{"gene":"USP36","run_date":"2026-04-28T23:00:23","timeline":{"discoveries":[{"year":2009,"finding":"USP36 localizes to nucleoli via a C-terminal basic amino acid stretch and deubiquitinates nucleolar proteins nucleophosmin/B23 and fibrillarin, stabilizing them against proteasomal degradation; RNAi depletion reduces rRNA transcription, processing, and nucleolar morphology, leading to reduced cell proliferation.","method":"Dominant-negative inhibition, RNAi knockdown, in vivo ubiquitination assay, microscopy, rRNA analysis","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (DN inhibition, RNAi, biochemical assays, morphological analysis) in single study with clear functional readouts","pmids":["19208757"],"is_preprint":false},{"year":2009,"finding":"A short basic amino acid motif (RGKEKKIKKFKREKRR) in the C-terminal region of USP36 serves as a nucleolar localization signal that interacts with the central acidic region of nucleophosmin/B23; knockdown of nucleophosmin/B23 reduces nucleolar USP36 and elevates fibrillarin ubiquitination, demonstrating nucleophosmin/B23 recruits USP36 to nucleoli.","method":"Deletion mutagenesis, co-immunoprecipitation, siRNA knockdown, ubiquitination assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — mutagenesis identifying localization signal combined with Co-IP and functional ubiquitination readout","pmids":["19679658"],"is_preprint":false},{"year":2005,"finding":"USP36 possesses deubiquitinase activity capable of cleaving ubiquitin from substrates; it contains a PEST motif and is itself polyubiquitinated.","method":"Ubiquitin cleavage assay, structural analysis, immunoprecipitation","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 — enzymatic activity confirmed in vitro plus IP for polyubiquitination, but single study with limited mechanistic depth","pmids":["15809067"],"is_preprint":false},{"year":2011,"finding":"USP36 interacts with the mitochondrial antioxidant enzyme SOD2, deubiquitinates it, and stabilizes it against proteasomal degradation, thereby extending SOD2 protein half-life.","method":"2-DE/MALDI-TOF MS identification, co-immunoprecipitation, yeast two-hybrid, ubiquitination assay, cycloheximide chase","journal":"Journal of cellular biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 — reciprocal Co-IP, yeast two-hybrid, and in vivo ubiquitination assay in single study","pmids":["21268071"],"is_preprint":false},{"year":2012,"finding":"Drosophila USP36 (dUsp36) controls selective autophagy activation in a p62/SQSTM1-dependent manner; loss of dUsp36 causes nuclear accumulation of ubiquitinated proteins including histone H2B, and cytoplasmic ubiquitinated proteins are cleared by p62-dependent autophagy. This function is conserved in human cells.","method":"Genetic loss-of-function (Drosophila mutants), RNAi in human cells, autophagy assays, p62 dependency epistasis","journal":"Autophagy","confidence":"High","confidence_rationale":"Tier 2 — epistasis in two organisms (fly genetics + human cell KD) with mechanistic pathway placement","pmids":["22622177"],"is_preprint":false},{"year":2015,"finding":"USP36 interacts with and deubiquitinates c-Myc in cells and in vitro, stabilizing c-Myc and counteracting SCF(Fbw7γ)-mediated degradation specifically in the nucleolus; USP36 interacts with nucleolar Fbw7γ but not nucleoplasmic Fbw7α, yet abolishes c-Myc degradation by both isoforms. USP36 is itself a c-Myc target gene, forming a positive feedback loop.","method":"Co-immunoprecipitation, in vitro deubiquitination assay, siRNA knockdown, cycloheximide chase, reporter assays, subcellular fractionation","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro deubiquitination assay plus multiple cellular assays, highly cited (186 citations), reciprocal Co-IPs","pmids":["25775507"],"is_preprint":false},{"year":2016,"finding":"USP36 binds to the E3 ubiquitin ligase Nedd4-2 and regulates Nedd4-2 substrate targeting: USP36 depletion increases TrkA·Nedd4-2 complex formation and TrkA ubiquitination, enhancing NGF-mediated TrkA signaling and PC12 cell differentiation; similarly, USP36 interferes with Nedd4-2-dependent Kv7.2/3 channel regulation. USP36 acts indirectly on TrkA (not directly deubiquitinating TrkA) through regulating Nedd4-2.","method":"siRNA screen, co-immunoprecipitation, surface biotinylation, signaling assays, PC12 differentiation assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — siRNA screen followed by reciprocal Co-IP and multiple functional readouts establishing indirect mechanism via Nedd4-2","pmids":["27445338"],"is_preprint":false},{"year":2017,"finding":"USP36 reduces ubiquitination of DEAH-box RNA helicase DHX33 and increases its stability; Usp36 knockout in mice is lethal at morula-to-blastocyst transition, with impaired ribosomal RNA synthesis and protein translation. DHX33 knockdown phenocopies USP36 loss in cancer cells.","method":"Conditional mouse knockout, ubiquitination assay, O-propargyl-puromycin incorporation, Northern blot, electron microscopy, cancer cell shRNA","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — in vivo mouse knockout with multiple orthogonal mechanistic readouts (EM, Northern blot, translation assay) plus epistasis via DHX33 KD","pmids":["29273634"],"is_preprint":false},{"year":2017,"finding":"USP36 is a novel histone H2B deubiquitinase: it interacts with H2B and deubiquitinates H2Bub1 in cells and in vitro. Depletion of USP36 increases H2Bub1 at the p21 gene locus, induces p21 expression, and inhibits cell proliferation.","method":"Co-immunoprecipitation, in vitro deubiquitination assay, ChIP, siRNA knockdown, cell proliferation assay","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro deubiquitination, ChIP at specific locus, and clear functional consequence","pmids":["29274341"],"is_preprint":false},{"year":2018,"finding":"USP36 controls the cellular localization of CHD7 in neuroblastoma: loss of 6p22lncRNAs (CASC15/NBAT1) modulates USP36 localization, affecting CHD7 stability and SOX9 expression.","method":"lncRNA loss-of-function, immunofluorescence/localization assay, CHD7 stability measurements, SOX9 expression analysis","journal":"Cancer cell","confidence":"Medium","confidence_rationale":"Tier 3 — localization-linked mechanism but mechanistic detail of USP36's direct action on CHD7 is indirect/inferred","pmids":["29533783"],"is_preprint":false},{"year":2018,"finding":"USP36 stabilizes PME-1 through its deubiquitinase activity, promoting ERK and Akt signaling pathways; USP36 depletion decreases PME-1 expression levels.","method":"Co-immunoprecipitation, ubiquitination assay, siRNA knockdown, pathway signaling assays","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 3 — single study with Co-IP and functional pathway readout but limited mechanistic depth","pmids":["29577269"],"is_preprint":false},{"year":2019,"finding":"USP36 knockdown impairs Parkin-dependent mitophagy by reducing Beclin-1 and ATG14L mRNA and protein levels; transfection of ATG14L restores mitophagy in USP36-silenced cells. USP36 knockdown also reduces H2B K120 monoubiquitination at transcriptionally active chromatin.","method":"siRNA knockdown, high-content imaging, mitophagy assay, mRNA/protein level measurements, rescue experiment with ATG14L transfection","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 2 — mechanistic rescue experiment placing USP36 upstream of Beclin-1/ATG14L in mitophagy","pmids":["31550441"],"is_preprint":false},{"year":2020,"finding":"USP36 is deubiquitinated following DNA replication stress, upregulates, and interacts with PrimPol; USP36 removes K29-linked polyubiquitin chains from PrimPol, increasing its protein stability. Depletion of USP36 causes replication stress defects and sensitizes cells to cisplatin and olaparib.","method":"Co-immunoprecipitation, mass spectrometry, in vivo and in vitro ubiquitination assay, siRNA knockdown, DNA fiber assay, drug sensitivity assay","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1–2 — specific ubiquitin linkage (K29) identified by in vitro assay plus multiple cellular functional readouts","pmids":["33237263"],"is_preprint":false},{"year":2021,"finding":"USP36 promotes nucleolar SUMOylation by interacting with SUMO2 and Ubc9, and directly mediates SUMOylation in cells and in vitro; specifically, USP36 promotes SUMOylation of snoRNP components Nop58, Nhp2, Nop56, and DKC1, enhancing their binding to snoRNAs. USP36 knockdown or deletion markedly impairs rRNA processing and translation.","method":"Co-immunoprecipitation, in vitro SUMOylation assay, overexpression/knockdown, rRNA processing analysis, translation assay","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution of SUMOylation combined with multiple cellular substrates and functional rRNA/translation readouts","pmids":["33852194"],"is_preprint":false},{"year":2021,"finding":"USP36 interacts with and deubiquitinates DOCK4, stabilizing it; elevated USP36/DOCK4 activates Wnt/β-catenin signaling and promotes epithelial-to-mesenchymal transition in diabetic renal tubular epithelial cells.","method":"Co-immunoprecipitation, ubiquitination assay, Western blot, EMT markers, Wnt/β-catenin signaling assay","journal":"Frontiers in cell and developmental biology","confidence":"Medium","confidence_rationale":"Tier 3 — single Co-IP and ubiquitination assay with functional pathway readout, single study","pmids":["33968925"],"is_preprint":false},{"year":2021,"finding":"USP36 cleaves the FUBI-eS30 fusion protein in vitro; purified USP36 processes FUBI-eS30 directly, and depletion of USP36 by RNAi or CRISPRi impairs FUBI-eS30 processing and late steps of cytoplasmic 40S maturation including 18S rRNA maturation and recycling of ribosome biogenesis factors.","method":"Differential affinity purification, in vitro cleavage assay with purified USP36, RNAi, CRISPRi, rRNA maturation analysis","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution with purified enzyme confirmed by two independent depletion approaches (RNAi + CRISPRi) with functional ribosome maturation readouts","pmids":["34318747"],"is_preprint":false},{"year":2021,"finding":"NBAT1/CASC15-003 lncRNAs post-translationally control MYCN protein stability through USP36, which functions as a deubiquitinase for MYCN; USP36 downregulation significantly reduces neuroblastoma tumor growth in xenograft models.","method":"Co-immunoprecipitation, immunoblotting, loss-of-function experiments, xenograft models, RNA-seq","journal":"Neuro-oncology advances","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP establishing direct interaction plus in vivo xenograft, but limited in vitro deubiquitination mechanistic detail","pmids":["34056606"],"is_preprint":false},{"year":2022,"finding":"USP36 associates with and deubiquitinates YAP, blocking K48-linked polyubiquitination and stabilizing YAP protein, thereby promoting Hippo/YAP signaling and esophageal squamous carcinoma progression; identified by DUB siRNA screening.","method":"DUB siRNA screening, co-immunoprecipitation, ubiquitination assay, siRNA knockdown, cell proliferation/invasion assays","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 — siRNA screen validated by Co-IP and specific ubiquitin linkage (K48) identified, single lab","pmids":["36470870"],"is_preprint":false},{"year":2023,"finding":"USP36 binds EXOSC10 in the nucleolus and mediates its SUMOylation at K583, without significantly altering EXOSC10 protein levels; K583R mutation impairs EXOSC10 binding to pre-rRNAs and fails to rescue defects in rRNA processing and cell growth caused by EXOSC10 knockdown.","method":"Co-immunoprecipitation, in vivo SUMOylation assay, site-directed mutagenesis (K583R), rRNA processing analysis, rescue experiments","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 — site-specific mutagenesis with functional rescue validation across rRNA processing and cell growth readouts","pmids":["36912080"],"is_preprint":false},{"year":2023,"finding":"Crystal structures of USP36 complexed with Fubi and ubiquitin reveal its dual ubiquitin/Fubi cleavage mechanism and substrate recognition; chemoproteomics identified USP16 as a second Fubi protease. The structures explain how other deubiquitinases are excluded from Fubi recognition.","method":"Crystal structure determination, chemoproteomics, Fubi C-terminal hydrolase measurements, in vitro activity assays","journal":"Nature chemical biology","confidence":"High","confidence_rationale":"Tier 1 — crystal structures with functional validation and chemoproteomics discovery of dual substrate specificity","pmids":["37443395"],"is_preprint":false},{"year":2023,"finding":"USP36 interacts with and stabilizes ALKBH5 via deubiquitination in glioblastoma; USP36 depletion impairs cell proliferation, self-renewal of glioblastoma stem cells, and sensitizes them to temozolomide. The USP36-ALKBH5 axis regulates ALKBH5-mediated gene expression.","method":"Mass spectrometry, co-immunoprecipitation, in vivo and in vitro ubiquitination assays, cell proliferation, neurosphere, and intracranial tumor assays","journal":"Neuro-oncology","confidence":"High","confidence_rationale":"Tier 1–2 — MS-based identification followed by Co-IP, in vitro ubiquitination assay, and in vivo intracranial model","pmids":["36239338"],"is_preprint":false},{"year":2023,"finding":"USP36 interacts with the Drosha-DGCR8 microprocessor complex and mediates DGCR8 SUMOylation specifically by SUMO2; this SUMOylation does not affect DGCR8 levels or Drosha-DGCR8 complex formation, but promotes DGCR8 binding to pri-miRNAs. Knockdown of USP36 attenuates pri-miRNA processing and reduces mature miRNA levels.","method":"Co-immunoprecipitation, in vivo SUMOylation assay, RNA immunoprecipitation (RIP) of pri-miRNAs, qPCR for mature miRNAs, siRNA knockdown","journal":"Cancer research communications","confidence":"High","confidence_rationale":"Tier 1–2 — biochemical SUMO-specific assay, RIP for substrate binding, and functional miRNA processing readout","pmids":["36950067"],"is_preprint":false},{"year":2023,"finding":"Ribotoxic stress activates the JNK-USP36 signaling axis, which stabilizes Snail1 in the nucleolus via deubiquitination; nucleolar Snail1 facilitates ribosome biogenesis and promotes tumor cell survival and resistance to homoharringtonine (HHT). Combination of HHT with JNK-USP36-Snail1 axis inhibition synergistically inhibits solid tumor growth.","method":"Co-immunoprecipitation, ubiquitination assay, JNK inhibition, nucleolar fractionation, rRNA analysis, in vivo tumor models","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal assays establishing kinase-DUB-substrate pathway with in vivo validation","pmids":["37833415"],"is_preprint":false},{"year":2024,"finding":"USP36 interacts with and removes K11-linked ubiquitin chains from cIAP1 and K48-linked ubiquitin chains from survivin, stabilizing both anti-apoptotic proteins. USP36 disrupts XIAP-SMAC complex formation and promotes RIP1 ubiquitination, inhibiting both intrinsic and extrinsic apoptosis in colorectal cancer cells.","method":"Gene silencing, co-immunoprecipitation, ubiquitination assay (linkage-specific), apoptosis assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — linkage-specific ubiquitin chain identification (K11 vs K48) with mechanistic downstream apoptosis pathway determination","pmids":["38876304"],"is_preprint":false},{"year":2024,"finding":"USP36 binds PARP1 and deubiquitinates it, increasing PARP1 protein stability in cardiomyocytes exposed to doxorubicin; the catalytically inactive mutant C131A fails to stabilize PARP1. Cardiac knockdown of USP36 by AAV9-shUSP36 preserves cardiac function in doxorubicin-treated mice.","method":"Co-immunoprecipitation, ubiquitination assay, catalytic mutant (C131A), AAV9 cardiac knockdown in mice, echocardiography","journal":"Cellular signalling","confidence":"High","confidence_rationale":"Tier 1–2 — catalytic mutant validation, in vivo AAV9 cardiac knockdown with functional cardiac readout","pmids":["38307305"],"is_preprint":false},{"year":2024,"finding":"USP36 deubiquitinates ERα and inhibits its K48-linked polyubiquitination, stabilizing ERα and enhancing its transcriptome; the C131A catalytic mutant fails to promote breast cancer progression. USP36 silencing destabilizes the tamoxifen-resistant ERα Y537S mutant and restores tamoxifen sensitivity.","method":"DUB siRNA library screening, co-immunoprecipitation, ubiquitination assay, catalytic mutant (C131A), xenograft models, RNA-seq, luciferase assay","journal":"Journal of experimental & clinical cancer research","confidence":"High","confidence_rationale":"Tier 1–2 — siRNA screen, K48-linkage-specific ubiquitination assay, catalytic mutant, and in vivo xenograft with therapeutic relevance","pmids":["39215346"],"is_preprint":false},{"year":2024,"finding":"USP36 interacts with and stabilizes RBM28 via deubiquitination at K162 residue; elevated RBM28 binds p53 to suppress its transcriptional activity, inactivating p53 signaling and promoting colorectal cancer progression.","method":"Co-immunoprecipitation, site-specific ubiquitination assay (K162), p53 transcription reporter, cell proliferation/invasion assays, in vivo","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1–2 — residue-specific ubiquitination site identified with mechanistic pathway connecting USP36 → RBM28 → p53 via multiple assays","pmids":["39343961"],"is_preprint":false},{"year":2024,"finding":"USP36 interacts with WDR5 and stabilizes it via deubiquitination; USP36 knockdown increases WDR5 ubiquitination and promotes its degradation, impairing osteoblast differentiation; WDR5 overexpression rescues osteogenic differentiation in USP36-deficient cells.","method":"Co-immunoprecipitation, ubiquitination assay, Western blot, Alizarin red staining, rescue experiment","journal":"Journal of orthopaedic surgery and research","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP, ubiquitination assay, and rescue experiment establishing functional axis, single study","pmids":["39152465"],"is_preprint":false},{"year":2024,"finding":"USP36 interacts with Las1L and Nol9, regulates their stability via deubiquitination, and mediates SUMOylation of Las1L at K565; the K565R mutant fails to rescue ITS2 rRNA processing defects caused by Las1L knockdown, demonstrating USP36-mediated Las1L SUMOylation is critical for pre-rRNA ITS2 processing.","method":"Co-immunoprecipitation, in vitro deubiquitination assay, in vivo SUMOylation assay, site-directed mutagenesis (K565R), rRNA processing analysis, rescue experiments","journal":"Cancer research communications","confidence":"High","confidence_rationale":"Tier 1 — site-specific mutagenesis with functional rescue at specific rRNA processing step","pmids":["39356143"],"is_preprint":false},{"year":2024,"finding":"USP36 interacts with and deubiquitinates MLLT3 in the nucleolus, stabilizing it and activating downstream HIF1α and Snai signaling; the germline USP36 variant K814N (rs3744797) upregulates USP36 expression by reducing m6A modification, facilitating MLLT3 stabilization and EGFR-TKI resistance.","method":"Co-immunoprecipitation, ubiquitination assay, m6A analysis, nucleolar fractionation, in vitro/in vivo proliferation and resistance assays","journal":"Clinical cancer research","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP and ubiquitination assay with functional in vivo data, but m6A-USP36 mechanism is correlative","pmids":["38261467"],"is_preprint":false},{"year":2025,"finding":"USP36 functions as a SUMO ligase for GNL3, mediating its SUMOylation; GNL3 SUMOylation is required for its interaction with the BLM-DNA2 helicase-nuclease complex and for DNA end resection in homologous recombination repair. SENP3 acts as the opposing SUMO protease for GNL3.","method":"Co-immunoprecipitation, SUMOylation assay, CRISPR mutagenesis (K196R), DNA end resection assay, RPA/RAD51 loading assay, epistasis with SENP3","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal assays in a preprint; not yet peer-reviewed","pmids":["bio_10.1101_2025.11.04.686352"],"is_preprint":true},{"year":2025,"finding":"In Drosophila, a CRISPR-induced catalytic mutation in dUSP36 produces viable adults with only minor growth defects but causes male infertility, demonstrating that dUSP36 deubiquitinating activity is dispensable for cell growth but essential for spermatogenesis; USP36 functions through both catalytic-dependent and catalytic-independent mechanisms.","method":"CRISPR/Cas9 catalytic mutation, in vivo viability and fertility assays, genetic epistasis","journal":"Genetics","confidence":"High","confidence_rationale":"Tier 1 — endogenous CRISPR catalytic mutation in vivo with clear phenotypic separation of functions","pmids":["40646716"],"is_preprint":false},{"year":2025,"finding":"Methylmalonylation of USP36 at K499 by MMA (methylmalonic acid) inhibits USP36-mediated deubiquitination and SUMOylation of SUFU, promoting Hedgehog signaling (GLI1 target genes) and M2 macrophage polarization in clear-cell renal cell carcinoma.","method":"Metabolomics, co-immunoprecipitation, in vitro deubiquitination/SUMOylation assays, K499 site-specific analysis, in vivo tumor models","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 — novel PTM (methylmalonylation) identified at specific residue with functional consequences on USP36 enzymatic activities, single study","pmids":["41398045"],"is_preprint":false},{"year":2025,"finding":"USP36 deubiquitinates and stabilizes APEX1 via cleavage of K48-linked ubiquitin chains; USP36 overexpression suppresses erastin-induced ferroptosis in melanoma cells, while USP36 deficiency increases ferroptosis. APEX1 knockdown abolishes the anti-ferroptotic effect of USP36.","method":"Co-immunoprecipitation, ubiquitination assay (K48-linkage specific), overexpression/knockdown, ferroptosis assays, xenograft model","journal":"Clinical and experimental medicine","confidence":"Medium","confidence_rationale":"Tier 2 — linkage-specific ubiquitination assay plus rescue experiment establishing USP36-APEX1 axis, single study","pmids":["41649582"],"is_preprint":false},{"year":2025,"finding":"FBL acts as a carrier for monoubiquitinated H2A (H2Aub) in nucleolar lysosome-like structures; USP36, FBL, Midnolin, and BMI1 form a complex regulating the balance between H2A monoubiquitination and deubiquitination during glucose starvation. Knockdown of USP36 causes S-phase cell cycle arrest and reduced cell viability.","method":"Protein complex screening, co-immunoprecipitation, glucose starvation assay, cell cycle analysis, viability assay","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 3 — preprint with Co-IP evidence for complex formation, mechanistic detail of USP36's specific role limited","pmids":["bio_10.1101_2025.06.30.662258"],"is_preprint":true},{"year":2025,"finding":"Oxidative stress (H2O2) promotes USP36 interaction with androgen receptor (AR) in prostate cancer cells; USP36 deubiquitinates and stabilizes AR, and USP36 knockdown abolishes H2O2-induced AR-PSA pathway activation.","method":"TurboID proximity biotin labeling/MS, co-immunoprecipitation, deubiquitination assay, siRNA knockdown, luciferase reporter assay","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 — proximity labeling MS identification validated by Co-IP and functional reporter assay, single study","pmids":["41298501"],"is_preprint":false},{"year":2025,"finding":"USP36 deubiquitinates and stabilizes SOD2 to preserve mitochondrial integrity in gastric cancer; resveratrol disrupts the USP36-SOD2 axis, reducing SOD2 stability, inducing mitochondrial dysfunction, and triggering autophagy and ferroptosis.","method":"Western blot, ubiquitination assay, functional assays (colony formation, Transwell), xenograft model, fluorescence staining for mitochondrial function","journal":"Gastric cancer","confidence":"Medium","confidence_rationale":"Tier 2 — ubiquitination assay and functional in vivo data, but primarily extends prior SOD2 findings","pmids":["40650854"],"is_preprint":false},{"year":2022,"finding":"PRL1 activates USP36-mediated Snail2 deubiquitination in glioblastoma; USP36 deubiquitinates Snail2, and PRL1 expression positively correlates with Snail2 levels, promoting EMT, invasion, and tumorigenicity.","method":"Co-immunoprecipitation, ubiquitination assay, overexpression/knockdown, in vitro and in vivo invasion/tumorigenicity assays","journal":"Frontiers in oncology","confidence":"Medium","confidence_rationale":"Tier 3 — Co-IP and ubiquitination assay, functional in vivo data, but mechanism of PRL1-USP36 activation unclear","pmids":["35111679"],"is_preprint":false},{"year":2024,"finding":"USP36 stabilizes KIF2C via K48-linked deubiquitination; USP36 overexpression suppresses erastin-induced ferroptosis in breast cancer cells, and KIF2C knockdown counteracts this anti-ferroptotic effect. USP36-deficient tumors show reduced proliferation and increased ferroptosis in vivo.","method":"Co-immunoprecipitation, K48-linkage ubiquitination assay, overexpression/knockdown, ferroptosis assays, xenograft model","journal":"Biochemical pharmacology","confidence":"Medium","confidence_rationale":"Tier 2 — linkage-specific ubiquitination assay with functional rescue and in vivo validation, single study","pmids":["40744233"],"is_preprint":false}],"current_model":"USP36 is a nucleolar deubiquitinating enzyme (DUB) that localizes to nucleoli via a basic amino acid nucleolar localization signal recruited by nucleophosmin/B23, where it deubiquitinates and stabilizes multiple substrates (c-Myc/MYCN, nucleophosmin/B23, fibrillarin, DHX33, SOD2, H2Bub1, PrimPol, and others) to regulate ribosome biogenesis, rRNA processing, and protein translation; beyond its canonical DUB activity, USP36 also acts as a non-canonical SUMO E3 ligase mediating group SUMOylation of snoRNP components (Nop58, Nhp2, Nop56, DKC1), EXOSC10 (K583), Las1L (K565), DGCR8, and GNL3, thereby coordinating multiple steps of ribosome biogenesis and miRNA processing; USP36 additionally functions as a FUBI-specific protease required for FUBI-eS30 cleavage and 40S subunit maturation, and its activity is regulated by post-translational modification including methylmalonylation at K499 that inhibits both its DUB and SUMO ligase functions, while Drosophila studies reveal that its catalytic activity is dispensable for cell growth but essential for spermatogenesis, indicating catalytic-independent functions."},"narrative":{"teleology":[{"year":2005,"claim":"Establishing that USP36 possesses deubiquitinase activity and is itself regulated by polyubiquitination provided the initial enzymatic characterization of this DUB.","evidence":"In vitro ubiquitin cleavage assay and immunoprecipitation","pmids":["15809067"],"confidence":"Medium","gaps":["No physiological substrate identified","No cellular context or localization determined","Regulation of USP36 stability via its own ubiquitination not characterized"]},{"year":2009,"claim":"Demonstrating that USP36 localizes to nucleoli via a basic amino acid motif, is recruited by nucleophosmin/B23, and deubiquitinates nucleolar proteins (B23, fibrillarin) established USP36 as a nucleolar DUB regulating rRNA biogenesis.","evidence":"Dominant-negative inhibition, RNAi, deletion mutagenesis, Co-IP, and rRNA processing analysis in human cells","pmids":["19208757","19679658"],"confidence":"High","gaps":["Structural basis of nucleolar targeting not resolved","Full spectrum of nucleolar substrates unknown","How USP36 activity is regulated remained unclear"]},{"year":2012,"claim":"Genetic studies in Drosophila revealed that loss of USP36 causes nuclear accumulation of ubiquitinated proteins including H2B and triggers p62-dependent selective autophagy, linking USP36 to ubiquitin homeostasis and autophagy regulation conserved across species.","evidence":"Drosophila loss-of-function mutants combined with RNAi in human cells and p62 epistasis","pmids":["22622177"],"confidence":"High","gaps":["Whether autophagy activation is a direct or compensatory consequence of USP36 loss","Identity of critical nuclear ubiquitinated substrates beyond H2B"]},{"year":2015,"claim":"Identification of c-Myc as a direct USP36 substrate, stabilized against SCF(Fbw7γ)-mediated degradation in the nucleolus with a positive feedback loop (USP36 is a c-Myc target gene), established USP36 as a key regulator of oncogenic signaling.","evidence":"In vitro deubiquitination, reciprocal Co-IP, cycloheximide chase, reporter assays, and subcellular fractionation","pmids":["25775507"],"confidence":"High","gaps":["How USP36-c-Myc feedback loop is terminated or restrained","Whether nucleolar versus nucleoplasmic c-Myc pools have distinct functional outcomes"]},{"year":2017,"claim":"Mouse knockout revealing embryonic lethality at morula-to-blastocyst transition, combined with identification of DHX33 and H2Bub1 as direct substrates, established USP36 as essential for early development, ribosome biogenesis, and epigenetic regulation.","evidence":"Conditional mouse knockout with EM, Northern blot, translation assay; in vitro deubiquitination of H2B with ChIP at p21 locus","pmids":["29273634","29274341"],"confidence":"High","gaps":["Which substrate(s) account for the morula-blastocyst lethality","Genome-wide landscape of H2Bub1 changes upon USP36 loss"]},{"year":2020,"claim":"Discovery that USP36 removes K29-linked polyubiquitin chains from PrimPol during replication stress extended USP36 function beyond nucleolar/ribosomal roles to DNA damage tolerance.","evidence":"Mass spectrometry, in vitro K29-linkage-specific deubiquitination, DNA fiber assay, cisplatin/olaparib sensitivity","pmids":["33237263"],"confidence":"High","gaps":["How USP36 is relocated or activated at replication forks","Whether other replication stress substrates exist"]},{"year":2021,"claim":"The paradigm-shifting discovery that USP36 acts as a non-canonical SUMO E3 ligase for snoRNP components (Nop58, Nhp2, Nop56, DKC1) revealed a dual enzymatic function — DUB and SUMO ligase — and explained its broad impact on rRNA processing and translation.","evidence":"In vitro SUMOylation reconstitution with purified components, Co-IP with SUMO2/Ubc9, rRNA processing and translation assays","pmids":["33852194"],"confidence":"High","gaps":["Structural basis for SUMO E3 ligase activity","Whether DUB and SUMO ligase activities are coordinated or independent on the same substrates"]},{"year":2021,"claim":"Identification of USP36 as a FUBI-specific protease required for FUBI-eS30 cleavage and 40S maturation, confirmed by crystal structures in 2023, established a unique dual ubiquitin/ubiquitin-like substrate specificity and explained USP36's role in cytoplasmic ribosome maturation.","evidence":"In vitro cleavage with purified USP36, RNAi/CRISPRi depletion, rRNA maturation analysis; crystal structures of USP36-Fubi and USP36-Ub complexes","pmids":["34318747","37443395"],"confidence":"High","gaps":["How USP36 transitions between nucleolar functions and cytoplasmic 40S maturation","Whether FUBI cleavage is rate-limiting for 40S assembly in vivo"]},{"year":2023,"claim":"Extension of the SUMO E3 ligase function to EXOSC10 (K583), DGCR8, and later Las1L (K565), with site-specific mutagenesis showing SUMOylation is required for pre-rRNA binding/processing and pri-miRNA processing, established USP36 as a central coordinator of nucleolar RNA metabolism through SUMOylation.","evidence":"Site-directed mutagenesis (K583R, K565R) with functional rescue of rRNA processing; RIP for pri-miRNA binding","pmids":["36912080","36950067","39356143"],"confidence":"High","gaps":["Full catalog of USP36 SUMOylation substrates","Whether a single domain mediates all SUMO E3 ligase activities","Interplay between USP36 SUMOylation and opposing SENP activities in steady state"]},{"year":2023,"claim":"Discovery that ribotoxic stress activates a JNK-USP36-Snail1 axis in the nucleolus, promoting ribosome biogenesis and drug resistance, placed USP36 as a stress-responsive effector integrating kinase signaling with nucleolar function.","evidence":"Co-IP, JNK inhibition, nucleolar fractionation, rRNA analysis, in vivo tumor models","pmids":["37833415"],"confidence":"High","gaps":["Whether JNK directly phosphorylates USP36 and at which site","Generalizability to other ribotoxic stress agents"]},{"year":2024,"claim":"Demonstrations that USP36 deubiquitinates anti-apoptotic proteins (cIAP1 via K11-linked, survivin via K48-linked chains), ERα, PARP1, and other substrates across cancer types broadened USP36's substrate repertoire to apoptosis, hormone signaling, and cardioprotection, with linkage-type specificity emerging as a recurring feature.","evidence":"Linkage-specific ubiquitination assays, catalytic mutant C131A validation, in vivo cardiac and xenograft models","pmids":["38876304","39215346","38307305"],"confidence":"High","gaps":["How USP36 achieves substrate selectivity among its many targets","Whether linkage specificity is substrate-determined or context-dependent"]},{"year":2025,"claim":"Identification of methylmalonylation at K499 as an inhibitory post-translational modification of both DUB and SUMO ligase activities, combined with Drosophila evidence that catalytic activity is dispensable for growth but essential for spermatogenesis, revealed regulatory complexity and catalytic-independent functions of USP36.","evidence":"Metabolomics with K499 site-specific analysis and in vitro activity assays; CRISPR catalytic mutation in Drosophila with viability/fertility phenotyping","pmids":["41398045","40646716"],"confidence":"Medium","gaps":["Structural basis of methylmalonylation-mediated inhibition","Identity of catalytic-independent binding partners mediating growth functions","Whether methylmalonylation occurs physiologically outside cancer contexts"]},{"year":null,"claim":"Key unresolved questions include the structural basis for USP36's dual DUB/SUMO E3 ligase activities, how substrate selectivity is achieved among its many targets, and the full scope of catalytic-independent functions implied by Drosophila genetics.","evidence":"","pmids":[],"confidence":"High","gaps":["No full-length USP36 structure available","Mechanism of substrate prioritization in the nucleolus unknown","Catalytic-independent interaction partners not identified","No disease-causing germline mutations reported in humans"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[0,2,5,7,8,12,15,19,23,24,25]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,5,8,12,15,23,24,25]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[13,18,21,28]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[6,13,21]}],"localization":[{"term_id":"GO:0005730","term_label":"nucleolus","supporting_discovery_ids":[0,1,5,13,18,22,28]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,4,8]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[0,13,15,18,21,28]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,5,7,13,15,19]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[15,19]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[12]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[23]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[4,11]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[8,25]}],"complexes":["snoRNP (via SUMOylation of Nop58/Nhp2/Nop56/DKC1)","Drosha-DGCR8 microprocessor (via DGCR8 SUMOylation)"],"partners":["NPM1","FBL","MYC","DHX33","DGCR8","PRIMPOL","EXOSC10","LAS1L"],"other_free_text":[]},"mechanistic_narrative":"USP36 is a nucleolar deubiquitinating enzyme with dual enzymatic functions — canonical ubiquitin hydrolase activity and non-canonical SUMO E3 ligase activity — that coordinates ribosome biogenesis, rRNA processing, and RNA metabolism. As a DUB, USP36 localizes to nucleoli via a basic amino acid motif recruited by nucleophosmin/B23, where it deubiquitinates and stabilizes nucleolar substrates including c-Myc, nucleophosmin/B23, fibrillarin, DHX33, and H2Bub1 to promote rRNA transcription, processing, and cell proliferation; it also functions as a FUBI-specific protease that cleaves FUBI-eS30, an activity required for cytoplasmic 40S ribosomal subunit maturation [PMID:19208757, PMID:25775507, PMID:34318747, PMID:29273634]. As a SUMO E3 ligase, USP36 mediates SUMOylation of snoRNP components (Nop58, Nhp2, Nop56, DKC1), EXOSC10 at K583, Las1L at K565, and DGCR8, thereby regulating snoRNA binding, ITS2 rRNA processing, and pri-miRNA processing, respectively [PMID:33852194, PMID:36912080, PMID:39356143, PMID:36950067]. Beyond ribosome biogenesis, USP36 stabilizes diverse substrates across multiple pathways — including PrimPol during replication stress, anti-apoptotic proteins cIAP1/survivin, ERα, and SOD2 — and its enzymatic activities are inhibited by methylmalonylation at K499, while Drosophila studies demonstrate that catalytic activity is dispensable for viability but essential for spermatogenesis [PMID:33237263, PMID:38876304, PMID:39215346, PMID:41398045, PMID:40646716]."},"prefetch_data":{"uniprot":{"accession":"Q9P275","full_name":"Ubiquitin carboxyl-terminal hydrolase 36","aliases":["Deubiquitinating enzyme 36","Ubiquitin thioesterase 36","Ubiquitin-specific-processing protease 36"],"length_aa":1123,"mass_kda":122.9,"function":"Deubiquitinase essential for the regulation of nucleolar structure and function (PubMed:19208757, PubMed:22902402, PubMed:29273634). Required for cell and organism viability (PubMed:19208757, PubMed:22902402, PubMed:29273634). Plays an important role in ribosomal RNA processing and protein synthesis, which is mediated, at least in part, through deubiquitination of DHX33, NPM1 and FBL, regulating their protein stability (PubMed:19208757, PubMed:22902402, PubMed:29273634, PubMed:36912080). Functions as a transcriptional repressor by deubiquiting histone H2B at the promoters of genes critical for cellular differentiation, such as CDKN1A, thereby preventing histone H3 'Lys-4' trimethylation (H3K4) (PubMed:29274341). Specifically deubiquitinates MYC in the nucleolus, leading to prevent MYC degradation by the proteasome: acts by specifically interacting with isoform 3 of FBXW7 (FBW7gamma) in the nucleolus and counteracting ubiquitination of MYC by the SCF(FBW7) complex (PubMed:25775507). In contrast, it does not interact with isoform 1 of FBXW7 (FBW7alpha) in the nucleoplasm (PubMed:25775507). Interacts to and regulates the actions of E3 ubiquitin-protein ligase NEDD4L over substrates such as NTRK1, KCNQ2 and KCNQ3, affecting their expression an functions (PubMed:27445338). Deubiquitinates SOD2, regulates SOD2 protein stability (PubMed:21268071). Deubiquitinase activity is required to control selective autophagy activation by ubiquitinated proteins (PubMed:22622177). Promotes CEP63 stabilization through 'Lys-48'-linked deubiquitination leading to increased stability (PubMed:35989368). Acts as a SUMO ligase to promote EXOSC10 sumoylation critical for the nucleolar RNA exosome function in rRNA processing (PubMed:36912080). Binds to pre-rRNAs (PubMed:36912080)","subcellular_location":"Nucleus, nucleolus; Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q9P275/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/USP36","classification":"Common Essential","n_dependent_lines":1199,"n_total_lines":1208,"dependency_fraction":0.9925496688741722},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000055483","cell_line_id":"CID001111","localizations":[{"compartment":"nucleolus_gc","grade":3}],"interactors":[{"gene":"DHX33","stoichiometry":4.0}],"url":"https://opencell.sf.czbiohub.org/target/CID001111","total_profiled":1310},"omim":[{"mim_id":"612543","title":"UBIQUITIN-SPECIFIC PROTEASE 36; USP36","url":"https://www.omim.org/entry/612543"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Nucleoli","reliability":"Enhanced"},{"location":"Nucleoli rim","reliability":"Enhanced"},{"location":"Nuclear speckles","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/USP36"},"hgnc":{"alias_symbol":["KIAA1453","FLJ12851"],"prev_symbol":[]},"alphafold":{"accession":"Q9P275","domains":[{"cath_id":"3.90.70.10","chopping":"121-421","consensus_level":"medium","plddt":92.7951,"start":121,"end":421}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9P275","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9P275-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9P275-F1-predicted_aligned_error_v6.png","plddt_mean":55.19},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=USP36","jax_strain_url":"https://www.jax.org/strain/search?query=USP36"},"sequence":{"accession":"Q9P275","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9P275.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9P275/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9P275"}},"corpus_meta":[{"pmid":"25775507","id":"PMC_25775507","title":"The nucleolar 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communications","url":"https://pubmed.ncbi.nlm.nih.gov/39067250","citation_count":0,"is_preprint":false},{"pmid":"41298501","id":"PMC_41298501","title":"Oxidative stress reactivates androgen receptor signaling via USP36 to drive castration resistance in prostate cancer.","date":"2025","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/41298501","citation_count":0,"is_preprint":false},{"pmid":"41649582","id":"PMC_41649582","title":"USP36 inhibits ferroptosis of melanoma cells by stabilizing APEX1.","date":"2026","source":"Clinical and experimental medicine","url":"https://pubmed.ncbi.nlm.nih.gov/41649582","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.11.04.686352","title":"GNL3 SUMOylation is essential for DNA double-strand break repair by homologous recombination","date":"2025-11-05","source":"bioRxiv","url":"https://doi.org/10.1101/2025.11.04.686352","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.06.30.662258","title":"FBL as a carrier mediate H2Aub degradation in the lysosome-like structures","date":"2025-07-03","source":"bioRxiv","url":"https://doi.org/10.1101/2025.06.30.662258","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.03.25.644999","title":"The Deubiquitinase USP36 Regulates Growth and Spermatogenesis Through Catalytic-Dependent and -Independent Mechanisms in <i>Drosophila</i>","date":"2025-03-27","source":"bioRxiv","url":"https://doi.org/10.1101/2025.03.25.644999","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":26651,"output_tokens":9384,"usd":0.110356},"stage2":{"model":"claude-opus-4-6","input_tokens":13288,"output_tokens":3717,"usd":0.239047},"total_usd":0.349403,"stage1_batch_id":"msgbatch_01BparvgRZ9S2LMidvaoo86K","stage2_batch_id":"msgbatch_01NkBfv9SVZWthhS7j6mU6G3","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2009,\n      \"finding\": \"USP36 localizes to nucleoli via a C-terminal basic amino acid stretch and deubiquitinates nucleolar proteins nucleophosmin/B23 and fibrillarin, stabilizing them against proteasomal degradation; RNAi depletion reduces rRNA transcription, processing, and nucleolar morphology, leading to reduced cell proliferation.\",\n      \"method\": \"Dominant-negative inhibition, RNAi knockdown, in vivo ubiquitination assay, microscopy, rRNA analysis\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (DN inhibition, RNAi, biochemical assays, morphological analysis) in single study with clear functional readouts\",\n      \"pmids\": [\"19208757\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"A short basic amino acid motif (RGKEKKIKKFKREKRR) in the C-terminal region of USP36 serves as a nucleolar localization signal that interacts with the central acidic region of nucleophosmin/B23; knockdown of nucleophosmin/B23 reduces nucleolar USP36 and elevates fibrillarin ubiquitination, demonstrating nucleophosmin/B23 recruits USP36 to nucleoli.\",\n      \"method\": \"Deletion mutagenesis, co-immunoprecipitation, siRNA knockdown, ubiquitination assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — mutagenesis identifying localization signal combined with Co-IP and functional ubiquitination readout\",\n      \"pmids\": [\"19679658\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"USP36 possesses deubiquitinase activity capable of cleaving ubiquitin from substrates; it contains a PEST motif and is itself polyubiquitinated.\",\n      \"method\": \"Ubiquitin cleavage assay, structural analysis, immunoprecipitation\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — enzymatic activity confirmed in vitro plus IP for polyubiquitination, but single study with limited mechanistic depth\",\n      \"pmids\": [\"15809067\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"USP36 interacts with the mitochondrial antioxidant enzyme SOD2, deubiquitinates it, and stabilizes it against proteasomal degradation, thereby extending SOD2 protein half-life.\",\n      \"method\": \"2-DE/MALDI-TOF MS identification, co-immunoprecipitation, yeast two-hybrid, ubiquitination assay, cycloheximide chase\",\n      \"journal\": \"Journal of cellular biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, yeast two-hybrid, and in vivo ubiquitination assay in single study\",\n      \"pmids\": [\"21268071\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Drosophila USP36 (dUsp36) controls selective autophagy activation in a p62/SQSTM1-dependent manner; loss of dUsp36 causes nuclear accumulation of ubiquitinated proteins including histone H2B, and cytoplasmic ubiquitinated proteins are cleared by p62-dependent autophagy. This function is conserved in human cells.\",\n      \"method\": \"Genetic loss-of-function (Drosophila mutants), RNAi in human cells, autophagy assays, p62 dependency epistasis\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — epistasis in two organisms (fly genetics + human cell KD) with mechanistic pathway placement\",\n      \"pmids\": [\"22622177\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"USP36 interacts with and deubiquitinates c-Myc in cells and in vitro, stabilizing c-Myc and counteracting SCF(Fbw7γ)-mediated degradation specifically in the nucleolus; USP36 interacts with nucleolar Fbw7γ but not nucleoplasmic Fbw7α, yet abolishes c-Myc degradation by both isoforms. USP36 is itself a c-Myc target gene, forming a positive feedback loop.\",\n      \"method\": \"Co-immunoprecipitation, in vitro deubiquitination assay, siRNA knockdown, cycloheximide chase, reporter assays, subcellular fractionation\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro deubiquitination assay plus multiple cellular assays, highly cited (186 citations), reciprocal Co-IPs\",\n      \"pmids\": [\"25775507\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"USP36 binds to the E3 ubiquitin ligase Nedd4-2 and regulates Nedd4-2 substrate targeting: USP36 depletion increases TrkA·Nedd4-2 complex formation and TrkA ubiquitination, enhancing NGF-mediated TrkA signaling and PC12 cell differentiation; similarly, USP36 interferes with Nedd4-2-dependent Kv7.2/3 channel regulation. USP36 acts indirectly on TrkA (not directly deubiquitinating TrkA) through regulating Nedd4-2.\",\n      \"method\": \"siRNA screen, co-immunoprecipitation, surface biotinylation, signaling assays, PC12 differentiation assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — siRNA screen followed by reciprocal Co-IP and multiple functional readouts establishing indirect mechanism via Nedd4-2\",\n      \"pmids\": [\"27445338\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"USP36 reduces ubiquitination of DEAH-box RNA helicase DHX33 and increases its stability; Usp36 knockout in mice is lethal at morula-to-blastocyst transition, with impaired ribosomal RNA synthesis and protein translation. DHX33 knockdown phenocopies USP36 loss in cancer cells.\",\n      \"method\": \"Conditional mouse knockout, ubiquitination assay, O-propargyl-puromycin incorporation, Northern blot, electron microscopy, cancer cell shRNA\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vivo mouse knockout with multiple orthogonal mechanistic readouts (EM, Northern blot, translation assay) plus epistasis via DHX33 KD\",\n      \"pmids\": [\"29273634\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"USP36 is a novel histone H2B deubiquitinase: it interacts with H2B and deubiquitinates H2Bub1 in cells and in vitro. Depletion of USP36 increases H2Bub1 at the p21 gene locus, induces p21 expression, and inhibits cell proliferation.\",\n      \"method\": \"Co-immunoprecipitation, in vitro deubiquitination assay, ChIP, siRNA knockdown, cell proliferation assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro deubiquitination, ChIP at specific locus, and clear functional consequence\",\n      \"pmids\": [\"29274341\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"USP36 controls the cellular localization of CHD7 in neuroblastoma: loss of 6p22lncRNAs (CASC15/NBAT1) modulates USP36 localization, affecting CHD7 stability and SOX9 expression.\",\n      \"method\": \"lncRNA loss-of-function, immunofluorescence/localization assay, CHD7 stability measurements, SOX9 expression analysis\",\n      \"journal\": \"Cancer cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — localization-linked mechanism but mechanistic detail of USP36's direct action on CHD7 is indirect/inferred\",\n      \"pmids\": [\"29533783\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"USP36 stabilizes PME-1 through its deubiquitinase activity, promoting ERK and Akt signaling pathways; USP36 depletion decreases PME-1 expression levels.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, siRNA knockdown, pathway signaling assays\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single study with Co-IP and functional pathway readout but limited mechanistic depth\",\n      \"pmids\": [\"29577269\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"USP36 knockdown impairs Parkin-dependent mitophagy by reducing Beclin-1 and ATG14L mRNA and protein levels; transfection of ATG14L restores mitophagy in USP36-silenced cells. USP36 knockdown also reduces H2B K120 monoubiquitination at transcriptionally active chromatin.\",\n      \"method\": \"siRNA knockdown, high-content imaging, mitophagy assay, mRNA/protein level measurements, rescue experiment with ATG14L transfection\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — mechanistic rescue experiment placing USP36 upstream of Beclin-1/ATG14L in mitophagy\",\n      \"pmids\": [\"31550441\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"USP36 is deubiquitinated following DNA replication stress, upregulates, and interacts with PrimPol; USP36 removes K29-linked polyubiquitin chains from PrimPol, increasing its protein stability. Depletion of USP36 causes replication stress defects and sensitizes cells to cisplatin and olaparib.\",\n      \"method\": \"Co-immunoprecipitation, mass spectrometry, in vivo and in vitro ubiquitination assay, siRNA knockdown, DNA fiber assay, drug sensitivity assay\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — specific ubiquitin linkage (K29) identified by in vitro assay plus multiple cellular functional readouts\",\n      \"pmids\": [\"33237263\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"USP36 promotes nucleolar SUMOylation by interacting with SUMO2 and Ubc9, and directly mediates SUMOylation in cells and in vitro; specifically, USP36 promotes SUMOylation of snoRNP components Nop58, Nhp2, Nop56, and DKC1, enhancing their binding to snoRNAs. USP36 knockdown or deletion markedly impairs rRNA processing and translation.\",\n      \"method\": \"Co-immunoprecipitation, in vitro SUMOylation assay, overexpression/knockdown, rRNA processing analysis, translation assay\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution of SUMOylation combined with multiple cellular substrates and functional rRNA/translation readouts\",\n      \"pmids\": [\"33852194\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"USP36 interacts with and deubiquitinates DOCK4, stabilizing it; elevated USP36/DOCK4 activates Wnt/β-catenin signaling and promotes epithelial-to-mesenchymal transition in diabetic renal tubular epithelial cells.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, Western blot, EMT markers, Wnt/β-catenin signaling assay\",\n      \"journal\": \"Frontiers in cell and developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single Co-IP and ubiquitination assay with functional pathway readout, single study\",\n      \"pmids\": [\"33968925\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"USP36 cleaves the FUBI-eS30 fusion protein in vitro; purified USP36 processes FUBI-eS30 directly, and depletion of USP36 by RNAi or CRISPRi impairs FUBI-eS30 processing and late steps of cytoplasmic 40S maturation including 18S rRNA maturation and recycling of ribosome biogenesis factors.\",\n      \"method\": \"Differential affinity purification, in vitro cleavage assay with purified USP36, RNAi, CRISPRi, rRNA maturation analysis\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with purified enzyme confirmed by two independent depletion approaches (RNAi + CRISPRi) with functional ribosome maturation readouts\",\n      \"pmids\": [\"34318747\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"NBAT1/CASC15-003 lncRNAs post-translationally control MYCN protein stability through USP36, which functions as a deubiquitinase for MYCN; USP36 downregulation significantly reduces neuroblastoma tumor growth in xenograft models.\",\n      \"method\": \"Co-immunoprecipitation, immunoblotting, loss-of-function experiments, xenograft models, RNA-seq\",\n      \"journal\": \"Neuro-oncology advances\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP establishing direct interaction plus in vivo xenograft, but limited in vitro deubiquitination mechanistic detail\",\n      \"pmids\": [\"34056606\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"USP36 associates with and deubiquitinates YAP, blocking K48-linked polyubiquitination and stabilizing YAP protein, thereby promoting Hippo/YAP signaling and esophageal squamous carcinoma progression; identified by DUB siRNA screening.\",\n      \"method\": \"DUB siRNA screening, co-immunoprecipitation, ubiquitination assay, siRNA knockdown, cell proliferation/invasion assays\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — siRNA screen validated by Co-IP and specific ubiquitin linkage (K48) identified, single lab\",\n      \"pmids\": [\"36470870\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"USP36 binds EXOSC10 in the nucleolus and mediates its SUMOylation at K583, without significantly altering EXOSC10 protein levels; K583R mutation impairs EXOSC10 binding to pre-rRNAs and fails to rescue defects in rRNA processing and cell growth caused by EXOSC10 knockdown.\",\n      \"method\": \"Co-immunoprecipitation, in vivo SUMOylation assay, site-directed mutagenesis (K583R), rRNA processing analysis, rescue experiments\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — site-specific mutagenesis with functional rescue validation across rRNA processing and cell growth readouts\",\n      \"pmids\": [\"36912080\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Crystal structures of USP36 complexed with Fubi and ubiquitin reveal its dual ubiquitin/Fubi cleavage mechanism and substrate recognition; chemoproteomics identified USP16 as a second Fubi protease. The structures explain how other deubiquitinases are excluded from Fubi recognition.\",\n      \"method\": \"Crystal structure determination, chemoproteomics, Fubi C-terminal hydrolase measurements, in vitro activity assays\",\n      \"journal\": \"Nature chemical biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structures with functional validation and chemoproteomics discovery of dual substrate specificity\",\n      \"pmids\": [\"37443395\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"USP36 interacts with and stabilizes ALKBH5 via deubiquitination in glioblastoma; USP36 depletion impairs cell proliferation, self-renewal of glioblastoma stem cells, and sensitizes them to temozolomide. The USP36-ALKBH5 axis regulates ALKBH5-mediated gene expression.\",\n      \"method\": \"Mass spectrometry, co-immunoprecipitation, in vivo and in vitro ubiquitination assays, cell proliferation, neurosphere, and intracranial tumor assays\",\n      \"journal\": \"Neuro-oncology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — MS-based identification followed by Co-IP, in vitro ubiquitination assay, and in vivo intracranial model\",\n      \"pmids\": [\"36239338\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"USP36 interacts with the Drosha-DGCR8 microprocessor complex and mediates DGCR8 SUMOylation specifically by SUMO2; this SUMOylation does not affect DGCR8 levels or Drosha-DGCR8 complex formation, but promotes DGCR8 binding to pri-miRNAs. Knockdown of USP36 attenuates pri-miRNA processing and reduces mature miRNA levels.\",\n      \"method\": \"Co-immunoprecipitation, in vivo SUMOylation assay, RNA immunoprecipitation (RIP) of pri-miRNAs, qPCR for mature miRNAs, siRNA knockdown\",\n      \"journal\": \"Cancer research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — biochemical SUMO-specific assay, RIP for substrate binding, and functional miRNA processing readout\",\n      \"pmids\": [\"36950067\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Ribotoxic stress activates the JNK-USP36 signaling axis, which stabilizes Snail1 in the nucleolus via deubiquitination; nucleolar Snail1 facilitates ribosome biogenesis and promotes tumor cell survival and resistance to homoharringtonine (HHT). Combination of HHT with JNK-USP36-Snail1 axis inhibition synergistically inhibits solid tumor growth.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, JNK inhibition, nucleolar fractionation, rRNA analysis, in vivo tumor models\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal assays establishing kinase-DUB-substrate pathway with in vivo validation\",\n      \"pmids\": [\"37833415\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP36 interacts with and removes K11-linked ubiquitin chains from cIAP1 and K48-linked ubiquitin chains from survivin, stabilizing both anti-apoptotic proteins. USP36 disrupts XIAP-SMAC complex formation and promotes RIP1 ubiquitination, inhibiting both intrinsic and extrinsic apoptosis in colorectal cancer cells.\",\n      \"method\": \"Gene silencing, co-immunoprecipitation, ubiquitination assay (linkage-specific), apoptosis assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — linkage-specific ubiquitin chain identification (K11 vs K48) with mechanistic downstream apoptosis pathway determination\",\n      \"pmids\": [\"38876304\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP36 binds PARP1 and deubiquitinates it, increasing PARP1 protein stability in cardiomyocytes exposed to doxorubicin; the catalytically inactive mutant C131A fails to stabilize PARP1. Cardiac knockdown of USP36 by AAV9-shUSP36 preserves cardiac function in doxorubicin-treated mice.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, catalytic mutant (C131A), AAV9 cardiac knockdown in mice, echocardiography\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — catalytic mutant validation, in vivo AAV9 cardiac knockdown with functional cardiac readout\",\n      \"pmids\": [\"38307305\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP36 deubiquitinates ERα and inhibits its K48-linked polyubiquitination, stabilizing ERα and enhancing its transcriptome; the C131A catalytic mutant fails to promote breast cancer progression. USP36 silencing destabilizes the tamoxifen-resistant ERα Y537S mutant and restores tamoxifen sensitivity.\",\n      \"method\": \"DUB siRNA library screening, co-immunoprecipitation, ubiquitination assay, catalytic mutant (C131A), xenograft models, RNA-seq, luciferase assay\",\n      \"journal\": \"Journal of experimental & clinical cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — siRNA screen, K48-linkage-specific ubiquitination assay, catalytic mutant, and in vivo xenograft with therapeutic relevance\",\n      \"pmids\": [\"39215346\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP36 interacts with and stabilizes RBM28 via deubiquitination at K162 residue; elevated RBM28 binds p53 to suppress its transcriptional activity, inactivating p53 signaling and promoting colorectal cancer progression.\",\n      \"method\": \"Co-immunoprecipitation, site-specific ubiquitination assay (K162), p53 transcription reporter, cell proliferation/invasion assays, in vivo\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — residue-specific ubiquitination site identified with mechanistic pathway connecting USP36 → RBM28 → p53 via multiple assays\",\n      \"pmids\": [\"39343961\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP36 interacts with WDR5 and stabilizes it via deubiquitination; USP36 knockdown increases WDR5 ubiquitination and promotes its degradation, impairing osteoblast differentiation; WDR5 overexpression rescues osteogenic differentiation in USP36-deficient cells.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, Western blot, Alizarin red staining, rescue experiment\",\n      \"journal\": \"Journal of orthopaedic surgery and research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP, ubiquitination assay, and rescue experiment establishing functional axis, single study\",\n      \"pmids\": [\"39152465\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP36 interacts with Las1L and Nol9, regulates their stability via deubiquitination, and mediates SUMOylation of Las1L at K565; the K565R mutant fails to rescue ITS2 rRNA processing defects caused by Las1L knockdown, demonstrating USP36-mediated Las1L SUMOylation is critical for pre-rRNA ITS2 processing.\",\n      \"method\": \"Co-immunoprecipitation, in vitro deubiquitination assay, in vivo SUMOylation assay, site-directed mutagenesis (K565R), rRNA processing analysis, rescue experiments\",\n      \"journal\": \"Cancer research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — site-specific mutagenesis with functional rescue at specific rRNA processing step\",\n      \"pmids\": [\"39356143\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP36 interacts with and deubiquitinates MLLT3 in the nucleolus, stabilizing it and activating downstream HIF1α and Snai signaling; the germline USP36 variant K814N (rs3744797) upregulates USP36 expression by reducing m6A modification, facilitating MLLT3 stabilization and EGFR-TKI resistance.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, m6A analysis, nucleolar fractionation, in vitro/in vivo proliferation and resistance assays\",\n      \"journal\": \"Clinical cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP and ubiquitination assay with functional in vivo data, but m6A-USP36 mechanism is correlative\",\n      \"pmids\": [\"38261467\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP36 functions as a SUMO ligase for GNL3, mediating its SUMOylation; GNL3 SUMOylation is required for its interaction with the BLM-DNA2 helicase-nuclease complex and for DNA end resection in homologous recombination repair. SENP3 acts as the opposing SUMO protease for GNL3.\",\n      \"method\": \"Co-immunoprecipitation, SUMOylation assay, CRISPR mutagenesis (K196R), DNA end resection assay, RPA/RAD51 loading assay, epistasis with SENP3\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal assays in a preprint; not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2025.11.04.686352\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In Drosophila, a CRISPR-induced catalytic mutation in dUSP36 produces viable adults with only minor growth defects but causes male infertility, demonstrating that dUSP36 deubiquitinating activity is dispensable for cell growth but essential for spermatogenesis; USP36 functions through both catalytic-dependent and catalytic-independent mechanisms.\",\n      \"method\": \"CRISPR/Cas9 catalytic mutation, in vivo viability and fertility assays, genetic epistasis\",\n      \"journal\": \"Genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — endogenous CRISPR catalytic mutation in vivo with clear phenotypic separation of functions\",\n      \"pmids\": [\"40646716\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Methylmalonylation of USP36 at K499 by MMA (methylmalonic acid) inhibits USP36-mediated deubiquitination and SUMOylation of SUFU, promoting Hedgehog signaling (GLI1 target genes) and M2 macrophage polarization in clear-cell renal cell carcinoma.\",\n      \"method\": \"Metabolomics, co-immunoprecipitation, in vitro deubiquitination/SUMOylation assays, K499 site-specific analysis, in vivo tumor models\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — novel PTM (methylmalonylation) identified at specific residue with functional consequences on USP36 enzymatic activities, single study\",\n      \"pmids\": [\"41398045\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP36 deubiquitinates and stabilizes APEX1 via cleavage of K48-linked ubiquitin chains; USP36 overexpression suppresses erastin-induced ferroptosis in melanoma cells, while USP36 deficiency increases ferroptosis. APEX1 knockdown abolishes the anti-ferroptotic effect of USP36.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay (K48-linkage specific), overexpression/knockdown, ferroptosis assays, xenograft model\",\n      \"journal\": \"Clinical and experimental medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — linkage-specific ubiquitination assay plus rescue experiment establishing USP36-APEX1 axis, single study\",\n      \"pmids\": [\"41649582\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"FBL acts as a carrier for monoubiquitinated H2A (H2Aub) in nucleolar lysosome-like structures; USP36, FBL, Midnolin, and BMI1 form a complex regulating the balance between H2A monoubiquitination and deubiquitination during glucose starvation. Knockdown of USP36 causes S-phase cell cycle arrest and reduced cell viability.\",\n      \"method\": \"Protein complex screening, co-immunoprecipitation, glucose starvation assay, cell cycle analysis, viability assay\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — preprint with Co-IP evidence for complex formation, mechanistic detail of USP36's specific role limited\",\n      \"pmids\": [\"bio_10.1101_2025.06.30.662258\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Oxidative stress (H2O2) promotes USP36 interaction with androgen receptor (AR) in prostate cancer cells; USP36 deubiquitinates and stabilizes AR, and USP36 knockdown abolishes H2O2-induced AR-PSA pathway activation.\",\n      \"method\": \"TurboID proximity biotin labeling/MS, co-immunoprecipitation, deubiquitination assay, siRNA knockdown, luciferase reporter assay\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — proximity labeling MS identification validated by Co-IP and functional reporter assay, single study\",\n      \"pmids\": [\"41298501\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP36 deubiquitinates and stabilizes SOD2 to preserve mitochondrial integrity in gastric cancer; resveratrol disrupts the USP36-SOD2 axis, reducing SOD2 stability, inducing mitochondrial dysfunction, and triggering autophagy and ferroptosis.\",\n      \"method\": \"Western blot, ubiquitination assay, functional assays (colony formation, Transwell), xenograft model, fluorescence staining for mitochondrial function\",\n      \"journal\": \"Gastric cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ubiquitination assay and functional in vivo data, but primarily extends prior SOD2 findings\",\n      \"pmids\": [\"40650854\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"PRL1 activates USP36-mediated Snail2 deubiquitination in glioblastoma; USP36 deubiquitinates Snail2, and PRL1 expression positively correlates with Snail2 levels, promoting EMT, invasion, and tumorigenicity.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, overexpression/knockdown, in vitro and in vivo invasion/tumorigenicity assays\",\n      \"journal\": \"Frontiers in oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — Co-IP and ubiquitination assay, functional in vivo data, but mechanism of PRL1-USP36 activation unclear\",\n      \"pmids\": [\"35111679\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP36 stabilizes KIF2C via K48-linked deubiquitination; USP36 overexpression suppresses erastin-induced ferroptosis in breast cancer cells, and KIF2C knockdown counteracts this anti-ferroptotic effect. USP36-deficient tumors show reduced proliferation and increased ferroptosis in vivo.\",\n      \"method\": \"Co-immunoprecipitation, K48-linkage ubiquitination assay, overexpression/knockdown, ferroptosis assays, xenograft model\",\n      \"journal\": \"Biochemical pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — linkage-specific ubiquitination assay with functional rescue and in vivo validation, single study\",\n      \"pmids\": [\"40744233\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"USP36 is a nucleolar deubiquitinating enzyme (DUB) that localizes to nucleoli via a basic amino acid nucleolar localization signal recruited by nucleophosmin/B23, where it deubiquitinates and stabilizes multiple substrates (c-Myc/MYCN, nucleophosmin/B23, fibrillarin, DHX33, SOD2, H2Bub1, PrimPol, and others) to regulate ribosome biogenesis, rRNA processing, and protein translation; beyond its canonical DUB activity, USP36 also acts as a non-canonical SUMO E3 ligase mediating group SUMOylation of snoRNP components (Nop58, Nhp2, Nop56, DKC1), EXOSC10 (K583), Las1L (K565), DGCR8, and GNL3, thereby coordinating multiple steps of ribosome biogenesis and miRNA processing; USP36 additionally functions as a FUBI-specific protease required for FUBI-eS30 cleavage and 40S subunit maturation, and its activity is regulated by post-translational modification including methylmalonylation at K499 that inhibits both its DUB and SUMO ligase functions, while Drosophila studies reveal that its catalytic activity is dispensable for cell growth but essential for spermatogenesis, indicating catalytic-independent functions.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"USP36 is a nucleolar deubiquitinating enzyme with dual enzymatic functions — canonical ubiquitin hydrolase activity and non-canonical SUMO E3 ligase activity — that coordinates ribosome biogenesis, rRNA processing, and RNA metabolism. As a DUB, USP36 localizes to nucleoli via a basic amino acid motif recruited by nucleophosmin/B23, where it deubiquitinates and stabilizes nucleolar substrates including c-Myc, nucleophosmin/B23, fibrillarin, DHX33, and H2Bub1 to promote rRNA transcription, processing, and cell proliferation; it also functions as a FUBI-specific protease that cleaves FUBI-eS30, an activity required for cytoplasmic 40S ribosomal subunit maturation [PMID:19208757, PMID:25775507, PMID:34318747, PMID:29273634]. As a SUMO E3 ligase, USP36 mediates SUMOylation of snoRNP components (Nop58, Nhp2, Nop56, DKC1), EXOSC10 at K583, Las1L at K565, and DGCR8, thereby regulating snoRNA binding, ITS2 rRNA processing, and pri-miRNA processing, respectively [PMID:33852194, PMID:36912080, PMID:39356143, PMID:36950067]. Beyond ribosome biogenesis, USP36 stabilizes diverse substrates across multiple pathways — including PrimPol during replication stress, anti-apoptotic proteins cIAP1/survivin, ERα, and SOD2 — and its enzymatic activities are inhibited by methylmalonylation at K499, while Drosophila studies demonstrate that catalytic activity is dispensable for viability but essential for spermatogenesis [PMID:33237263, PMID:38876304, PMID:39215346, PMID:41398045, PMID:40646716].\",\n  \"teleology\": [\n    {\n      \"year\": 2005,\n      \"claim\": \"Establishing that USP36 possesses deubiquitinase activity and is itself regulated by polyubiquitination provided the initial enzymatic characterization of this DUB.\",\n      \"evidence\": \"In vitro ubiquitin cleavage assay and immunoprecipitation\",\n      \"pmids\": [\"15809067\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No physiological substrate identified\", \"No cellular context or localization determined\", \"Regulation of USP36 stability via its own ubiquitination not characterized\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Demonstrating that USP36 localizes to nucleoli via a basic amino acid motif, is recruited by nucleophosmin/B23, and deubiquitinates nucleolar proteins (B23, fibrillarin) established USP36 as a nucleolar DUB regulating rRNA biogenesis.\",\n      \"evidence\": \"Dominant-negative inhibition, RNAi, deletion mutagenesis, Co-IP, and rRNA processing analysis in human cells\",\n      \"pmids\": [\"19208757\", \"19679658\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of nucleolar targeting not resolved\", \"Full spectrum of nucleolar substrates unknown\", \"How USP36 activity is regulated remained unclear\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Genetic studies in Drosophila revealed that loss of USP36 causes nuclear accumulation of ubiquitinated proteins including H2B and triggers p62-dependent selective autophagy, linking USP36 to ubiquitin homeostasis and autophagy regulation conserved across species.\",\n      \"evidence\": \"Drosophila loss-of-function mutants combined with RNAi in human cells and p62 epistasis\",\n      \"pmids\": [\"22622177\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether autophagy activation is a direct or compensatory consequence of USP36 loss\", \"Identity of critical nuclear ubiquitinated substrates beyond H2B\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Identification of c-Myc as a direct USP36 substrate, stabilized against SCF(Fbw7γ)-mediated degradation in the nucleolus with a positive feedback loop (USP36 is a c-Myc target gene), established USP36 as a key regulator of oncogenic signaling.\",\n      \"evidence\": \"In vitro deubiquitination, reciprocal Co-IP, cycloheximide chase, reporter assays, and subcellular fractionation\",\n      \"pmids\": [\"25775507\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How USP36-c-Myc feedback loop is terminated or restrained\", \"Whether nucleolar versus nucleoplasmic c-Myc pools have distinct functional outcomes\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Mouse knockout revealing embryonic lethality at morula-to-blastocyst transition, combined with identification of DHX33 and H2Bub1 as direct substrates, established USP36 as essential for early development, ribosome biogenesis, and epigenetic regulation.\",\n      \"evidence\": \"Conditional mouse knockout with EM, Northern blot, translation assay; in vitro deubiquitination of H2B with ChIP at p21 locus\",\n      \"pmids\": [\"29273634\", \"29274341\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which substrate(s) account for the morula-blastocyst lethality\", \"Genome-wide landscape of H2Bub1 changes upon USP36 loss\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Discovery that USP36 removes K29-linked polyubiquitin chains from PrimPol during replication stress extended USP36 function beyond nucleolar/ribosomal roles to DNA damage tolerance.\",\n      \"evidence\": \"Mass spectrometry, in vitro K29-linkage-specific deubiquitination, DNA fiber assay, cisplatin/olaparib sensitivity\",\n      \"pmids\": [\"33237263\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How USP36 is relocated or activated at replication forks\", \"Whether other replication stress substrates exist\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"The paradigm-shifting discovery that USP36 acts as a non-canonical SUMO E3 ligase for snoRNP components (Nop58, Nhp2, Nop56, DKC1) revealed a dual enzymatic function — DUB and SUMO ligase — and explained its broad impact on rRNA processing and translation.\",\n      \"evidence\": \"In vitro SUMOylation reconstitution with purified components, Co-IP with SUMO2/Ubc9, rRNA processing and translation assays\",\n      \"pmids\": [\"33852194\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis for SUMO E3 ligase activity\", \"Whether DUB and SUMO ligase activities are coordinated or independent on the same substrates\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identification of USP36 as a FUBI-specific protease required for FUBI-eS30 cleavage and 40S maturation, confirmed by crystal structures in 2023, established a unique dual ubiquitin/ubiquitin-like substrate specificity and explained USP36's role in cytoplasmic ribosome maturation.\",\n      \"evidence\": \"In vitro cleavage with purified USP36, RNAi/CRISPRi depletion, rRNA maturation analysis; crystal structures of USP36-Fubi and USP36-Ub complexes\",\n      \"pmids\": [\"34318747\", \"37443395\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How USP36 transitions between nucleolar functions and cytoplasmic 40S maturation\", \"Whether FUBI cleavage is rate-limiting for 40S assembly in vivo\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Extension of the SUMO E3 ligase function to EXOSC10 (K583), DGCR8, and later Las1L (K565), with site-specific mutagenesis showing SUMOylation is required for pre-rRNA binding/processing and pri-miRNA processing, established USP36 as a central coordinator of nucleolar RNA metabolism through SUMOylation.\",\n      \"evidence\": \"Site-directed mutagenesis (K583R, K565R) with functional rescue of rRNA processing; RIP for pri-miRNA binding\",\n      \"pmids\": [\"36912080\", \"36950067\", \"39356143\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full catalog of USP36 SUMOylation substrates\", \"Whether a single domain mediates all SUMO E3 ligase activities\", \"Interplay between USP36 SUMOylation and opposing SENP activities in steady state\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Discovery that ribotoxic stress activates a JNK-USP36-Snail1 axis in the nucleolus, promoting ribosome biogenesis and drug resistance, placed USP36 as a stress-responsive effector integrating kinase signaling with nucleolar function.\",\n      \"evidence\": \"Co-IP, JNK inhibition, nucleolar fractionation, rRNA analysis, in vivo tumor models\",\n      \"pmids\": [\"37833415\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether JNK directly phosphorylates USP36 and at which site\", \"Generalizability to other ribotoxic stress agents\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Demonstrations that USP36 deubiquitinates anti-apoptotic proteins (cIAP1 via K11-linked, survivin via K48-linked chains), ERα, PARP1, and other substrates across cancer types broadened USP36's substrate repertoire to apoptosis, hormone signaling, and cardioprotection, with linkage-type specificity emerging as a recurring feature.\",\n      \"evidence\": \"Linkage-specific ubiquitination assays, catalytic mutant C131A validation, in vivo cardiac and xenograft models\",\n      \"pmids\": [\"38876304\", \"39215346\", \"38307305\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How USP36 achieves substrate selectivity among its many targets\", \"Whether linkage specificity is substrate-determined or context-dependent\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identification of methylmalonylation at K499 as an inhibitory post-translational modification of both DUB and SUMO ligase activities, combined with Drosophila evidence that catalytic activity is dispensable for growth but essential for spermatogenesis, revealed regulatory complexity and catalytic-independent functions of USP36.\",\n      \"evidence\": \"Metabolomics with K499 site-specific analysis and in vitro activity assays; CRISPR catalytic mutation in Drosophila with viability/fertility phenotyping\",\n      \"pmids\": [\"41398045\", \"40646716\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis of methylmalonylation-mediated inhibition\", \"Identity of catalytic-independent binding partners mediating growth functions\", \"Whether methylmalonylation occurs physiologically outside cancer contexts\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the structural basis for USP36's dual DUB/SUMO E3 ligase activities, how substrate selectivity is achieved among its many targets, and the full scope of catalytic-independent functions implied by Drosophila genetics.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No full-length USP36 structure available\", \"Mechanism of substrate prioritization in the nucleolus unknown\", \"Catalytic-independent interaction partners not identified\", \"No disease-causing germline mutations reported in humans\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [0, 2, 5, 7, 8, 12, 15, 19, 23, 24, 25]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 5, 8, 12, 15, 23, 24, 25]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [13, 18, 21, 28]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [6, 13, 21]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005730\", \"supporting_discovery_ids\": [0, 1, 5, 13, 18, 22, 28]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 4, 8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [0, 13, 15, 18, 21, 28]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 5, 7, 13, 15, 19]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [15, 19]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [12]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [23]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [4, 11]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [8, 25]}\n    ],\n    \"complexes\": [\n      \"snoRNP (via SUMOylation of Nop58/Nhp2/Nop56/DKC1)\",\n      \"Drosha-DGCR8 microprocessor (via DGCR8 SUMOylation)\"\n    ],\n    \"partners\": [\n      \"NPM1\",\n      \"FBL\",\n      \"MYC\",\n      \"DHX33\",\n      \"DGCR8\",\n      \"PRIMPOL\",\n      \"EXOSC10\",\n      \"LAS1L\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}