection by inducing premature parasite egress downstream of IFNγ signaling."},"narrative":{"mechanistic_narrative":"PLAAT4 (TIG3/RARRES3) is a Ca2+-independent phospholipase A1/A2 and acyl protein thioesterase that functions as a tumor suppressor and effector of keratinocyte terminal differentiation [PMID:19615464, PMID:24867881]. Its catalytic activity resides in the N-terminal hydrophilic domain (residues ~1–134), which is sufficient for phospholipase A2 activity and adopts an H-REV107-like fold, while the C-terminal hydrophobic domain dictates subcellular localization rather than catalysis [PMID:20100577, PMID:25871522, PMID:11078805]. The protein partitions between two functional sites: at membranes its C-terminal domain drives plasma membrane targeting where it binds and activates type I transglutaminase to promote cornified envelope formation [PMID:11078805, PMID:17762858, PMID:18612777], whereas an N-terminal motif directs it to the centrosome, where it co-localizes with γ-tubulin and pericentrin, suppresses centrosome separation, alters microtubule dynamics, and restrains proliferation while triggering apoptotic markers [PMID:24401997, PMID:21858038, PMID:22427689]. As an acyl protein thioesterase, RARRES3 deacylates Wnt proteins and LRP6 to suppress Wnt/β-catenin signaling, EMT, and cancer stem cell properties downstream of p53, and its enzymatic activity limits breast cancer lung metastasis [PMID:25361079, PMID:24867881]. Its expression is induced by retinoids through RAR-dependent transcription and is repressed by MEK-ERK signaling, by the histone modifiers G9a (H3K9me2) and KDM2A (H3K36me2 demethylation), and by the transcription factor BCL6 [PMID:12879006, PMID:15856468, PMID:28532996, PMID:35697678, PMID:40777995]. Additional effector interactions include RPLP0 (mediating cell cycle arrest and apoptosis) and MTDH (suppressing metastasis), and RARRES3 functions as an IFNγ-stimulated gene that restricts Toxoplasma gondii by inducing premature parasite egress [PMID:31131438, PMID:26269758, PMID:34871166].","teleology":[{"year":2000,"claim":"Established that the C-terminal hydrophobic domain is required for membrane/perinuclear localization and for the protein's growth-suppressive activity, the first link between localization and function.","evidence":"GFP-fusion localization and colony formation assays of full-length vs. C-terminal truncation mutants in CHO, T47D and HaCaT cells","pmids":["11078805"],"confidence":"High","gaps":["Did not define the catalytic basis of growth suppression","Did not identify molecular partners"]},{"year":2003,"claim":"Defined the upstream transcriptional control, showing TIG3 induction by retinoic acid is RAR-dependent and accompanies suppression of transformed growth.","evidence":"RAR antagonist treatment and RT-PCR with anchorage-independent colony assays in HNSCC and NSCLC cells","pmids":["12879006"],"confidence":"Medium","gaps":["Direct RAR binding to the promoter not demonstrated","Did not connect transcription to a downstream enzymatic mechanism"]},{"year":2005,"claim":"Identified MEK-ERK signaling as a negative regulator of TIG3 expression, with an additional IFNγ-reversible MEK-independent route in some cells.","evidence":"MEK inhibition, IFNγ treatment, RT-PCR and in situ hybridization in ovarian carcinoma cells","pmids":["15856468"],"confidence":"Medium","gaps":["Did not identify the transcription factor coupling MEK-ERK to the locus","MEK-independent mechanism left undefined"]},{"year":2008,"claim":"Provided a molecular effector mechanism for differentiation by showing TIG3 binds and activates type I transglutaminase to drive cornified envelope formation.","evidence":"Co-immunoprecipitation, transglutaminase activity assay and epidermal immunofluorescence (with reciprocal co-precipitation and truncation mapping in companion work)","pmids":["18612777","17762858"],"confidence":"Medium","gaps":["Did not establish whether phospholipase activity is required for TG1 activation","Structural basis of the 112–164 interaction interface unresolved"]},{"year":2009,"claim":"Defined the core biochemical activity, showing recombinant TIG3 is a Ca2+-independent phospholipase with PLA1 predominating over PLA2 and minor acyltransferase activity.","evidence":"In vitro enzymatic assays with purified recombinant protein and substrate specificity profiling","pmids":["19615464"],"confidence":"High","gaps":["Physiological substrate(s) in cells not identified","Did not link enzymatic output to specific cellular phenotypes"]},{"year":2010,"claim":"Localized catalysis to the N-terminal hydrophilic domain, separating enzymatic function from the localization role of the C-terminus.","evidence":"Truncated N-terminal domain expression, in vitro phospholipase assay and limited proteolysis mapping","pmids":["20100577"],"confidence":"High","gaps":["Catalytic residues not pinpointed in this study","No structure available at this stage"]},{"year":2012,"claim":"Resolved the centrosomal mechanism, showing pericentrosomal TIG3 colocalizes with γ-tubulin/pericentrin, alters microtubule nucleation and tubulin modifications, and blocks centrosome separation to limit proliferation.","evidence":"Immunofluorescence co-localization, microtubule dynamics and tubulin-modification assays, centrosome separation counting and proliferation assays (building on 2011 SCC-13 findings)","pmids":["22427689","21858038"],"confidence":"Medium","gaps":["Direct centrosomal binding partner not identified","Whether phospholipase activity drives the microtubule effects unresolved"]},{"year":2013,"claim":"Established dual localization signals, mapping an N-terminal (1–135) centrosome-targeting segment distinct from the C-terminal membrane/mitochondrial signal.","evidence":"GFP-fusion isolated-domain constructs, fluorescence microscopy and subcellular fractionation in keratinocytes","pmids":["24401997"],"confidence":"Medium","gaps":["The trans-acting factors that read each targeting signal are unknown","Switch between membrane and centrosome pools not characterized"]},{"year":2014,"claim":"Demonstrated that phospholipase activity itself is anti-metastatic and uncovered a distinct thioesterase activity that deacylates Wnt/LRP6 to suppress Wnt signaling and EMT downstream of p53.","evidence":"Active-site mutant rescue with in vivo metastasis models; co-IP of RARRES3 with Wnt/LRP6, active-site mutagenesis, acylation and EMT/stemness assays, p53 modulation","pmids":["24867881","25361079"],"confidence":"High","gaps":["Direct demonstration of thioester bond cleavage on endogenous Wnt in vivo limited","Relationship between PLA and thioesterase active sites unresolved"]},{"year":2015,"claim":"Solved the NMR structure of the N-terminal domain and identified MTDH and (later) RPLP0 as effector partners, while showing the NTD-CTD modulatory logic differs from the H-REV107 paralog.","evidence":"NMR solution structure with domain cell-death assays; co-IP and in vivo metastasis assays for MTDH; Y2H/co-IP/co-localization with functional phenocopy for RPLP0","pmids":["25871522","26269758","31131438"],"confidence":"Medium","gaps":["No full-length protein structure","Mechanistic basis of RPLP0 and MTDH downregulation not defined"]},{"year":2017,"claim":"Identified epigenetic silencing of RARRES3 by G9a-mediated H3K9me2 as a route to cancer progression, and an immune-regulatory role as an endogenous inhibitor of immunoproteasome expression under IRF1/RORA control.","evidence":"ChIP for H3K9me2 at the locus with genetic/pharmacological G9a inactivation and in vivo HCC models; siRNA knockdown with immunoproteasome subunit readouts and IRF1/RORA analysis","pmids":["28532996","28051153"],"confidence":"High","gaps":["Mechanism linking RARRES3 to immunoproteasome levels not defined","Direct vs. indirect transcriptional effects of G9a not fully separated"]},{"year":2021,"claim":"Extended RARRES3 function to cell-autonomous immunity, showing this IFNγ-stimulated gene restricts Toxoplasma gondii by inducing premature parasite egress.","evidence":"Overexpression screen of 414 ISGs with T. gondii infection and egress assays across multiple human cell lines","pmids":["34871166"],"confidence":"Medium","gaps":["Molecular mechanism triggering egress unknown","Role of phospholipase vs. thioesterase activity in restriction not tested"]},{"year":2022,"claim":"Added a second epigenetic repressor, placing RARRES3 downstream of KDM2A-mediated H3K36me2 demethylation in bladder cancer malignancy.","evidence":"ChIP for H3K36me2, KDM2A knockdown, RARRES3 knockdown epistasis, xenografts and KDM2A inhibitor + ATRA combination","pmids":["35697678"],"confidence":"High","gaps":["How H3K36me2 demethylation represses transcription mechanistically not detailed","Interplay with G9a/H3K9me2 regulation untested"]},{"year":2025,"claim":"Broadened post-translational and transcriptional regulation and the partner landscape, identifying CRABP2 as a destabilizer, BCL6 as a transcriptional repressor coupling loss to PI3K/AKT activation, and a TIG3-derived peptide binding the KRAS G12V Switch II.","evidence":"Co-IP and protein stability assays with rescue for CRABP2; CUT&Tag/RNA-seq with knockdown/overexpression and AKT readouts for BCL6; X-ray crystallography of a TIG3 peptide–KRAS G12V complex with viability assays","pmids":["40657374","40777995","40752582"],"confidence":"Medium","gaps":["KRAS interaction is peptide-based, not full-length protein","Whether CRABP2-driven degradation and BCL6 repression act in the same tumors unknown"]},{"year":null,"claim":"How the phospholipase and thioesterase activities are partitioned between the membrane and centrosomal pools, and which activity drives each phenotype (differentiation, proliferation control, Wnt suppression, pathogen restriction), remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No full-length structure linking catalytic sites to localization signals","Endogenous lipid and protein substrates in each compartment unidentified","Causal requirement of specific catalytic activity for centrosomal and anti-parasitic functions untested"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[0,1,9,10]},{"term_id":"GO:0140098","term_label":"catalytic activity, acting on RNA","supporting_discovery_ids":[0,1]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[10]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[0]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[2,7,8]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[5,6,7,8]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[2]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[10,21]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[3,4]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[5,6,16]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[17,15]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[9,14,18]}],"complexes":[],"partners":["TGM1","LRP6","MTDH","RPLP0","CRABP2","KRAS"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9UL19","full_name":"Phospholipase A and acyltransferase 4","aliases":["HRAS-like suppressor 4","HRSL4","RAR-responsive protein TIG3","Retinoic acid receptor responder protein 3","Retinoid-inducible gene 1 protein","Tazarotene-induced gene 3 protein"],"length_aa":164,"mass_kda":18.2,"function":"Exhibits both phospholipase A1/2 and acyltransferase activities (PubMed:19615464, PubMed:22605381, PubMed:22825852, PubMed:26503625). Shows phospholipase A1 (PLA1) and A2 (PLA2), 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)","subcellular_location":"Membrane","url":"https://www.uniprot.org/uniprotkb/Q9UL19/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PLAAT4","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/PLAAT4","total_profiled":1310},"omim":[{"mim_id":"605092","title":"PHOSPHOLIPASE A AND ACYLTRANSFERASE 4; PLAAT4","url":"https://www.omim.org/entry/605092"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/PLAAT4"},"hgnc":{"alias_symbol":["TIG3","HRASLS4","PLAAT-4","RIG1"],"prev_symbol":["RARRES3"]},"alphafold":{"accession":"Q9UL19","domains":[{"cath_id":"3.90.1720.10","chopping":"13-128","consensus_level":"high","plddt":83.1901,"start":13,"end":128}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UL19","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UL19-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UL19-F1-predicted_aligned_error_v6.png","plddt_mean":75.69},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PLAAT4","jax_strain_url":"https://www.jax.org/strain/search?query=PLAAT4"},"sequence":{"accession":"Q9UL19","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9UL19.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9UL19/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UL19"}},"corpus_meta":[{"pmid":"28532996","id":"PMC_28532996","title":"Histone 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Proteins and proteomics","url":"https://pubmed.ncbi.nlm.nih.gov/40752582","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":17921,"output_tokens":6250,"usd":0.073757,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":15048,"output_tokens":4778,"usd":0.097345,"stage2_stop_reason":"end_turn"},"total_usd":0.171102,"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\": \"Purified recombinant TIG3 (PLAAT4) functions as a Ca2+-independent phospholipase A1/2 with maximal activity of 0.53 µmol/min/mg, active on phosphatidylcholines and phosphatidylethanolamines, with PLA1 activity predominating over PLA2. TIG3 also catalyzes N-acylation of PE and O-acylation of lyso-PC at relatively low rates.\",\n      \"method\": \"In vitro enzymatic assay with purified recombinant protein; substrate specificity profiling\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct in vitro reconstitution with purified recombinant protein, quantitative activity measurements, replicated across multiple substrates\",\n      \"pmids\": [\"19615464\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The N-terminal hydrophilic region of TIG3 (residues 1–134) is sufficient for Ca2+-independent phospholipase A2 enzymatic activity, while the C-terminal hydrophobic region is important for cellular localization rather than catalysis.\",\n      \"method\": \"Expression and purification of truncated N-terminal domain; in vitro phospholipase activity assay; limited proteolysis mapping structural domain boundaries\",\n      \"journal\": \"Protein expression and purification\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro enzymatic assay with domain-truncation constructs, limited proteolysis structural mapping, single lab\",\n      \"pmids\": [\"20100577\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"The C-terminal hydrophobic domain of TIG3 is required for perinuclear/membrane localization and for full growth-suppressive activity. Truncated TIG3 lacking this domain (TIG3 1–134) redistributes to the cytoplasm and shows partial loss of colony-suppression activity.\",\n      \"method\": \"Vector-mediated expression of full-length vs. C-terminal truncation mutants; GFP-fusion localization by fluorescence microscopy; colony formation assay in CHO, T47D and HaCaT cells\",\n      \"journal\": \"International journal of oncology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization experiment with functional consequence, multiple cell lines, domain-truncation mutagenesis, two orthogonal readouts (localization + colony formation)\",\n      \"pmids\": [\"11078805\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"TIG3 interacts with type I transglutaminase (TG1) through a domain spanning amino acids 112–164. The N-terminal conserved region of TIG3 is required for keratinocyte differentiation; its removal converts TIG3 into a proapoptotic protein characterized by cell rounding, membrane blebbing, cytochrome c release, and caspase-3/PARP cleavage. Loss of the N-terminal region also shifts TIG3 to increased membrane association.\",\n      \"method\": \"Co-precipitation of TG1 with TIG3 truncation mutants; apoptosis assays (cytochrome c release, caspase-3/PARP cleavage, p53/p21 levels); fluorescence localization of mutant series\",\n      \"journal\": \"The Journal of investigative dermatology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-precipitation, multiple truncation mutants, multiple orthogonal apoptosis readouts, functional differentiation assay; replicated in context of prior TG1-interaction work\",\n      \"pmids\": [\"17762858\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"TIG3 interacts with and activates type I transglutaminase (TG1) to promote cornified envelope formation during keratinocyte terminal differentiation. TIG3 expression in the suprabasal epidermis is associated with TG1 activation.\",\n      \"method\": \"Co-immunoprecipitation; transglutaminase activity assay; immunofluorescence localization in epidermis\",\n      \"journal\": \"Amino acids\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP and activity assay from single lab; consistent with companion papers on the same interaction\",\n      \"pmids\": [\"18612777\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"TIG3 localizes near the centrosome in squamous cell carcinoma cells, and pericentrosomal accumulation of TIG3 alters microtubule and microfilament organization, drives pericentrosomal organelle clustering (a hallmark of apoptosis), reduces cyclin D1/E/A, increases p21, elevates Bax, reduces Bcl-XL, and promotes cleavage of procaspase-3/-9 and PARP.\",\n      \"method\": \"Fluorescence microscopy of GFP-TIG3; organelle distribution assays; Western blot for cell-cycle and apoptosis markers in SCC-13 cells expressing TIG3\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization with functional consequence, multiple downstream markers, single lab\",\n      \"pmids\": [\"21858038\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"TIG3 colocalizes with γ-tubulin and pericentrin at the centrosome, alters microtubule nucleation and anterograde growth, increases α-tubulin acetylation and detyrosination, increases insoluble tubulin, drives formation of a peripheral microtubule ring, suppresses centrosome separation (but not duplication), and reduces cell proliferation.\",\n      \"method\": \"Immunofluorescence co-localization with centrosome markers; microtubule dynamics assays; tubulin modification Western blots; centrosome separation counting; proliferation assays\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization with functional consequences, multiple orthogonal readouts, single lab\",\n      \"pmids\": [\"22427689\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"The C-terminal hydrophobic domain of TIG3 targets intact TIG3 to the plasma membrane but, when isolated independently, localizes to mitochondria. A segment within the N-terminal hydrophilic region (amino acids 1–135) is necessary and sufficient for centrosomal targeting, indicating dual localization signals for membrane vs. centrosome functions.\",\n      \"method\": \"GFP-fusion constructs of isolated domains; fluorescence microscopy in keratinocytes; subcellular fractionation\",\n      \"journal\": \"The Journal of investigative dermatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization experiment with domain dissection, functional consequence inferred from prior work; single lab\",\n      \"pmids\": [\"24401997\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"TIG3 distributes to the cell membrane (where it activates TG1 for terminal differentiation) and to the centrosome (where it inhibits centrosome separation during mitosis and alters microtubule function), establishing two spatially distinct mechanisms for controlling keratinocyte proliferation and survival.\",\n      \"method\": \"Immunofluorescence localization; TG1 activity assay; centrosome separation assay; cell proliferation assay (review/synthesis of prior experimental work)\",\n      \"journal\": \"The Journal of investigative dermatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — review synthesizing direct experimental findings from the same lab across multiple papers; each individual claim is from prior Tier-2 work\",\n      \"pmids\": [\"24599174\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"RARRES3 phospholipase A1/A2 enzymatic activity contributes to tumor cell differentiation; loss of this activity promotes lung metastasis of breast cancer cells. RARRES3 downregulation also facilitates adhesion of tumor cells to the lung parenchyma.\",\n      \"method\": \"Loss-of-function (shRNA knockdown) and re-expression of wild-type vs. catalytically inactive RARRES3 mutants; in vitro adhesion assays; in vivo lung metastasis mouse models\",\n      \"journal\": \"EMBO molecular medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — active-site mutant rescue experiment linking enzymatic activity to biological phenotype, combined with in vivo metastasis model; multiple orthogonal methods\",\n      \"pmids\": [\"24867881\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"RARRES3 acts as an acyl protein thioesterase that binds Wnt proteins and LRP6, modulates their acylation status, and thereby suppresses Wnt/β-catenin signaling, epithelial-mesenchymal transition, and cancer stem cell properties. Mutation of conserved active-site residues abolishes this deacylation activity. p53 induces RARRES3 expression, linking p53 to Wnt pathway regulation through protein deacylation.\",\n      \"method\": \"Co-immunoprecipitation of RARRES3 with Wnt proteins and LRP6; active-site mutagenesis; acylation status assays; EMT and cancer stem cell phenotype assays; p53 modulation experiments in breast cancer cells\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — active-site mutagenesis combined with co-IP and functional pathway assays; novel enzymatic mechanism (thioesterase) with multiple orthogonal readouts; single lab\",\n      \"pmids\": [\"25361079\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"RARRES3 interacts with MTDH (metadherin/AEG-1) as determined by co-immunoprecipitation, and their interaction is inversely correlated; RARRES3 suppresses EMT and metastasis of colorectal cancer cells in vitro and in vivo through this suppression of MTDH.\",\n      \"method\": \"Co-immunoprecipitation; knockdown and re-expression in CRC cell lines; transwell/wound healing migration assays; tail-vein xenograft metastasis model\",\n      \"journal\": \"American journal of cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — co-IP binding partner identification combined with functional in vivo assay; single lab, limited mechanistic depth\",\n      \"pmids\": [\"26269758\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The NMR solution structure of the TIG3 N-terminal domain (NTD) is similar in overall fold to H-REV107 NTD, but the CTD-binding regions on the NTD differ between TIG3 and H-REV107. The TIG3 NTD enhances cell death induced by the CTD, while the H-REV107 NTD is inhibitory; the flexible main loop of H-REV107, but not TIG3, is critical for this NTD-CTD modulatory function.\",\n      \"method\": \"NMR solution structure determination; cell death assays with domain constructs; domain-interaction studies in HeLa cells\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structure with functional validation (cell death assay), domain-swap and deletion analysis; single lab\",\n      \"pmids\": [\"25871522\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Overexpression of TIG3 in HCC Hep3B cells suppresses tumor growth in vitro and in vivo via inhibition of ERK1/2 signaling, promoting apoptosis and inhibiting proliferation and migration.\",\n      \"method\": \"TIG3 cDNA overexpression; Western blot for pERK1/2; apoptosis assays; nude mouse xenograft model\",\n      \"journal\": \"Tumour biology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single overexpression experiment with Western blot for pathway marker; no direct mechanistic link between TIG3 and ERK demonstrated; single lab, single method\",\n      \"pmids\": [\"26951515\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"G9a histone methyltransferase epigenetically silences RARRES3 through H3K9 di-methylation at the RARRES3 locus, and this silencing is a key downstream mechanism by which G9a promotes HCC progression. Inactivation of G9a (RNAi, CRISPR, or pharmacological) restores RARRES3 expression and suppresses HCC cell proliferation and metastasis.\",\n      \"method\": \"ChIP assay (H3K9me2 at RARRES3 promoter); RNA-seq; G9a shRNA/CRISPR KO; pharmacological inhibition; in vivo nude mouse model\",\n      \"journal\": \"Journal of hepatology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — ChIP directly linking G9a to H3K9me2 at RARRES3 locus, confirmed by multiple independent genetic and pharmacological loss-of-function approaches, in vitro and in vivo\",\n      \"pmids\": [\"28532996\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"RARRES3 knockdown increases transcript and protein levels of immunoproteasome subunits (but not constitutive proteasome subunits) in mammary epithelial and breast cancer cell lines, identifying RARRES3 as an endogenous inhibitor of immunoproteasome expression. RARRES3 expression is regulated by IRF1 and is sensitive to RORA depletion.\",\n      \"method\": \"RARRES3 siRNA knockdown; Western blot and RT-qPCR for immunoproteasome subunits; RORA depletion; IRF1 functional analysis\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — knockdown with protein/mRNA readout in two cell line types; no direct binding or mechanistic reconstitution; single lab\",\n      \"pmids\": [\"28051153\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"PLAAT4 physically interacts with the ribosomal protein RPLP0, as identified by yeast two-hybrid screening and confirmed by co-immunoprecipitation and co-localization. PLAAT4 expression suppresses RPLP0 protein levels; cells expressing PLAAT4 or with RPLP0 silenced show similar patterns of decreased cell viability/proliferation, increased cell death, and reduced levels of cell-cycle-associated and anti-apoptotic proteins, indicating that RPLP0 downregulation mediates PLAAT4-induced cell cycle arrest and apoptosis.\",\n      \"method\": \"Yeast two-hybrid screening; co-immunoprecipitation; co-localization by fluorescence microscopy; RPLP0 siRNA knockdown; cell viability/death assays; Western blot for cell-cycle and apoptosis proteins\",\n      \"journal\": \"Cell biochemistry and biophysics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — interaction confirmed by three methods (Y2H, Co-IP, co-localization); functional phenocopy of PLAAT4 OE by RPLP0 KD; single lab\",\n      \"pmids\": [\"31131438\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"RARRES3 restricts Toxoplasma gondii infection in human cells by inducing premature egress of the parasite. RARRES3 is an IFNγ-stimulated gene whose individual expression is sufficient to restrict parasite growth across multiple human cell lines.\",\n      \"method\": \"Overexpression screen of 414 IFNγ-induced ISGs; T. gondii infection assays; parasite egress assays in multiple human cell lines\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional overexpression screen with validation in multiple cell lines; defined cellular mechanism (premature egress); single lab\",\n      \"pmids\": [\"34871166\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"KDM2A (H3K36me2 demethylase) suppresses RARRES3 expression via demethylation of H3K36me2 at the RARRES3 promoter. RARRES3 knockdown attenuates the inhibitory effects of KDM2A depletion on bladder cancer cell malignant phenotypes, placing RARRES3 downstream of KDM2A in an epigenetic regulatory axis.\",\n      \"method\": \"ChIP for H3K36me2 at RARRES3 promoter; KDM2A knockdown; RARRES3 knockdown rescue epistasis; xenograft mouse model; KDM2A inhibitor + ATRA combination treatment\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct ChIP showing histone mark at RARRES3 promoter, genetic epistasis (RARRES3 KD rescues KDM2A KD phenotype), in vivo validation; single lab\",\n      \"pmids\": [\"35697678\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CRABP2 physically binds PLAAT4 and decreases its protein stability; inhibition of PLAAT4 reverses the suppression of NSCLC cell malignant phenotypes and lipid droplet formation caused by CRABP2 knockdown, defining a CRABP2/PLAAT4-mediated lipid metabolic axis in lung cancer progression.\",\n      \"method\": \"Co-immunoprecipitation of CRABP2 and PLAAT4; protein stability assay; PLAAT4 knockdown rescue experiment; lipid droplet quantification; xenograft mouse model\",\n      \"journal\": \"Journal of Cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Weak — Co-IP binding confirmed, genetic epistasis via PLAAT4 KD rescue, functional lipid droplet and xenograft readouts; single lab, single paper\",\n      \"pmids\": [\"40657374\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"A peptide derived from TIG3 binds near the Switch II domain of KRAS G12V with moderate affinity, induces conformational changes in KRAS G12V as determined by X-ray crystallography, and reduces viability of cancer cell lines harboring KRAS G12V mutation.\",\n      \"method\": \"X-ray crystallography of TIG3 peptide–KRAS G12V complex; binding affinity measurement; cell viability assay\",\n      \"journal\": \"Biochimica et biophysica acta. Proteins and proteomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — crystal structure provides direct structural evidence of interaction; functional cell viability data; single lab, single paper, peptide-based interaction not full-length protein\",\n      \"pmids\": [\"40752582\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"BCL6 transcriptionally represses PLAAT4 expression in high-grade serous ovarian cancer (HGSOC), and BCL6-mediated downregulation of PLAAT4 activates the PI3K/AKT signaling pathway to promote tumor cell proliferation, invasion, and migration in vitro and in vivo.\",\n      \"method\": \"CUT&Tag + RNA-seq to identify BCL6 target genes; PLAAT4 knockdown and overexpression; Western blot for PI3K/AKT and EMT markers; xenograft and abdominal metastasis mouse models\",\n      \"journal\": \"Frontiers in pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CUT&Tag identifies direct BCL6 binding to PLAAT4 locus; genetic epistasis with functional rescue; in vivo model; single lab\",\n      \"pmids\": [\"40777995\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"TIG3 mRNA induction by ATRA in head and neck and lung carcinoma cells is blocked by pan-RAR antagonist AGN193109 and RARα antagonist Ro 41-5253, demonstrating that TIG3 transcription is regulated through retinoid receptors (RAR-dependent). Induction of TIG3 by ATRA is associated with suppression of anchorage-independent colony formation.\",\n      \"method\": \"Pharmacological RAR antagonist treatment; RT-PCR; anchorage-independent colony formation assay in HNSCC and NSCLC cell lines\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — receptor antagonist experiments defining upstream regulatory pathway, multiple cell lines, functional colony suppression readout; single lab\",\n      \"pmids\": [\"12879006\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"TIG3 expression in ovarian carcinoma cells is negatively regulated by an activated MEK-ERK signaling pathway; specific MEK inhibition restores TIG3 mRNA and is correlated with growth inhibition. In a subset of ovarian carcinoma cells, TIG3 suppression is MEK-ERK-independent but can be partially reversed by IFNγ, indicating multiple upstream regulatory mechanisms.\",\n      \"method\": \"MEK-ERK pathway inhibition with small-molecule MEK inhibitors; IFNγ treatment; RT-PCR; cell growth assay; in situ hybridization\",\n      \"journal\": \"International journal of cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — pharmacological epistasis with MEK inhibitors in multiple cell lines; mechanistic pathway placement; single lab\",\n      \"pmids\": [\"15856468\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PLAAT4/TIG3/RARRES3 is a Ca2+-independent phospholipase A1/A2 and acyl protein thioesterase (LRAT-family) whose N-terminal domain harbors catalytic activity while its C-terminal hydrophobic domain drives plasma membrane localization; at the membrane it activates type I transglutaminase to promote keratinocyte terminal differentiation, and at the centrosome (directed by an N-terminal motif) it inhibits centrosome separation and microtubule dynamics to suppress cell proliferation; its thioesterase activity deacylates Wnt proteins and LRP6 to suppress Wnt/β-catenin signaling and EMT; its expression is induced by retinoids through RAR-dependent transcription and suppressed epigenetically by G9a (H3K9me2) and KDM2A (H3K36me2 demethylation), as well as transcriptionally by BCL6; it interacts with RPLP0 to mediate cell cycle arrest and apoptosis, and with MTDH to suppress metastasis; and it restricts Toxoplasma gondii infection by inducing premature parasite egress downstream of IFNγ signaling.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PLAAT4 (TIG3/RARRES3) is a Ca2+-independent phospholipase A1/A2 and acyl protein thioesterase that functions as a tumor suppressor and effector of keratinocyte terminal differentiation [#0, #9]. Its catalytic activity resides in the N-terminal hydrophilic domain (residues ~1–134), which is sufficient for phospholipase A2 activity and adopts an H-REV107-like fold, while the C-terminal hydrophobic domain dictates subcellular localization rather than catalysis [#1, #12, #2]. The protein partitions between two functional sites: at membranes its C-terminal domain drives plasma membrane targeting where it binds and activates type I transglutaminase to promote cornified envelope formation [#2, #3, #4], whereas an N-terminal motif directs it to the centrosome, where it co-localizes with γ-tubulin and pericentrin, suppresses centrosome separation, alters microtubule dynamics, and restrains proliferation while triggering apoptotic markers [#7, #5, #6]. As an acyl protein thioesterase, RARRES3 deacylates Wnt proteins and LRP6 to suppress Wnt/β-catenin signaling, EMT, and cancer stem cell properties downstream of p53, and its enzymatic activity limits breast cancer lung metastasis [#10, #9]. Its expression is induced by retinoids through RAR-dependent transcription and is repressed by MEK-ERK signaling, by the histone modifiers G9a (H3K9me2) and KDM2A (H3K36me2 demethylation), and by the transcription factor BCL6 [#22, #23, #14, #18, #21]. Additional effector interactions include RPLP0 (mediating cell cycle arrest and apoptosis) and MTDH (suppressing metastasis), and RARRES3 functions as an IFNγ-stimulated gene that restricts Toxoplasma gondii by inducing premature parasite egress [#16, #11, #17].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Established that the C-terminal hydrophobic domain is required for membrane/perinuclear localization and for the protein's growth-suppressive activity, the first link between localization and function.\",\n      \"evidence\": \"GFP-fusion localization and colony formation assays of full-length vs. C-terminal truncation mutants in CHO, T47D and HaCaT cells\",\n      \"pmids\": [\"11078805\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the catalytic basis of growth suppression\", \"Did not identify molecular partners\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Defined the upstream transcriptional control, showing TIG3 induction by retinoic acid is RAR-dependent and accompanies suppression of transformed growth.\",\n      \"evidence\": \"RAR antagonist treatment and RT-PCR with anchorage-independent colony assays in HNSCC and NSCLC cells\",\n      \"pmids\": [\"12879006\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct RAR binding to the promoter not demonstrated\", \"Did not connect transcription to a downstream enzymatic mechanism\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Identified MEK-ERK signaling as a negative regulator of TIG3 expression, with an additional IFNγ-reversible MEK-independent route in some cells.\",\n      \"evidence\": \"MEK inhibition, IFNγ treatment, RT-PCR and in situ hybridization in ovarian carcinoma cells\",\n      \"pmids\": [\"15856468\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not identify the transcription factor coupling MEK-ERK to the locus\", \"MEK-independent mechanism left undefined\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Provided a molecular effector mechanism for differentiation by showing TIG3 binds and activates type I transglutaminase to drive cornified envelope formation.\",\n      \"evidence\": \"Co-immunoprecipitation, transglutaminase activity assay and epidermal immunofluorescence (with reciprocal co-precipitation and truncation mapping in companion work)\",\n      \"pmids\": [\"18612777\", \"17762858\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not establish whether phospholipase activity is required for TG1 activation\", \"Structural basis of the 112–164 interaction interface unresolved\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Defined the core biochemical activity, showing recombinant TIG3 is a Ca2+-independent phospholipase with PLA1 predominating over PLA2 and minor acyltransferase activity.\",\n      \"evidence\": \"In vitro enzymatic assays with purified recombinant protein and substrate specificity profiling\",\n      \"pmids\": [\"19615464\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological substrate(s) in cells not identified\", \"Did not link enzymatic output to specific cellular phenotypes\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Localized catalysis to the N-terminal hydrophilic domain, separating enzymatic function from the localization role of the C-terminus.\",\n      \"evidence\": \"Truncated N-terminal domain expression, in vitro phospholipase assay and limited proteolysis mapping\",\n      \"pmids\": [\"20100577\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Catalytic residues not pinpointed in this study\", \"No structure available at this stage\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Resolved the centrosomal mechanism, showing pericentrosomal TIG3 colocalizes with γ-tubulin/pericentrin, alters microtubule nucleation and tubulin modifications, and blocks centrosome separation to limit proliferation.\",\n      \"evidence\": \"Immunofluorescence co-localization, microtubule dynamics and tubulin-modification assays, centrosome separation counting and proliferation assays (building on 2011 SCC-13 findings)\",\n      \"pmids\": [\"22427689\", \"21858038\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct centrosomal binding partner not identified\", \"Whether phospholipase activity drives the microtubule effects unresolved\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Established dual localization signals, mapping an N-terminal (1–135) centrosome-targeting segment distinct from the C-terminal membrane/mitochondrial signal.\",\n      \"evidence\": \"GFP-fusion isolated-domain constructs, fluorescence microscopy and subcellular fractionation in keratinocytes\",\n      \"pmids\": [\"24401997\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"The trans-acting factors that read each targeting signal are unknown\", \"Switch between membrane and centrosome pools not characterized\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Demonstrated that phospholipase activity itself is anti-metastatic and uncovered a distinct thioesterase activity that deacylates Wnt/LRP6 to suppress Wnt signaling and EMT downstream of p53.\",\n      \"evidence\": \"Active-site mutant rescue with in vivo metastasis models; co-IP of RARRES3 with Wnt/LRP6, active-site mutagenesis, acylation and EMT/stemness assays, p53 modulation\",\n      \"pmids\": [\"24867881\", \"25361079\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct demonstration of thioester bond cleavage on endogenous Wnt in vivo limited\", \"Relationship between PLA and thioesterase active sites unresolved\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Solved the NMR structure of the N-terminal domain and identified MTDH and (later) RPLP0 as effector partners, while showing the NTD-CTD modulatory logic differs from the H-REV107 paralog.\",\n      \"evidence\": \"NMR solution structure with domain cell-death assays; co-IP and in vivo metastasis assays for MTDH; Y2H/co-IP/co-localization with functional phenocopy for RPLP0\",\n      \"pmids\": [\"25871522\", \"26269758\", \"31131438\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No full-length protein structure\", \"Mechanistic basis of RPLP0 and MTDH downregulation not defined\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Identified epigenetic silencing of RARRES3 by G9a-mediated H3K9me2 as a route to cancer progression, and an immune-regulatory role as an endogenous inhibitor of immunoproteasome expression under IRF1/RORA control.\",\n      \"evidence\": \"ChIP for H3K9me2 at the locus with genetic/pharmacological G9a inactivation and in vivo HCC models; siRNA knockdown with immunoproteasome subunit readouts and IRF1/RORA analysis\",\n      \"pmids\": [\"28532996\", \"28051153\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism linking RARRES3 to immunoproteasome levels not defined\", \"Direct vs. indirect transcriptional effects of G9a not fully separated\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Extended RARRES3 function to cell-autonomous immunity, showing this IFNγ-stimulated gene restricts Toxoplasma gondii by inducing premature parasite egress.\",\n      \"evidence\": \"Overexpression screen of 414 ISGs with T. gondii infection and egress assays across multiple human cell lines\",\n      \"pmids\": [\"34871166\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular mechanism triggering egress unknown\", \"Role of phospholipase vs. thioesterase activity in restriction not tested\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Added a second epigenetic repressor, placing RARRES3 downstream of KDM2A-mediated H3K36me2 demethylation in bladder cancer malignancy.\",\n      \"evidence\": \"ChIP for H3K36me2, KDM2A knockdown, RARRES3 knockdown epistasis, xenografts and KDM2A inhibitor + ATRA combination\",\n      \"pmids\": [\"35697678\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How H3K36me2 demethylation represses transcription mechanistically not detailed\", \"Interplay with G9a/H3K9me2 regulation untested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Broadened post-translational and transcriptional regulation and the partner landscape, identifying CRABP2 as a destabilizer, BCL6 as a transcriptional repressor coupling loss to PI3K/AKT activation, and a TIG3-derived peptide binding the KRAS G12V Switch II.\",\n      \"evidence\": \"Co-IP and protein stability assays with rescue for CRABP2; CUT&Tag/RNA-seq with knockdown/overexpression and AKT readouts for BCL6; X-ray crystallography of a TIG3 peptide–KRAS G12V complex with viability assays\",\n      \"pmids\": [\"40657374\", \"40777995\", \"40752582\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"KRAS interaction is peptide-based, not full-length protein\", \"Whether CRABP2-driven degradation and BCL6 repression act in the same tumors unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the phospholipase and thioesterase activities are partitioned between the membrane and centrosomal pools, and which activity drives each phenotype (differentiation, proliferation control, Wnt suppression, pathogen restriction), remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No full-length structure linking catalytic sites to localization signals\", \"Endogenous lipid and protein substrates in each compartment unidentified\", \"Causal requirement of specific catalytic activity for centrosomal and anti-parasitic functions untested\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [0, 1, 9, 10]},\n      {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [10]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [2, 7, 8]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [5, 6, 7, 8]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [10, 21]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [3, 4]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [5, 6, 16]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [17, 15]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [9, 14, 18]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"TGM1\", \"LRP6\", \"MTDH\", \"RPLP0\", \"CRABP2\", \"KRAS\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}