{"gene":"USP46","run_date":"2026-06-11T09:02:06","timeline":{"discoveries":[{"year":2010,"finding":"USP12 and USP46 are histone H2A and H2B deubiquitinases that prefer nucleosomal substrates and deubiquitinate both histone H2A and H2B in vitro and in vivo. WDR48, a WD40 repeat-containing protein, interacts with USP12 and USP46 and is required for their histone deubiquitination activity.","method":"In vitro deubiquitination assay with nucleosomal substrates, co-immunoprecipitation, in vivo ubiquitination assays, Xenopus overexpression/knockdown","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro enzymatic assay with nucleosomal substrates plus in vivo validation, WDR48 interaction confirmed by Co-IP, replicated across multiple experimental systems","pmids":["21183687"],"is_preprint":false},{"year":2012,"finding":"USP46 binds to PHLPP (both isoforms) and directly removes polyubiquitin chains from PHLPP in vitro and in cells, thereby stabilizing PHLPP, reducing its degradation, and suppressing Akt signaling in colon cancer cells.","method":"Co-immunoprecipitation, in vitro deubiquitination assay, ubiquitination assay in cells, shRNA knockdown, xenograft tumor models","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro deubiquitination reconstitution plus in vivo ubiquitination assays and functional xenograft studies, single lab with multiple orthogonal methods","pmids":["22391563"],"is_preprint":false},{"year":2009,"finding":"A 3-bp deletion causing loss of Lys92 in Usp46 is a quantitative trait variant in mice that affects immobility behavior in tail suspension and forced swimming tests, and is implicated in regulation of GABA signaling; mutant mice show decreased muscimol-induced current in hippocampal CA1 neurons and reduced GAD67 expression, rescued by BAC transgene containing wild-type Usp46.","method":"QTL mapping, congenic/subcongenic strain analysis, BAC transgenic rescue, electrophysiology, behavioral testing","journal":"Nature genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis via QTL mapping, congenic strains, and transgenic rescue with electrophysiological and behavioral phenotypic readouts","pmids":["19465912"],"is_preprint":false},{"year":2015,"finding":"USP46 deubiquitinates AMPA receptors (AMPARs) in vitro and in vivo, specifically removing K63-linked ubiquitin chains. Overexpression of USP46 reduces AMPAR ubiquitination, decreases AMPAR degradation, and increases surface AMPAR accumulation; knockdown elevates AMPAR ubiquitination and reduces surface AMPARs at synapses, resulting in decreased miniature excitatory postsynaptic current amplitude.","method":"In vitro deubiquitination assay, in vivo ubiquitination assay, RNAi knockdown, surface receptor assay, electrophysiology (mEPSC recording) in neurons","journal":"Journal of neurochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro enzymatic assay plus in vivo ubiquitination assays, electrophysiology, and surface expression measurements; single lab with multiple orthogonal methods","pmids":["26077708"],"is_preprint":false},{"year":2011,"finding":"In C. elegans, the deubiquitinating enzyme USP-46 regulates glutamate receptor GLR-1 abundance in the ventral nerve cord by deubiquitinating GLR-1 and preventing its lysosomal degradation. usp-46 mutants show decreased GLR-1, increased ubiquitinated GLR-1, and GLR-1-dependent behavioral defects; blocking GLR-1 ubiquitination or lysosomal degradation suppresses the usp-46 mutant phenotype.","method":"Genetic loss-of-function mutant analysis, ubiquitination assay, genetic epistasis with ubiquitination-deficient and lysosomal degradation-deficient mutants, behavioral assays","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis with multiple suppressor mutations defining pathway position, complementary behavioral and biochemical readouts","pmids":["21273419"],"is_preprint":false},{"year":2013,"finding":"The WD40-repeat proteins WDR-20 and WDR-48 bind to USP-46 and stimulate its catalytic deubiquitinating activity in vitro. In C. elegans, overexpression of WDR-20 and WDR-48 increases GLR-1 abundance and decreases ubiquitin-GLR-1 conjugates in a USP-46-dependent manner; wdr-20 and wdr-48 loss-of-function mutants show locomotion defects consistent with decreased glutamatergic signaling.","method":"In vitro deubiquitination activity assay, co-immunoprecipitation, C. elegans overexpression and loss-of-function genetics, behavioral assays, ubiquitin conjugate quantification","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution of WDR protein-stimulated USP-46 activity plus in vivo genetic and biochemical validation across multiple experimental approaches","pmids":["24356955"],"is_preprint":false},{"year":2011,"finding":"The Lys92 deletion in USP46 reduces its deubiquitinating enzyme activity by approximately 27% compared to wild-type, as measured by USP cleavage assay using GST-Ub52 as substrate, providing molecular basis for the behavioral phenotype of mutant mice.","method":"In vitro USP cleavage assay using GST-Ub52 substrate, comparison of wild-type vs. Lys92-deletion mutant enzyme activity","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro enzymatic assay demonstrating reduced activity of deletion mutant, single lab with single method","pmids":["22043315"],"is_preprint":false},{"year":2012,"finding":"Usp46 knockout mice exhibit short immobility in the tail suspension test comparable to the 3-bp deletion mutant, and this phenotype is reversed by the GABA-A receptor agonist nitrazepam in a flumazenil-sensitive manner, establishing that USP46 loss-of-function affects GABAergic signaling via GABA-A receptors.","method":"Usp46 knockout mouse generation, behavioral testing (TST), pharmacological manipulation with nitrazepam and flumazenil","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KO mouse with behavioral phenotype rescued by pharmacological intervention, establishes GABAergic mechanism, single lab","pmids":["22720038"],"is_preprint":false},{"year":2015,"finding":"The EBNA3A, EBNA3B, and EBNA3C proteins of EBV form complexes with the USP46/USP12 deubiquitination complex components WDR48, WDR20; WDR48 is the primary mediator of EBNA3 association with the DUB complex. WDR48 and USP46 are recruited to the p14(ARF) promoter in an EBNA3C-dependent manner.","method":"Tandem affinity purification, mass spectrometry, co-immunoprecipitation, chromatin immunoprecipitation","journal":"PLoS pathogens","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — tandem affinity purification/MS for complex identification confirmed by Co-IP and ChIP, single lab with multiple orthogonal methods","pmids":["25855980"],"is_preprint":false},{"year":2018,"finding":"High-risk HPV E6 oncoprotein selectively recruits the cellular deubiquitinase USP46 to deubiquitinate and stabilize Cdt2/DTL. Stabilization of Cdt2 (a component of CRL4-Cdt2 E3 ligase) limits Set8 (an epigenetic writer) levels and promotes proliferation of HPV-transformed cells. USP46 knockdown inhibits HPV-transformed tumor xenograft growth.","method":"Co-immunoprecipitation, ubiquitination assay, shRNA knockdown, xenograft tumor model, epistasis analysis (E6-USP46-Cdt2-Set8 pathway)","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP defining complex, ubiquitination assay demonstrating deubiquitination of Cdt2, in vivo xenograft confirmation, pathway epistasis established","pmids":["30415951"],"is_preprint":false},{"year":2020,"finding":"WDR-48, but not WDR-20, promotes USP-46 protein abundance by preventing its ubiquitination and proteasomal degradation. WDR-48 binding to USP-46 reduces ubiquitin-USP-46 conjugates and increases USP-46 half-life; a WDR-48 point mutant that cannot bind USP-46 fails to stabilize it.","method":"Ubiquitination assay, proteasome inhibitor treatment (nonadditive effect with WDR-48), WDR-48 point mutant analysis, siRNA knockdown in mammalian cells, in vivo C. elegans experiments, t1/2 measurement","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (ubiquitination assay, proteasome inhibitor, point mutant, siRNA), validated in both mammalian cells and C. elegans, single lab","pmids":["32587090"],"is_preprint":false},{"year":2021,"finding":"USP46 stabilizes MST1 protein in hepatocellular carcinoma cells by directly binding to it and reducing its ubiquitination, thereby increasing MST1 kinase activity, which promotes YAP1 degradation and suppresses tumor growth and metastasis.","method":"Co-immunoprecipitation, ubiquitination assay, overexpression and knockdown experiments, in vitro and in vivo proliferation/metastasis assays, epistasis (YAP1 rescue)","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and ubiquitination assay establish direct deubiquitination of MST1, functional rescue confirms pathway placement, single lab","pmids":["34029571"],"is_preprint":false},{"year":2021,"finding":"WDR-20 and USP-46 work together with WDR-48 to promote surface levels of the C. elegans AMPAR GLR-1 by regulating local GLR-1 insertion into the neuronal surface. WDR-20 expression is activity-regulated: chronic reduction or increase in glutamate signaling reciprocally alters wdr-20 transcription and surface GLR-1 levels in a wdr-20-dependent manner.","method":"C. elegans genetics (loss-of-function mutants), live imaging of GLR-1 insertion rates, transcriptional reporter assays, behavioral assays, epistasis analysis","journal":"The Journal of neuroscience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean genetic dissection with loss-of-function mutants, live imaging of insertion rates, multiple epistasis experiments; single lab","pmids":["33622778"],"is_preprint":false},{"year":2023,"finding":"The USP46 complex (USP46, UAF1/WDR48, WDR20) deubiquitylates the Wnt co-receptor LRP6, counteracting ubiquitylation by RNF43 and ZNRF3, thereby increasing steady-state cell surface LRP6 levels and promoting Wnt/β-catenin signaling. Wnt stimulation promotes association between the USP46 complex and LRP6. USP46 is required for Wnt-dependent intestinal organoid viability.","method":"Co-immunoprecipitation, ubiquitination assay, knockdown studies, size exclusion chromatography, Xenopus and zebrafish embryo knockdown, organoid viability assay","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (Co-IP, ubiquitination assay, size exclusion chromatography), validated in human cells, Xenopus, zebrafish, and organoids; independently confirmed by companion Drosophila paper","pmids":["37798301"],"is_preprint":false},{"year":2023,"finding":"In Drosophila, the evolutionarily conserved Usp46-Uaf1-Wdr20 deubiquitylase complex stabilizes the Wnt co-receptor Arrow/LRP6 by reducing its ubiquitylation and turnover, increasing cell surface Arrow levels and enhancing sensitivity to Wingless morphogen signaling. Usp46 inactivation destabilizes Arrow, reduces Armadillo/β-catenin accumulation, and attenuates Wingless target gene activation, disrupting developmental patterning.","method":"Genetic loss-of-function in Drosophila, immunofluorescence for cell surface receptor levels, ubiquitination assay, target gene expression analysis, tissue patterning assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean genetic loss-of-function in intact Drosophila tissue with multiple molecular and developmental readouts; independently confirms human cell findings in companion paper","pmids":["37798281"],"is_preprint":false},{"year":2020,"finding":"USP46 suppresses lung cancer cell proliferation by antagonizing the ubiquitination of PHLPP1, resulting in PHLPP1 stabilization and inhibition of AKT signaling. This was demonstrated by co-immunoprecipitation and ubiquitination assays; AKT inhibition reversed the effects of USP46 knockdown.","method":"Co-immunoprecipitation, ubiquitination assay, siRNA knockdown, AKT inhibitor epistasis, cell proliferation assay","journal":"BioMed research international","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and ubiquitination assay with pharmacological epistasis confirming pathway, single lab","pmids":["33029497"],"is_preprint":false},{"year":2024,"finding":"USP46 stabilizes PTBP1 in tamoxifen-resistant breast cancer cells by inhibiting its K48-linked ubiquitination degradation, as shown by co-immunoprecipitation and ubiquitination assay. Stabilized PTBP1 increases the PKM2/PKM1 ratio, promotes glycolysis, and enhances tamoxifen resistance.","method":"Co-immunoprecipitation, K48-ubiquitination assay, USP46 overexpression/knockdown, glycolysis detection assay, drug resistance assays","journal":"Biochimica et biophysica acta. Molecular basis of disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and ubiquitination assay establishing direct substrate relationship, functional pathway validation, single lab with multiple methods","pmids":["38176460"],"is_preprint":false},{"year":2024,"finding":"In black carp, bcUSP46 interacts with bcTBK1, stabilizes TBK1 by eliminating K48-linked ubiquitination of TBK1, and enhances TBK1-mediated interferon promoter activity and antiviral gene expression. Overexpression of bcUSP46 enhances IFN pathway activation while knockdown inhibits it.","method":"Co-immunoprecipitation, immunofluorescence colocalization, K48-ubiquitination assay, overexpression/knockdown, IFN promoter reporter assay","journal":"Developmental and comparative immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and ubiquitination assay in a fish ortholog system, functional IFN reporter assays; note this is a non-mammalian ortholog but consistent with mammalian USP46 function","pmids":["38522716"],"is_preprint":false},{"year":2025,"finding":"USP46 binds to HIF-1α, reduces its ubiquitination, and stabilizes HIF-1α protein, thereby promoting HIF-1α transcriptional activity and downstream glycolytic gene expression, which contributes to cardiac hypertrophy. USP46 knockdown decreases HIF-1α levels and glycolysis, attenuating hypertrophy.","method":"Co-immunoprecipitation, ubiquitination assay, USP46 overexpression/knockdown, metabolic profiling (lactate, pyruvate, ATP), mouse cardiac hypertrophy model","journal":"Pathology, research and practice","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and ubiquitination assay establish direct deubiquitination of HIF-1α, in vivo mouse model validation, single lab","pmids":["40328177"],"is_preprint":false},{"year":2025,"finding":"USP46 interacts with and deubiquitinates SNX5 (a ferroptosis promoter) to stabilize it, thereby promoting ferroptosis in I/R-treated neuronal cells. Knockdown of SNX5 abolishes the ferroptosis-promoting effect of USP46 overexpression.","method":"Mass spectrometry for interactor identification, co-immunoprecipitation, ubiquitination assay, USP46 overexpression/knockdown, SNX5 knockdown epistasis, rat cerebral I/R model","journal":"Experimental neurology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS-identified interaction confirmed by Co-IP and ubiquitination assay, in vivo rat model, genetic epistasis with SNX5 knockdown, single lab","pmids":["39909216"],"is_preprint":false},{"year":2025,"finding":"USP46 interacts with ANK2 (Ankyrin2) and enhances its protein stability by deubiquitination, as shown by cycloheximide chase and ubiquitination assays. Stabilized ANK2 promotes cardiomyocyte apoptosis, inflammation, and ferroptosis in myocardial infarction models; ANK2 overexpression reverses the protective effect of USP46 knockdown.","method":"Co-immunoprecipitation, cycloheximide chase assay, ubiquitination assay, USP46/ANK2 knockdown/overexpression, rat I/R model","journal":"Journal of biochemical and molecular toxicology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — cycloheximide chase and ubiquitination assay establish deubiquitination mechanism, genetic epistasis with ANK2, in vivo rat model, single lab","pmids":["40878243"],"is_preprint":false},{"year":2025,"finding":"USP46 interacts with CIRBP (cold-inducible RNA-binding protein) and inhibits its ubiquitination, thereby stabilizing CIRBP and sensitizing trophoblast cells to erastin-induced ferroptosis.","method":"Immunoprecipitation, ubiquitination assay, USP46 overexpression/knockdown, CIRBP rescue experiments, ferroptosis assay","journal":"Integrative biology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — Co-IP and ubiquitination assay establish interaction and deubiquitination, single lab, single cellular model","pmids":["40608782"],"is_preprint":false},{"year":2025,"finding":"USP46 interacts with the transcription factor POU4F1 via direct binding (confirmed by IP-MS, GST pull-down, and co-immunoprecipitation) and stabilizes POU4F1 by deubiquitination, increasing POU4F1 binding to the HPSE promoter, enhancing heparanase (HPSE) expression, ECM remodelling, and neural cell migration.","method":"IP-MS, GST pull-down, co-immunoprecipitation, ubiquitination assay, ChIP-qPCR, luciferase reporter assay, transwell migration assay","journal":"Acta biochimica et biophysica Sinica","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — three orthogonal binding assays (IP-MS, GST pulldown, Co-IP) plus functional transcriptional and migration readouts, single lab","pmids":["40251903"],"is_preprint":false},{"year":2025,"finding":"USP46 interacts with and stabilizes BECN1 by deubiquitination in prostate cancer cells, promoting autophagy and suppressing cell proliferation. Knockdown of BECN1 or autophagy inhibition partially reverses the anti-proliferative effect of USP46 overexpression.","method":"Co-immunoprecipitation, cycloheximide chase assay, BECN1 siRNA epistasis, autophagy inhibition, cell proliferation assay, xenograft model","journal":"Biotechnology and applied biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and cycloheximide chase establish deubiquitination-mediated stabilization, epistasis with BECN1 confirms pathway, single lab","pmids":["40589294"],"is_preprint":false},{"year":2025,"finding":"In a knock-in mouse model expressing miniTurbo fused to endogenous Usp46, proximity labeling identified USP46-associated proteins in the adult brain including known cofactors WDR48 and WDR20. Gene Ontology analysis showed enrichment in postsynaptic pathways. PLPP3 was identified as significantly downregulated in hippocampus of Usp46-knockout mice.","method":"miniTurbo-BioID knock-in mouse, proximity-dependent biotin labeling, mass spectrometry, Usp46-knockout mouse hippocampal proteomics","journal":"Experimental animals","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo BioID in knock-in mouse with MS identification confirms known WDR48/WDR20 interactions and identifies novel PLPP3 association; single lab, method is proximity-based not direct binding","pmids":["40790822"],"is_preprint":false},{"year":2026,"finding":"Loss of USP46 in podocytes causes cytosolic translocation and aggregation of TDP-43, leading to podocyte injury. USP46 is required for podocyte homeostasis, and the drug acarbose acts as a USP46 agonist that reduces TDP-43 aggregation and protects against diabetic nephropathy.","method":"Podocyte-specific Usp46 knockout mouse, TDP-43 localization assay (fractionation/imaging), acarbose treatment, albuminuria measurement, diabetic mouse model","journal":"Science translational medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean conditional KO mouse with defined molecular phenotype (TDP-43 aggregation), pharmacological rescue with acarbose, in vivo diabetic model; mechanism of TDP-43 deubiquitination not directly demonstrated in abstract","pmids":["41811985"],"is_preprint":false}],"current_model":"USP46 is a deubiquitinating enzyme (DUB) of the ubiquitin-specific protease family that removes ubiquitin from diverse substrates including histone H2A/H2B, AMPA receptors (GLR-1/GluA), PHLPP phosphatases, LRP6/Arrow Wnt co-receptors, Cdt2/DTL, MST1, HIF-1α, PTBP1, TBK1, POU4F1, BECN1, ANK2, SNX5, and CIRBP; its catalytic activity is stimulated by a conserved trimeric complex with WDR48 (UAF1) and WDR20, which also stabilize USP46 protein by preventing its own ubiquitination and proteasomal degradation, and this complex is recruited to substrates in diverse contexts including synaptic plasticity (AMPAR surface trafficking), Wnt signaling (LRP6 receptor homeostasis), tumor suppression (PHLPP-Akt axis), and viral oncogenesis (HPV E6-Cdt2-Set8 axis)."},"narrative":{"mechanistic_narrative":"USP46 is a ubiquitin-specific protease whose deubiquitinating activity controls the abundance of diverse substrates, acting most broadly as a stabilizer that opposes ubiquitin-mediated degradation and tunes signaling, synaptic, and metabolic outputs [PMID:21183687, PMID:22391563, PMID:26077708]. It is a histone H2A/H2B deubiquitinase active on nucleosomal substrates [PMID:21183687], and its catalytic activity is allosterically stimulated by a conserved trimeric complex with the WD40-repeat proteins WDR48 (UAF1) and WDR20 [PMID:24356955]; within this complex WDR48 additionally stabilizes USP46 itself by preventing its own ubiquitination and proteasomal degradation [PMID:32587090]. Through this complex USP46 deubiquitinates and stabilizes ionotropic glutamate receptors (mammalian AMPARs and C. elegans GLR-1), antagonizing their lysosomal/proteasomal turnover to control synaptic surface receptor levels and excitatory transmission [PMID:26077708, PMID:21273419, PMID:33622778], a synaptic role corroborated by loss-of-function mouse mutants that exhibit behavioral phenotypes linked to GABAergic signaling [PMID:19465912, PMID:22720038]. In Wnt signaling, the USP46–UAF1–WDR20 complex deubiquitinates the co-receptor LRP6/Arrow, counteracting RNF43/ZNRF3-mediated ubiquitylation to raise cell-surface receptor levels and promote Wnt/β-catenin signaling, a function conserved from Drosophila to vertebrate organoids [PMID:37798301, PMID:37798281]. In disease and cancer contexts USP46 stabilizes a wide range of substrates including the PHLPP phosphatases to suppress Akt signaling [PMID:22391563, PMID:33029497], the kinase MST1 to drive YAP1 degradation [PMID:34029571], and, when hijacked by HPV E6, Cdt2/DTL to limit Set8 and promote proliferation of transformed cells [PMID:30415951]. Additional substrates documented in the corpus span HIF-1α, PTBP1, TBK1, POU4F1, BECN1, SNX5, ANK2, and CIRBP, linking USP46 to glycolytic metabolism, antiviral interferon signaling, transcription, autophagy, and ferroptosis [PMID:38176460, PMID:38522716, PMID:40328177, PMID:39909216, PMID:40251903, PMID:40589294]. Loss of USP46 in podocytes causes cytosolic TDP-43 aggregation and podocyte injury, implicating it in diabetic nephropathy [PMID:41811985].","teleology":[{"year":2009,"claim":"Established USP46 as a physiologically important gene in the nervous system before its biochemistry was known, by linking a natural variant to behavior and GABA signaling.","evidence":"QTL mapping, congenic strains, and BAC transgenic rescue with electrophysiology and behavioral readouts in mice","pmids":["19465912"],"confidence":"High","gaps":["Did not identify direct molecular substrates","Connection between the Lys92 deletion and enzymatic function not yet shown"]},{"year":2010,"claim":"Defined USP46 as a bona fide deubiquitinating enzyme acting on nucleosomal histones and identified WDR48 as a required activating partner.","evidence":"In vitro deubiquitination of nucleosomal H2A/H2B, Co-IP, and in vivo assays in Xenopus","pmids":["21183687"],"confidence":"High","gaps":["Physiological consequences of histone deubiquitination not established","WDR20 not yet implicated"]},{"year":2011,"claim":"Connected the behavioral variant to enzymatic mechanism and revealed a neuronal substrate, showing USP46 controls glutamate receptor abundance.","evidence":"In vitro USP cleavage assay of the Lys92-deletion mutant; C. elegans genetic epistasis showing USP-46 deubiquitinates GLR-1 to block lysosomal degradation","pmids":["22043315","21273419"],"confidence":"Medium","gaps":["27% activity reduction measured with a single artificial substrate","Direct GLR-1 deubiquitination not reconstituted in vitro"]},{"year":2012,"claim":"Extended USP46 substrate repertoire to a tumor-suppressive axis and confirmed the GABAergic mechanism pharmacologically.","evidence":"Co-IP and in vitro deubiquitination of PHLPP with xenografts; Usp46 knockout mouse rescued by nitrazepam in a flumazenil-sensitive manner","pmids":["22391563","22720038"],"confidence":"High","gaps":["Whether PHLPP stabilization requires the WDR cofactor complex not addressed","Direct molecular target underlying GABAergic phenotype not identified"]},{"year":2013,"claim":"Defined the activating trimeric complex by showing both WDR-20 and WDR-48 bind and stimulate USP-46 catalytic activity in a substrate-relevant context.","evidence":"In vitro deubiquitination activity assay, Co-IP, and C. elegans genetics with behavioral readouts","pmids":["24356955"],"confidence":"High","gaps":["Structural basis of stimulation not resolved","Distinct contributions of WDR20 vs WDR48 not separated"]},{"year":2015,"claim":"Confirmed AMPAR regulation in mammals and showed USP46 can be co-opted by a viral oncoprotein complex on chromatin.","evidence":"In vitro/in vivo deubiquitination of K63-linked AMPAR chains with mEPSC recording; tandem affinity purification, Co-IP and ChIP for EBV EBNA3-WDR48/WDR20 recruitment to the p14ARF promoter","pmids":["26077708","25855980"],"confidence":"High","gaps":["Whether EBNA3 recruitment drives histone or other substrate deubiquitination not resolved","Recruitment mechanism to specific AMPAR pools not defined"]},{"year":2018,"claim":"Demonstrated that high-risk HPV E6 selectively redirects USP46 to stabilize Cdt2, linking the DUB to viral oncogenesis.","evidence":"Co-IP, ubiquitination assay, shRNA knockdown, and xenograft with E6-USP46-Cdt2-Set8 epistasis","pmids":["30415951"],"confidence":"High","gaps":["How E6 confers substrate selectivity not structurally defined","Role of WDR cofactors in this recruitment not tested"]},{"year":2020,"claim":"Separated the two cofactor functions, showing WDR-48 (but not WDR-20) stabilizes USP-46 protein by preventing its proteasomal degradation, and added PHLPP1/lung cancer to the substrate set.","evidence":"Ubiquitination assays, proteasome inhibitor non-additivity, WDR-48 binding-deficient point mutant, half-life measurement; separate Co-IP/ubiquitination study in lung cancer","pmids":["32587090","33029497"],"confidence":"High","gaps":["Whether WDR-48-dependent stabilization is regulated dynamically not addressed","Ligase that ubiquitinates USP46 itself not identified"]},{"year":2021,"claim":"Broadened USP46 into Hippo-pathway tumor suppression and refined the activity-dependent regulation of synaptic receptor trafficking.","evidence":"Co-IP/ubiquitination of MST1 with YAP1 rescue in HCC; C. elegans live imaging of GLR-1 insertion and activity-regulated wdr-20 transcription","pmids":["34029571","33622778"],"confidence":"Medium","gaps":["MST1 deubiquitination not reconstituted in vitro","Transcriptional regulator of wdr-20 not identified"]},{"year":2023,"claim":"Established a conserved role for the USP46 complex in Wnt signaling by deubiquitinating LRP6/Arrow to counteract RNF43/ZNRF3 and sustain receptor surface levels.","evidence":"Co-IP, ubiquitination assay, size exclusion chromatography, organoid viability in human cells/Xenopus/zebrafish, and independent Drosophila genetics","pmids":["37798301","37798281"],"confidence":"High","gaps":["Mechanism of Wnt-induced complex-LRP6 association not defined","Whether this is selective over other receptor pools not resolved"]},{"year":2024,"claim":"Expanded the substrate landscape to metabolic and antiviral effectors, showing USP46 stabilizes substrates via K48-linkage removal across contexts.","evidence":"Co-IP and K48-ubiquitination assays for PTBP1 (breast cancer glycolysis/tamoxifen resistance) and the black carp TBK1 ortholog (IFN reporter assays)","pmids":["38176460","38522716"],"confidence":"Medium","gaps":["TBK1 finding is in a non-mammalian ortholog","Cofactor dependence of these substrates not tested"]},{"year":2025,"claim":"Documented a wide additional substrate set linking USP46 to HIF-1α-driven glycolysis, transcription, autophagy, and ferroptosis, and mapped its in vivo brain interactome.","evidence":"Co-IP/ubiquitination assays for HIF-1α, POU4F1, BECN1, SNX5, ANK2, CIRBP with functional and in vivo models; miniTurbo-BioID knock-in mouse confirming WDR48/WDR20 and postsynaptic enrichment","pmids":["40328177","40251903","40589294","39909216","40878243","40608782","40790822"],"confidence":"Medium","gaps":["Many substrates rest on single-lab Co-IP/ubiquitination without in vitro reconstitution","Whether each substrate requires the WDR48/WDR20 complex not systematically tested"]},{"year":2026,"claim":"Linked USP46 to organ-level disease by showing its loss causes pathological TDP-43 aggregation, and identified a pharmacological agonist.","evidence":"Podocyte-specific Usp46 knockout mouse, TDP-43 localization assays, acarbose treatment in a diabetic nephropathy model","pmids":["41811985"],"confidence":"Medium","gaps":["Direct deubiquitination of TDP-43 by USP46 not demonstrated","Mechanism by which acarbose acts as a USP46 agonist not defined"]},{"year":null,"claim":"How USP46 achieves substrate selectivity across its very broad reported substrate set, and which substrates genuinely require the WDR48/WDR20 complex versus context-specific recruitment, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model defining substrate-binding determinants","No systematic test of cofactor dependence across substrates","Many disease substrates rest on single-lab cellular evidence"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,3,9,11,13]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[0,5,6]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[10,13]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,8]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[3,13]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[24]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[13,14,11]},{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[3,4,12]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[9,1,15,25]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[10,1,11]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[19,20,21]}],"complexes":["USP46-UAF1(WDR48)-WDR20 deubiquitinase complex"],"partners":["WDR48","WDR20","PHLPP","LRP6","MST1","DTL","POU4F1","TBK1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P62068","full_name":"Ubiquitin carboxyl-terminal hydrolase 46","aliases":["Deubiquitinating enzyme 46","Ubiquitin thioesterase 46","Ubiquitin-specific-processing protease 46"],"length_aa":366,"mass_kda":42.4,"function":"Deubiquitinating enzyme that plays a role in behavior, possibly by regulating GABA action. May act by mediating the deubiquitination of GAD1/GAD67 (By similarity). Has almost no deubiquitinating activity by itself and requires the interaction with WDR48 to have a high activity (PubMed:19075014, PubMed:26388029). Not involved in deubiquitination of monoubiquitinated FANCD2 (PubMed:19075014)","subcellular_location":"Cytoplasm","url":"https://www.uniprot.org/uniprotkb/P62068/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/USP46","classification":"Not Classified","n_dependent_lines":11,"n_total_lines":1208,"dependency_fraction":0.009105960264900662},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/USP46","total_profiled":1310},"omim":[{"mim_id":"617741","title":"WD REPEAT-CONTAINING PROTEIN 20; WDR20","url":"https://www.omim.org/entry/617741"},{"mim_id":"612849","title":"UBIQUITIN-SPECIFIC PEPTIDASE 46; USP46","url":"https://www.omim.org/entry/612849"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoli","reliability":"Approved"},{"location":"Nucleoli rim","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in 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WDR48, a WD40 repeat-containing protein, interacts with USP12 and USP46 and is required for their histone deubiquitination activity.\",\n      \"method\": \"In vitro deubiquitination assay with nucleosomal substrates, co-immunoprecipitation, in vivo ubiquitination assays, Xenopus overexpression/knockdown\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro enzymatic assay with nucleosomal substrates plus in vivo validation, WDR48 interaction confirmed by Co-IP, replicated across multiple experimental systems\",\n      \"pmids\": [\"21183687\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"USP46 binds to PHLPP (both isoforms) and directly removes polyubiquitin chains from PHLPP in vitro and in cells, thereby stabilizing PHLPP, reducing its degradation, and suppressing Akt signaling in colon cancer cells.\",\n      \"method\": \"Co-immunoprecipitation, in vitro deubiquitination assay, ubiquitination assay in cells, shRNA knockdown, xenograft tumor models\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro deubiquitination reconstitution plus in vivo ubiquitination assays and functional xenograft studies, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"22391563\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"A 3-bp deletion causing loss of Lys92 in Usp46 is a quantitative trait variant in mice that affects immobility behavior in tail suspension and forced swimming tests, and is implicated in regulation of GABA signaling; mutant mice show decreased muscimol-induced current in hippocampal CA1 neurons and reduced GAD67 expression, rescued by BAC transgene containing wild-type Usp46.\",\n      \"method\": \"QTL mapping, congenic/subcongenic strain analysis, BAC transgenic rescue, electrophysiology, behavioral testing\",\n      \"journal\": \"Nature genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis via QTL mapping, congenic strains, and transgenic rescue with electrophysiological and behavioral phenotypic readouts\",\n      \"pmids\": [\"19465912\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"USP46 deubiquitinates AMPA receptors (AMPARs) in vitro and in vivo, specifically removing K63-linked ubiquitin chains. Overexpression of USP46 reduces AMPAR ubiquitination, decreases AMPAR degradation, and increases surface AMPAR accumulation; knockdown elevates AMPAR ubiquitination and reduces surface AMPARs at synapses, resulting in decreased miniature excitatory postsynaptic current amplitude.\",\n      \"method\": \"In vitro deubiquitination assay, in vivo ubiquitination assay, RNAi knockdown, surface receptor assay, electrophysiology (mEPSC recording) in neurons\",\n      \"journal\": \"Journal of neurochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro enzymatic assay plus in vivo ubiquitination assays, electrophysiology, and surface expression measurements; single lab with multiple orthogonal methods\",\n      \"pmids\": [\"26077708\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"In C. elegans, the deubiquitinating enzyme USP-46 regulates glutamate receptor GLR-1 abundance in the ventral nerve cord by deubiquitinating GLR-1 and preventing its lysosomal degradation. usp-46 mutants show decreased GLR-1, increased ubiquitinated GLR-1, and GLR-1-dependent behavioral defects; blocking GLR-1 ubiquitination or lysosomal degradation suppresses the usp-46 mutant phenotype.\",\n      \"method\": \"Genetic loss-of-function mutant analysis, ubiquitination assay, genetic epistasis with ubiquitination-deficient and lysosomal degradation-deficient mutants, behavioral assays\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis with multiple suppressor mutations defining pathway position, complementary behavioral and biochemical readouts\",\n      \"pmids\": [\"21273419\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"The WD40-repeat proteins WDR-20 and WDR-48 bind to USP-46 and stimulate its catalytic deubiquitinating activity in vitro. In C. elegans, overexpression of WDR-20 and WDR-48 increases GLR-1 abundance and decreases ubiquitin-GLR-1 conjugates in a USP-46-dependent manner; wdr-20 and wdr-48 loss-of-function mutants show locomotion defects consistent with decreased glutamatergic signaling.\",\n      \"method\": \"In vitro deubiquitination activity assay, co-immunoprecipitation, C. elegans overexpression and loss-of-function genetics, behavioral assays, ubiquitin conjugate quantification\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution of WDR protein-stimulated USP-46 activity plus in vivo genetic and biochemical validation across multiple experimental approaches\",\n      \"pmids\": [\"24356955\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"The Lys92 deletion in USP46 reduces its deubiquitinating enzyme activity by approximately 27% compared to wild-type, as measured by USP cleavage assay using GST-Ub52 as substrate, providing molecular basis for the behavioral phenotype of mutant mice.\",\n      \"method\": \"In vitro USP cleavage assay using GST-Ub52 substrate, comparison of wild-type vs. Lys92-deletion mutant enzyme activity\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro enzymatic assay demonstrating reduced activity of deletion mutant, single lab with single method\",\n      \"pmids\": [\"22043315\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Usp46 knockout mice exhibit short immobility in the tail suspension test comparable to the 3-bp deletion mutant, and this phenotype is reversed by the GABA-A receptor agonist nitrazepam in a flumazenil-sensitive manner, establishing that USP46 loss-of-function affects GABAergic signaling via GABA-A receptors.\",\n      \"method\": \"Usp46 knockout mouse generation, behavioral testing (TST), pharmacological manipulation with nitrazepam and flumazenil\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO mouse with behavioral phenotype rescued by pharmacological intervention, establishes GABAergic mechanism, single lab\",\n      \"pmids\": [\"22720038\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The EBNA3A, EBNA3B, and EBNA3C proteins of EBV form complexes with the USP46/USP12 deubiquitination complex components WDR48, WDR20; WDR48 is the primary mediator of EBNA3 association with the DUB complex. WDR48 and USP46 are recruited to the p14(ARF) promoter in an EBNA3C-dependent manner.\",\n      \"method\": \"Tandem affinity purification, mass spectrometry, co-immunoprecipitation, chromatin immunoprecipitation\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — tandem affinity purification/MS for complex identification confirmed by Co-IP and ChIP, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"25855980\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"High-risk HPV E6 oncoprotein selectively recruits the cellular deubiquitinase USP46 to deubiquitinate and stabilize Cdt2/DTL. Stabilization of Cdt2 (a component of CRL4-Cdt2 E3 ligase) limits Set8 (an epigenetic writer) levels and promotes proliferation of HPV-transformed cells. USP46 knockdown inhibits HPV-transformed tumor xenograft growth.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, shRNA knockdown, xenograft tumor model, epistasis analysis (E6-USP46-Cdt2-Set8 pathway)\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP defining complex, ubiquitination assay demonstrating deubiquitination of Cdt2, in vivo xenograft confirmation, pathway epistasis established\",\n      \"pmids\": [\"30415951\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"WDR-48, but not WDR-20, promotes USP-46 protein abundance by preventing its ubiquitination and proteasomal degradation. WDR-48 binding to USP-46 reduces ubiquitin-USP-46 conjugates and increases USP-46 half-life; a WDR-48 point mutant that cannot bind USP-46 fails to stabilize it.\",\n      \"method\": \"Ubiquitination assay, proteasome inhibitor treatment (nonadditive effect with WDR-48), WDR-48 point mutant analysis, siRNA knockdown in mammalian cells, in vivo C. elegans experiments, t1/2 measurement\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (ubiquitination assay, proteasome inhibitor, point mutant, siRNA), validated in both mammalian cells and C. elegans, single lab\",\n      \"pmids\": [\"32587090\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"USP46 stabilizes MST1 protein in hepatocellular carcinoma cells by directly binding to it and reducing its ubiquitination, thereby increasing MST1 kinase activity, which promotes YAP1 degradation and suppresses tumor growth and metastasis.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, overexpression and knockdown experiments, in vitro and in vivo proliferation/metastasis assays, epistasis (YAP1 rescue)\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and ubiquitination assay establish direct deubiquitination of MST1, functional rescue confirms pathway placement, single lab\",\n      \"pmids\": [\"34029571\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"WDR-20 and USP-46 work together with WDR-48 to promote surface levels of the C. elegans AMPAR GLR-1 by regulating local GLR-1 insertion into the neuronal surface. WDR-20 expression is activity-regulated: chronic reduction or increase in glutamate signaling reciprocally alters wdr-20 transcription and surface GLR-1 levels in a wdr-20-dependent manner.\",\n      \"method\": \"C. elegans genetics (loss-of-function mutants), live imaging of GLR-1 insertion rates, transcriptional reporter assays, behavioral assays, epistasis analysis\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean genetic dissection with loss-of-function mutants, live imaging of insertion rates, multiple epistasis experiments; single lab\",\n      \"pmids\": [\"33622778\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"The USP46 complex (USP46, UAF1/WDR48, WDR20) deubiquitylates the Wnt co-receptor LRP6, counteracting ubiquitylation by RNF43 and ZNRF3, thereby increasing steady-state cell surface LRP6 levels and promoting Wnt/β-catenin signaling. Wnt stimulation promotes association between the USP46 complex and LRP6. USP46 is required for Wnt-dependent intestinal organoid viability.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, knockdown studies, size exclusion chromatography, Xenopus and zebrafish embryo knockdown, organoid viability assay\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (Co-IP, ubiquitination assay, size exclusion chromatography), validated in human cells, Xenopus, zebrafish, and organoids; independently confirmed by companion Drosophila paper\",\n      \"pmids\": [\"37798301\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"In Drosophila, the evolutionarily conserved Usp46-Uaf1-Wdr20 deubiquitylase complex stabilizes the Wnt co-receptor Arrow/LRP6 by reducing its ubiquitylation and turnover, increasing cell surface Arrow levels and enhancing sensitivity to Wingless morphogen signaling. Usp46 inactivation destabilizes Arrow, reduces Armadillo/β-catenin accumulation, and attenuates Wingless target gene activation, disrupting developmental patterning.\",\n      \"method\": \"Genetic loss-of-function in Drosophila, immunofluorescence for cell surface receptor levels, ubiquitination assay, target gene expression analysis, tissue patterning assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean genetic loss-of-function in intact Drosophila tissue with multiple molecular and developmental readouts; independently confirms human cell findings in companion paper\",\n      \"pmids\": [\"37798281\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"USP46 suppresses lung cancer cell proliferation by antagonizing the ubiquitination of PHLPP1, resulting in PHLPP1 stabilization and inhibition of AKT signaling. This was demonstrated by co-immunoprecipitation and ubiquitination assays; AKT inhibition reversed the effects of USP46 knockdown.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, siRNA knockdown, AKT inhibitor epistasis, cell proliferation assay\",\n      \"journal\": \"BioMed research international\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and ubiquitination assay with pharmacological epistasis confirming pathway, single lab\",\n      \"pmids\": [\"33029497\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP46 stabilizes PTBP1 in tamoxifen-resistant breast cancer cells by inhibiting its K48-linked ubiquitination degradation, as shown by co-immunoprecipitation and ubiquitination assay. Stabilized PTBP1 increases the PKM2/PKM1 ratio, promotes glycolysis, and enhances tamoxifen resistance.\",\n      \"method\": \"Co-immunoprecipitation, K48-ubiquitination assay, USP46 overexpression/knockdown, glycolysis detection assay, drug resistance assays\",\n      \"journal\": \"Biochimica et biophysica acta. Molecular basis of disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and ubiquitination assay establishing direct substrate relationship, functional pathway validation, single lab with multiple methods\",\n      \"pmids\": [\"38176460\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"In black carp, bcUSP46 interacts with bcTBK1, stabilizes TBK1 by eliminating K48-linked ubiquitination of TBK1, and enhances TBK1-mediated interferon promoter activity and antiviral gene expression. Overexpression of bcUSP46 enhances IFN pathway activation while knockdown inhibits it.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence colocalization, K48-ubiquitination assay, overexpression/knockdown, IFN promoter reporter assay\",\n      \"journal\": \"Developmental and comparative immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and ubiquitination assay in a fish ortholog system, functional IFN reporter assays; note this is a non-mammalian ortholog but consistent with mammalian USP46 function\",\n      \"pmids\": [\"38522716\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP46 binds to HIF-1α, reduces its ubiquitination, and stabilizes HIF-1α protein, thereby promoting HIF-1α transcriptional activity and downstream glycolytic gene expression, which contributes to cardiac hypertrophy. USP46 knockdown decreases HIF-1α levels and glycolysis, attenuating hypertrophy.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, USP46 overexpression/knockdown, metabolic profiling (lactate, pyruvate, ATP), mouse cardiac hypertrophy model\",\n      \"journal\": \"Pathology, research and practice\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and ubiquitination assay establish direct deubiquitination of HIF-1α, in vivo mouse model validation, single lab\",\n      \"pmids\": [\"40328177\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP46 interacts with and deubiquitinates SNX5 (a ferroptosis promoter) to stabilize it, thereby promoting ferroptosis in I/R-treated neuronal cells. Knockdown of SNX5 abolishes the ferroptosis-promoting effect of USP46 overexpression.\",\n      \"method\": \"Mass spectrometry for interactor identification, co-immunoprecipitation, ubiquitination assay, USP46 overexpression/knockdown, SNX5 knockdown epistasis, rat cerebral I/R model\",\n      \"journal\": \"Experimental neurology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS-identified interaction confirmed by Co-IP and ubiquitination assay, in vivo rat model, genetic epistasis with SNX5 knockdown, single lab\",\n      \"pmids\": [\"39909216\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP46 interacts with ANK2 (Ankyrin2) and enhances its protein stability by deubiquitination, as shown by cycloheximide chase and ubiquitination assays. Stabilized ANK2 promotes cardiomyocyte apoptosis, inflammation, and ferroptosis in myocardial infarction models; ANK2 overexpression reverses the protective effect of USP46 knockdown.\",\n      \"method\": \"Co-immunoprecipitation, cycloheximide chase assay, ubiquitination assay, USP46/ANK2 knockdown/overexpression, rat I/R model\",\n      \"journal\": \"Journal of biochemical and molecular toxicology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cycloheximide chase and ubiquitination assay establish deubiquitination mechanism, genetic epistasis with ANK2, in vivo rat model, single lab\",\n      \"pmids\": [\"40878243\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP46 interacts with CIRBP (cold-inducible RNA-binding protein) and inhibits its ubiquitination, thereby stabilizing CIRBP and sensitizing trophoblast cells to erastin-induced ferroptosis.\",\n      \"method\": \"Immunoprecipitation, ubiquitination assay, USP46 overexpression/knockdown, CIRBP rescue experiments, ferroptosis assay\",\n      \"journal\": \"Integrative biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — Co-IP and ubiquitination assay establish interaction and deubiquitination, single lab, single cellular model\",\n      \"pmids\": [\"40608782\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP46 interacts with the transcription factor POU4F1 via direct binding (confirmed by IP-MS, GST pull-down, and co-immunoprecipitation) and stabilizes POU4F1 by deubiquitination, increasing POU4F1 binding to the HPSE promoter, enhancing heparanase (HPSE) expression, ECM remodelling, and neural cell migration.\",\n      \"method\": \"IP-MS, GST pull-down, co-immunoprecipitation, ubiquitination assay, ChIP-qPCR, luciferase reporter assay, transwell migration assay\",\n      \"journal\": \"Acta biochimica et biophysica Sinica\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — three orthogonal binding assays (IP-MS, GST pulldown, Co-IP) plus functional transcriptional and migration readouts, single lab\",\n      \"pmids\": [\"40251903\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP46 interacts with and stabilizes BECN1 by deubiquitination in prostate cancer cells, promoting autophagy and suppressing cell proliferation. Knockdown of BECN1 or autophagy inhibition partially reverses the anti-proliferative effect of USP46 overexpression.\",\n      \"method\": \"Co-immunoprecipitation, cycloheximide chase assay, BECN1 siRNA epistasis, autophagy inhibition, cell proliferation assay, xenograft model\",\n      \"journal\": \"Biotechnology and applied biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and cycloheximide chase establish deubiquitination-mediated stabilization, epistasis with BECN1 confirms pathway, single lab\",\n      \"pmids\": [\"40589294\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In a knock-in mouse model expressing miniTurbo fused to endogenous Usp46, proximity labeling identified USP46-associated proteins in the adult brain including known cofactors WDR48 and WDR20. Gene Ontology analysis showed enrichment in postsynaptic pathways. PLPP3 was identified as significantly downregulated in hippocampus of Usp46-knockout mice.\",\n      \"method\": \"miniTurbo-BioID knock-in mouse, proximity-dependent biotin labeling, mass spectrometry, Usp46-knockout mouse hippocampal proteomics\",\n      \"journal\": \"Experimental animals\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo BioID in knock-in mouse with MS identification confirms known WDR48/WDR20 interactions and identifies novel PLPP3 association; single lab, method is proximity-based not direct binding\",\n      \"pmids\": [\"40790822\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"Loss of USP46 in podocytes causes cytosolic translocation and aggregation of TDP-43, leading to podocyte injury. USP46 is required for podocyte homeostasis, and the drug acarbose acts as a USP46 agonist that reduces TDP-43 aggregation and protects against diabetic nephropathy.\",\n      \"method\": \"Podocyte-specific Usp46 knockout mouse, TDP-43 localization assay (fractionation/imaging), acarbose treatment, albuminuria measurement, diabetic mouse model\",\n      \"journal\": \"Science translational medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean conditional KO mouse with defined molecular phenotype (TDP-43 aggregation), pharmacological rescue with acarbose, in vivo diabetic model; mechanism of TDP-43 deubiquitination not directly demonstrated in abstract\",\n      \"pmids\": [\"41811985\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"USP46 is a deubiquitinating enzyme (DUB) of the ubiquitin-specific protease family that removes ubiquitin from diverse substrates including histone H2A/H2B, AMPA receptors (GLR-1/GluA), PHLPP phosphatases, LRP6/Arrow Wnt co-receptors, Cdt2/DTL, MST1, HIF-1α, PTBP1, TBK1, POU4F1, BECN1, ANK2, SNX5, and CIRBP; its catalytic activity is stimulated by a conserved trimeric complex with WDR48 (UAF1) and WDR20, which also stabilize USP46 protein by preventing its own ubiquitination and proteasomal degradation, and this complex is recruited to substrates in diverse contexts including synaptic plasticity (AMPAR surface trafficking), Wnt signaling (LRP6 receptor homeostasis), tumor suppression (PHLPP-Akt axis), and viral oncogenesis (HPV E6-Cdt2-Set8 axis).\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"USP46 is a ubiquitin-specific protease whose deubiquitinating activity controls the abundance of diverse substrates, acting most broadly as a stabilizer that opposes ubiquitin-mediated degradation and tunes signaling, synaptic, and metabolic outputs [#0, #1, #3]. It is a histone H2A/H2B deubiquitinase active on nucleosomal substrates [#0], and its catalytic activity is allosterically stimulated by a conserved trimeric complex with the WD40-repeat proteins WDR48 (UAF1) and WDR20 [#5]; within this complex WDR48 additionally stabilizes USP46 itself by preventing its own ubiquitination and proteasomal degradation [#10]. Through this complex USP46 deubiquitinates and stabilizes ionotropic glutamate receptors (mammalian AMPARs and C. elegans GLR-1), antagonizing their lysosomal/proteasomal turnover to control synaptic surface receptor levels and excitatory transmission [#3, #4, #12], a synaptic role corroborated by loss-of-function mouse mutants that exhibit behavioral phenotypes linked to GABAergic signaling [#2, #7]. In Wnt signaling, the USP46–UAF1–WDR20 complex deubiquitinates the co-receptor LRP6/Arrow, counteracting RNF43/ZNRF3-mediated ubiquitylation to raise cell-surface receptor levels and promote Wnt/\\u03b2-catenin signaling, a function conserved from Drosophila to vertebrate organoids [#13, #14]. In disease and cancer contexts USP46 stabilizes a wide range of substrates including the PHLPP phosphatases to suppress Akt signaling [#1, #15], the kinase MST1 to drive YAP1 degradation [#11], and, when hijacked by HPV E6, Cdt2/DTL to limit Set8 and promote proliferation of transformed cells [#9]. Additional substrates documented in the corpus span HIF-1\\u03b1, PTBP1, TBK1, POU4F1, BECN1, SNX5, ANK2, and CIRBP, linking USP46 to glycolytic metabolism, antiviral interferon signaling, transcription, autophagy, and ferroptosis [#16, #17, #18, #19, #22, #23]. Loss of USP46 in podocytes causes cytosolic TDP-43 aggregation and podocyte injury, implicating it in diabetic nephropathy [#25].\",\n  \"teleology\": [\n    {\n      \"year\": 2009,\n      \"claim\": \"Established USP46 as a physiologically important gene in the nervous system before its biochemistry was known, by linking a natural variant to behavior and GABA signaling.\",\n      \"evidence\": \"QTL mapping, congenic strains, and BAC transgenic rescue with electrophysiology and behavioral readouts in mice\",\n      \"pmids\": [\"19465912\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify direct molecular substrates\", \"Connection between the Lys92 deletion and enzymatic function not yet shown\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Defined USP46 as a bona fide deubiquitinating enzyme acting on nucleosomal histones and identified WDR48 as a required activating partner.\",\n      \"evidence\": \"In vitro deubiquitination of nucleosomal H2A/H2B, Co-IP, and in vivo assays in Xenopus\",\n      \"pmids\": [\"21183687\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological consequences of histone deubiquitination not established\", \"WDR20 not yet implicated\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Connected the behavioral variant to enzymatic mechanism and revealed a neuronal substrate, showing USP46 controls glutamate receptor abundance.\",\n      \"evidence\": \"In vitro USP cleavage assay of the Lys92-deletion mutant; C. elegans genetic epistasis showing USP-46 deubiquitinates GLR-1 to block lysosomal degradation\",\n      \"pmids\": [\"22043315\", \"21273419\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"27% activity reduction measured with a single artificial substrate\", \"Direct GLR-1 deubiquitination not reconstituted in vitro\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Extended USP46 substrate repertoire to a tumor-suppressive axis and confirmed the GABAergic mechanism pharmacologically.\",\n      \"evidence\": \"Co-IP and in vitro deubiquitination of PHLPP with xenografts; Usp46 knockout mouse rescued by nitrazepam in a flumazenil-sensitive manner\",\n      \"pmids\": [\"22391563\", \"22720038\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether PHLPP stabilization requires the WDR cofactor complex not addressed\", \"Direct molecular target underlying GABAergic phenotype not identified\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Defined the activating trimeric complex by showing both WDR-20 and WDR-48 bind and stimulate USP-46 catalytic activity in a substrate-relevant context.\",\n      \"evidence\": \"In vitro deubiquitination activity assay, Co-IP, and C. elegans genetics with behavioral readouts\",\n      \"pmids\": [\"24356955\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of stimulation not resolved\", \"Distinct contributions of WDR20 vs WDR48 not separated\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Confirmed AMPAR regulation in mammals and showed USP46 can be co-opted by a viral oncoprotein complex on chromatin.\",\n      \"evidence\": \"In vitro/in vivo deubiquitination of K63-linked AMPAR chains with mEPSC recording; tandem affinity purification, Co-IP and ChIP for EBV EBNA3-WDR48/WDR20 recruitment to the p14ARF promoter\",\n      \"pmids\": [\"26077708\", \"25855980\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether EBNA3 recruitment drives histone or other substrate deubiquitination not resolved\", \"Recruitment mechanism to specific AMPAR pools not defined\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Demonstrated that high-risk HPV E6 selectively redirects USP46 to stabilize Cdt2, linking the DUB to viral oncogenesis.\",\n      \"evidence\": \"Co-IP, ubiquitination assay, shRNA knockdown, and xenograft with E6-USP46-Cdt2-Set8 epistasis\",\n      \"pmids\": [\"30415951\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How E6 confers substrate selectivity not structurally defined\", \"Role of WDR cofactors in this recruitment not tested\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Separated the two cofactor functions, showing WDR-48 (but not WDR-20) stabilizes USP-46 protein by preventing its proteasomal degradation, and added PHLPP1/lung cancer to the substrate set.\",\n      \"evidence\": \"Ubiquitination assays, proteasome inhibitor non-additivity, WDR-48 binding-deficient point mutant, half-life measurement; separate Co-IP/ubiquitination study in lung cancer\",\n      \"pmids\": [\"32587090\", \"33029497\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether WDR-48-dependent stabilization is regulated dynamically not addressed\", \"Ligase that ubiquitinates USP46 itself not identified\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Broadened USP46 into Hippo-pathway tumor suppression and refined the activity-dependent regulation of synaptic receptor trafficking.\",\n      \"evidence\": \"Co-IP/ubiquitination of MST1 with YAP1 rescue in HCC; C. elegans live imaging of GLR-1 insertion and activity-regulated wdr-20 transcription\",\n      \"pmids\": [\"34029571\", \"33622778\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"MST1 deubiquitination not reconstituted in vitro\", \"Transcriptional regulator of wdr-20 not identified\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Established a conserved role for the USP46 complex in Wnt signaling by deubiquitinating LRP6/Arrow to counteract RNF43/ZNRF3 and sustain receptor surface levels.\",\n      \"evidence\": \"Co-IP, ubiquitination assay, size exclusion chromatography, organoid viability in human cells/Xenopus/zebrafish, and independent Drosophila genetics\",\n      \"pmids\": [\"37798301\", \"37798281\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of Wnt-induced complex-LRP6 association not defined\", \"Whether this is selective over other receptor pools not resolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Expanded the substrate landscape to metabolic and antiviral effectors, showing USP46 stabilizes substrates via K48-linkage removal across contexts.\",\n      \"evidence\": \"Co-IP and K48-ubiquitination assays for PTBP1 (breast cancer glycolysis/tamoxifen resistance) and the black carp TBK1 ortholog (IFN reporter assays)\",\n      \"pmids\": [\"38176460\", \"38522716\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"TBK1 finding is in a non-mammalian ortholog\", \"Cofactor dependence of these substrates not tested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Documented a wide additional substrate set linking USP46 to HIF-1\\u03b1-driven glycolysis, transcription, autophagy, and ferroptosis, and mapped its in vivo brain interactome.\",\n      \"evidence\": \"Co-IP/ubiquitination assays for HIF-1\\u03b1, POU4F1, BECN1, SNX5, ANK2, CIRBP with functional and in vivo models; miniTurbo-BioID knock-in mouse confirming WDR48/WDR20 and postsynaptic enrichment\",\n      \"pmids\": [\"40328177\", \"40251903\", \"40589294\", \"39909216\", \"40878243\", \"40608782\", \"40790822\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Many substrates rest on single-lab Co-IP/ubiquitination without in vitro reconstitution\", \"Whether each substrate requires the WDR48/WDR20 complex not systematically tested\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Linked USP46 to organ-level disease by showing its loss causes pathological TDP-43 aggregation, and identified a pharmacological agonist.\",\n      \"evidence\": \"Podocyte-specific Usp46 knockout mouse, TDP-43 localization assays, acarbose treatment in a diabetic nephropathy model\",\n      \"pmids\": [\"41811985\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct deubiquitination of TDP-43 by USP46 not demonstrated\", \"Mechanism by which acarbose acts as a USP46 agonist not defined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How USP46 achieves substrate selectivity across its very broad reported substrate set, and which substrates genuinely require the WDR48/WDR20 complex versus context-specific recruitment, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model defining substrate-binding determinants\", \"No systematic test of cofactor dependence across substrates\", \"Many disease substrates rest on single-lab cellular evidence\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 3, 9, 11, 13]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [0, 5, 6]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [10, 13]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 8]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [3, 13]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [24]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [13, 14, 11]},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [3, 4, 12]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [9, 1, 15, 25]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [10, 1, 11]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [19, 20, 21]}\n    ],\n    \"complexes\": [\"USP46-UAF1(WDR48)-WDR20 deubiquitinase complex\"],\n    \"partners\": [\"WDR48\", \"WDR20\", \"PHLPP\", \"LRP6\", \"MST1\", \"DTL\", \"POU4F1\", \"TBK1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}