{"gene":"USP36","run_date":"2026-06-11T09:02:06","timeline":{"discoveries":[{"year":2009,"finding":"USP36 localizes to nucleoli via a C-terminal basic amino acid region and deubiquitinates nucleolar proteins nucleophosmin/B23 and fibrillarin, stabilizing them against ubiquitylation-mediated proteasomal degradation. RNAi-mediated depletion reduces rRNA transcription and processing, nucleolar morphology, and cell proliferation.","method":"Dominant-negative inhibition, RNAi knockdown, co-immunoprecipitation, in vitro deubiquitylation assay, subcellular fractionation/imaging","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods including dominant-negative, RNAi, in vitro deubiquitylation, and functional readouts in a single study","pmids":["19208757"],"is_preprint":false},{"year":2009,"finding":"Nucleophosmin/B23 recruits USP36 to nucleoli by interacting with a specific basic amino acid motif (RGKEKKIKKFKREKRR) in USP36's C-terminal region. Knockdown of nucleophosmin/B23 reduces nucleolar USP36 levels and elevates fibrillarin ubiquitylation.","method":"Nucleolar localization signal mapping, Co-IP, RNAi knockdown, ubiquitylation assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, domain mapping, and functional consequence shown in single study with multiple orthogonal methods","pmids":["19679658"],"is_preprint":false},{"year":2005,"finding":"USP36 possesses deubiquitinase activity (cleaves ubiquitin from substrates), contains a PEST motif, and is itself polyubiquitinated.","method":"RT-PCR cloning, ubiquitin-cleavage activity assay, immunoprecipitation, structural domain analysis","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — in vitro DUB activity assay and Co-IP, single lab, single study","pmids":["15809067"],"is_preprint":false},{"year":2011,"finding":"USP36 interacts with mitochondrial antioxidant enzyme SOD2, reduces its polyubiquitination, and stabilizes it against proteasomal degradation, extending its half-life.","method":"2D-gel electrophoresis, MALDI-TOF/MS identification, co-immunoprecipitation, yeast two-hybrid, ubiquitination assay","journal":"Journal of cellular biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP plus yeast two-hybrid plus in vivo ubiquitination assay, single lab","pmids":["21268071"],"is_preprint":false},{"year":2012,"finding":"USP36 (dUsp36) loss of function in Drosophila and human cells activates p62/SQSTM1-dependent selective autophagy of ubiquitinated cargo; dUSP36 loss leads to nuclear accumulation of ubiquitinated proteins (including histone H2B) and cytoplasmic ubiquitinated aggregates eliminated by autophagy. dUSP36 is not part of the TOR signaling pathway.","method":"Drosophila loss-of-function genetics, RNAi in human cells, p62/SQSTM1 requirement epistasis, fluorescence imaging of ubiquitin aggregates","journal":"Autophagy","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis in Drosophila plus RNAi in human cells, conservation demonstrated, negative TOR epistasis established","pmids":["22622177"],"is_preprint":false},{"year":2015,"finding":"USP36 is a nucleolar deubiquitinase for c-Myc: it interacts with and deubiquitinates c-Myc in cells and in vitro, stabilizing it against SCF(Fbw7)-mediated degradation in the nucleolus. USP36 interacts with nucleolar Fbw7γ but not nucleoplasmic Fbw7α, yet abolishes c-Myc degradation by both. USP36 is also a c-Myc target gene, forming a positive feedback loop.","method":"Co-immunoprecipitation, in vitro deubiquitination assay, knockdown, c-Myc stability assay, isoform-specific interaction mapping","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro deubiquitination plus cellular Co-IP plus epistasis with Fbw7 isoforms, multiple orthogonal approaches","pmids":["25775507"],"is_preprint":false},{"year":2016,"finding":"USP36 binds Nedd4-2 and regulates its association with TrkA neurotrophin receptor indirectly (USP36 does not directly deubiquitinate TrkA). Depletion of USP36 increases TrkA–Nedd4-2 complex formation, enhancing TrkA ubiquitination; USP36 overexpression disrupts this complex. USP36 also interferes with Nedd4-2-dependent regulation of Kv7.2/3 channels.","method":"siRNA screen, Co-IP, overexpression/depletion, TrkA ubiquitination assay, PC12 differentiation assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, functional rescue, multiple substrate contexts, single lab","pmids":["27445338"],"is_preprint":false},{"year":2017,"finding":"USP36 deubiquitinates and stabilizes the DEAH-box RNA helicase DHX33 in the nucleolus. Usp36 knockout in mice is lethal at the morula-to-blastocyst transition. USP36 depletion impairs rRNA synthesis, protein translation, cell proliferation, and induces apoptosis and cell cycle arrest in human cancer cells.","method":"Usp36 knockout mouse model, electron microscopy, Northern blot, O-propargyl-puromycin protein synthesis assay, ubiquitination assay, Co-IP","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vivo mouse KO model, multiple orthogonal methods (EM, Northern blot, protein synthesis), mechanistic link to DHX33 via ubiquitination assay","pmids":["29273634"],"is_preprint":false},{"year":2017,"finding":"USP36 is a histone H2B deubiquitinase: it interacts with H2B and deubiquitinates monoubiquitinated H2B (H2Bub1) in cells and in vitro. USP36 depletion increases H2Bub1 at the p21 locus gene body, inducing p21 expression and inhibiting cell proliferation.","method":"Co-IP, in vitro deubiquitination assay, overexpression, knockdown, ChIP at p21 locus","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — in vitro DUB assay plus ChIP functional linkage, single lab","pmids":["29274341"],"is_preprint":false},{"year":2018,"finding":"USP36 controls cellular localization of CHD7; in neuroblastoma, lncRNAs CASC15/NBAT1 modulate USP36 localization, which in turn regulates CHD7 stability and downstream SOX9 expression.","method":"Loss-of-function experiments, co-immunoprecipitation, immunoblotting, localization studies","journal":"Cancer cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, cellular imaging, functional knockdown with mechanistic readout, single lab","pmids":["29533783"],"is_preprint":false},{"year":2018,"finding":"USP36 deubiquitinates and stabilizes PME-1 (protein phosphatase methylesterase 1), thereby promoting ERK and Akt signaling pathways.","method":"Co-IP, USP36 knockdown/overexpression, ubiquitination assay, western blot for ERK/Akt activation","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — Co-IP and ubiquitination assay, single lab, single study","pmids":["29577269"],"is_preprint":false},{"year":2019,"finding":"USP36 knockdown impairs Parkin-dependent mitophagy by reducing mRNA and protein levels of Beclin-1 and ATG14L (without altering nucleolar USP36 localization during mitophagy); restoration of ATG14L rescues mitophagy in USP36-silenced cells.","method":"High-content imaging siRNA screen, knockdown, immunofluorescence, western blot, ATG14L overexpression rescue","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA screen plus rescue experiment plus mechanistic pathway linking, single lab","pmids":["31550441"],"is_preprint":false},{"year":2020,"finding":"USP36 interacts with PrimPol, deubiquitinates K29-linked polyubiquitin chains on PrimPol, and increases PrimPol protein stability in response to DNA replication stress. USP36 is itself deubiquitinated following replication stress, facilitating its upregulation.","method":"Co-IP, mass spectrometry, in vivo and in vitro ubiquitination assays, replication stress assays, cisplatin/olaparib sensitivity","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — Co-IP plus MS plus in vitro ubiquitination assay plus specific linkage identification (K29), multiple orthogonal methods","pmids":["33237263"],"is_preprint":false},{"year":2021,"finding":"USP36 promotes nucleolar SUMOylation: it interacts with SUMO2 and Ubc9, directly mediates SUMOylation in cells and in vitro, and specifically SUMOylates snoRNP components Nop58, Nhp2, Nop56, and DKC1, promoting their binding to snoRNAs. USP36 knockdown markedly impairs rRNA processing and translation.","method":"Overexpression, knockdown, genetic deletion, Co-IP, in vitro SUMOylation assay, snoRNA binding assay, rRNA processing assay","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro SUMOylation reconstitution plus cellular studies plus rRNA processing functional readout, multiple substrates confirmed","pmids":["33852194"],"is_preprint":false},{"year":2021,"finding":"USP36 cleaves the ribosomal fusion protein FUBI-eS30: purified USP36 cuts FUBI-eS30 in vitro, and USP36 depletion by RNAi or CRISPRi impairs FUBI-eS30 processing and late steps of cytoplasmic 40S maturation including 18S rRNA maturation and recycling of biogenesis factors.","method":"Differential affinity purification, RNAi, CRISPRi, in vitro processing assay with purified USP36, rRNA processing analysis","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with purified enzyme plus genetic depletion, orthogonal methods confirming processing role","pmids":["34318747"],"is_preprint":false},{"year":2021,"finding":"USP36 deubiquitinates and stabilizes DOCK4 (a guanine nucleotide exchange factor); increased USP36-DOCK4 stabilization activates Wnt/β-catenin signaling and promotes epithelial-to-mesenchymal transition in diabetic renal tubular epithelial cells.","method":"Co-IP, deubiquitination assay, knockdown/overexpression, Wnt pathway reporter","journal":"Frontiers in cell and developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — Co-IP plus deubiquitination assay, single lab, single study","pmids":["33968925"],"is_preprint":false},{"year":2022,"finding":"USP36 deubiquitinates and stabilizes YAP by blocking K48-linked polyubiquitination, thereby promoting Hippo/YAP signaling in esophageal squamous carcinoma.","method":"siRNA DUB screening, Co-IP, ubiquitination assay, knockdown/overexpression, western blot","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — Co-IP and ubiquitination assay, single lab, single study","pmids":["36470870"],"is_preprint":false},{"year":2022,"finding":"USP36 activates Snail2 deubiquitination in glioblastoma, downstream of PRL1 oncogene; PRL1 promotes EMT and invasion by activating USP36-mediated Snail2 stabilization.","method":"Co-IP, ubiquitination assay, knockdown/overexpression, in vitro and in vivo tumorigenesis assays","journal":"Frontiers in oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — Co-IP plus ubiquitination assay, single lab, single study","pmids":["35111679"],"is_preprint":false},{"year":2023,"finding":"USP36 interacts with EXOSC10 (nucleolar RNA exosome subunit) and mediates its SUMOylation at K583. K583 SUMOylation is required for EXOSC10 binding to pre-rRNAs; K583R mutant fails to rescue rRNA processing and cell growth defects upon EXOSC10 knockdown. USP36 does not significantly regulate EXOSC10 protein levels.","method":"Co-IP, in vitro SUMOylation assay, mutant rescue, rRNA processing assay, RNA binding assay","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro SUMOylation plus site-specific mutagenesis plus rescue experiments, single lab with multiple orthogonal methods","pmids":["36912080"],"is_preprint":false},{"year":2023,"finding":"USP36 deubiquitinates and stabilizes ALKBH5, regulating ALKBH5-mediated gene expression in glioblastoma. Depletion of USP36 impairs glioma stem cell self-renewal, proliferation, and sensitizes cells to temozolomide.","method":"DUB siRNA library screen, mass spectrometry, Co-IP, in vivo and in vitro ubiquitination assays, neurosphere formation, intracranial tumor growth","journal":"Neuro-oncology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — MS-identified interaction, Co-IP, in vitro ubiquitination, in vivo tumor model, multiple orthogonal methods","pmids":["36239338"],"is_preprint":false},{"year":2023,"finding":"Crystal structures of USP36 in complex with ubiquitin and Fubi reveal the substrate recognition mechanism for dual ubiquitin/Fubi cleavage activity. USP16 was also identified as having dual ubiquitin/Fubi cleavage activity by chemoproteomics; both USP16 and USP36 participate in Fubi-S30 maturation.","method":"Crystal structure determination, chemoproteomics, Fubi C-terminal hydrolase activity assay, substrate specificity analysis","journal":"Nature chemical biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structures of USP36 with both substrates, biochemical activity measurements, mechanistic explanation of specificity","pmids":["37443395"],"is_preprint":false},{"year":2023,"finding":"Ribotoxic stress activates JNK, which in turn activates USP36, leading to stabilization of Snail1 in the nucleolus. Nucleolar Snail1 facilitates ribosome biogenesis and tumor cell survival, and mediates solid tumor resistance to homoharringtonine (HHT).","method":"Co-IP, knockdown, JNK inhibition, HHT treatment, ribosome biogenesis assay, combination therapy in vivo","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus epistasis (JNK-USP36-Snail1) plus in vivo rescue, single lab","pmids":["37833415"],"is_preprint":false},{"year":2023,"finding":"USP36 associates with the Drosha-DGCR8 microprocessor complex and mediates DGCR8 SUMOylation by SUMO2. This SUMOylation promotes DGCR8 binding to pri-miRNAs without affecting DGCR8 levels or the Drosha-DGCR8 complex. USP36 knockdown attenuates pri-miRNA processing and reduces mature miRNA levels.","method":"Co-IP, in vitro/in vivo SUMOylation assay, SUMO site mapping, RNA immunoprecipitation, knockdown, cell proliferation assay","journal":"Cancer research communications","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro SUMOylation, RNA binding assay, functional rescue with SUMOylation mutant, multiple orthogonal approaches","pmids":["36950067"],"is_preprint":false},{"year":2024,"finding":"USP36 binds PARP1 and mediates its deubiquitination, increasing PARP1 protein stability in cardiomyocytes exposed to doxorubicin. The catalytically inactive C131A mutant of USP36 fails to stabilize PARP1, confirming catalytic dependence. Cardiac knockdown of USP36 (rAAV9-shUSP36) preserved cardiac function in a doxorubicin-induced cardiomyopathy mouse model.","method":"Co-IP, ubiquitination assay, catalytic mutant (C131A), rAAV9 in vivo knockdown, cardiac function assessment","journal":"Cellular signalling","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — catalytic mutant validation plus in vivo mouse model, multiple orthogonal methods","pmids":["38307305"],"is_preprint":false},{"year":2024,"finding":"USP36 deubiquitinates survivin (removing K48-linked chains) and cIAP1 (removing K11-linked chains), stabilizing both anti-apoptotic proteins. USP36 disrupts XIAP-SMAC complex formation and promotes RIP1 ubiquitination, thereby inhibiting both intrinsic and extrinsic apoptosis in colorectal cancer cells.","method":"siRNA gene silencing, Co-IP, ubiquitin linkage-specific assays, apoptosis assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus linkage-specific ubiquitination assays, functional apoptosis readouts, single lab","pmids":["38876304"],"is_preprint":false},{"year":2024,"finding":"USP36 deubiquitinates ERα, inhibiting K48-linked polyubiquitination and stabilizing ERα protein; the catalytically inactive C131A mutant fails to promote breast cancer progression through ERα signaling. USP36 knockdown destabilizes the tamoxifen-resistant ERα mutant (Y537S).","method":"DUB siRNA library screen, Co-IP, ubiquitination assay, catalytic mutant (C131A), xenograft model, RNA sequencing","journal":"Journal of experimental & clinical cancer research : CR","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — siRNA screen, catalytic mutant validation, in vivo xenograft, multiple orthogonal approaches","pmids":["39215346"],"is_preprint":false},{"year":2024,"finding":"USP36 deubiquitinates and stabilizes RBM28 at K162; upregulated RBM28 binds p53 and suppresses its transcriptional activity, inactivating the p53 signaling pathway in colorectal cancer.","method":"Co-IP, ubiquitination assay (site-specific K162), knockdown/overexpression, p53 transcription reporter","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with site-specific mutagenesis plus functional epistasis, single lab","pmids":["39343961"],"is_preprint":false},{"year":2024,"finding":"USP36 SUMOylates Las1L at K565; K565 SUMOylation is essential for ITS2 pre-rRNA processing but not for Las1L protein levels or Las1L-Nol9 complex formation. USP36 also deubiquitinates Las1L and Nol9, regulating their stability.","method":"Co-IP, in vitro SUMOylation assay, K565R mutant rescue, ITS2 processing assay, ubiquitination assay","journal":"Cancer research communications","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro SUMOylation, site-specific mutagenesis with functional rescue, multiple substrates, orthogonal DUB assay","pmids":["39356143"],"is_preprint":false},{"year":2024,"finding":"USP36 interacts with WDR5 and stabilizes it via deubiquitination; USP36 knockdown increases WDR5 ubiquitination and degradation, impairing osteogenic differentiation. Overexpression of WDR5 rescues the differentiation defects in USP36-deficient osteoblasts.","method":"Co-IP, ubiquitination assay, knockdown, Alizarin red staining, WDR5 rescue overexpression","journal":"Journal of orthopaedic surgery and research","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — Co-IP plus ubiquitination assay plus rescue, single lab, single study","pmids":["39152465"],"is_preprint":false},{"year":2024,"finding":"A germline USP36 variant (rs3744797, K814N) stabilizes MLLT3 via deubiquitination in the nucleolus, activating HIF1α and Snai downstream signaling and conferring resistance to EGFR-TKIs in non-small cell lung cancer.","method":"m6A-genome-wide-variant analysis, Co-IP, ubiquitination assay, in vitro/in vivo functional assays, patient cohort","journal":"Clinical cancer research : an official journal of the American Association for Cancer Research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus ubiquitination assay plus in vivo model, single lab, single study","pmids":["38261467"],"is_preprint":false},{"year":2025,"finding":"MMA (methylmalonic acid) induces methylmalonylation of USP36 at K499, inhibiting USP36-mediated deubiquitination and SUMOylation of SUFU, thereby promoting GLI1 expression and Hedgehog pathway activation in clear-cell renal cell carcinoma macrophages.","method":"Metabolomic profiling, site-specific modification analysis, ubiquitination and SUMOylation assays, in vitro and in vivo functional experiments","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple biochemical assays plus in vivo validation, novel PTM on USP36 itself, single lab","pmids":["41398045"],"is_preprint":false},{"year":2025,"finding":"In Drosophila, catalytically inactive dUSP36 (CRISPR knock-in) allows survival to adulthood with minor growth defects, demonstrating that the deubiquitinating activity is dispensable for cell growth but essential for spermatogenesis, revealing both catalytic-dependent and catalytic-independent mechanisms.","method":"CRISPR/Cas9 catalytic mutation knock-in, null mutant comparison, viability and fertility assays","journal":"Genetics","confidence":"High","confidence_rationale":"Tier 1 / Moderate — CRISPR knock-in of catalytic mutant with null comparison in whole organism, rigorous genetic dissection of catalytic vs. non-catalytic functions","pmids":["40646716"],"is_preprint":false},{"year":2025,"finding":"USP36 stabilizes APEX1 by removing K48-linked ubiquitin chains, suppressing ferroptosis in melanoma cells. USP36 knockdown sensitizes xenograft tumors to ferroptosis, and APEX1 knockdown abolishes USP36's anti-ferroptotic effect.","method":"Co-IP, ubiquitination assay (K48-specific), overexpression/knockdown, erastin treatment, xenograft model","journal":"Clinical and experimental medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — Co-IP plus K48-specific ubiquitination assay plus rescue, single lab, single study","pmids":["41649582"],"is_preprint":false},{"year":2025,"finding":"USP36 functions as a SUMO ligase for GNL3 (nucleolar GTP-binding protein), while SENP3 deSUMOylates GNL3. GNL3 SUMOylation is required for interaction with the BLM-DNA2 complex and DNA end resection in homologous recombination repair.","method":"Co-IP, in vitro SUMOylation assay, CRISPR/RNAi knockdown, DNA end resection assay, epistasis with SENP3","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro SUMOylation plus functional epistasis, preprint not yet peer-reviewed","pmids":[],"is_preprint":true},{"year":2025,"finding":"USP36 stabilizes KIF2C by removing K48-linked ubiquitin chains, suppressing ferroptosis in breast cancer cells. USP36-deficient xenograft tumors show reduced proliferation and increased ferroptosis.","method":"Co-IP, K48-specific ubiquitination assay, knockdown/overexpression, erastin ferroptosis assay, xenograft model","journal":"Biochemical pharmacology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — Co-IP plus ubiquitination assay, in vivo xenograft, single lab, single study","pmids":["40744233"],"is_preprint":false},{"year":2025,"finding":"USP36 deubiquitinates and stabilizes AR (androgen receptor) in prostate cancer cells under oxidative stress (H2O2); USP36–AR interaction is induced by H2O2 and knockdown of USP36 abolishes H2O2-induced activation of the AR-PSA pathway.","method":"TurboID proximity labeling plus mass spectrometry, Co-IP, ubiquitination assay, dual-luciferase reporter, knockdown","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — proximity labeling MS for discovery, Co-IP for validation, functional pathway reporter, single lab","pmids":["41298501"],"is_preprint":false},{"year":2025,"finding":"FBL (fibrillarin) acts as a carrier regulating the balance between BMI1-mediated H2A monoubiquitination and USP36-mediated H2Aub deubiquitination in nucleolar lysosome-like structures during glucose starvation; USP36, FBL, Midnolin, and BMI1 form a complex responsible for H2Aub degradation.","method":"Protein interaction screening, Co-IP, knockdown, cell cycle and viability assays","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 3 / Weak — Co-IP with complex formation claim, preprint not peer-reviewed, single lab","pmids":[],"is_preprint":true}],"current_model":"USP36 is a nucleolar deubiquitinating enzyme (and SUMO ligase) that localizes to the nucleolus via a C-terminal basic NLS recruited by nucleophosmin/B23, where it stabilizes diverse substrates (c-Myc, nucleophosmin/B23, fibrillarin, DHX33, SOD2, PrimPol, ALKBH5, and others) by removing polyubiquitin chains, while simultaneously acting as a non-canonical SUMO E3 ligase for multiple nucleolar proteins (Nop58, Nhp2, EXOSC10, DGCR8, Las1L, GNL3), thereby coordinating rRNA processing, ribosome biogenesis, miRNA biogenesis, DNA repair, and selective autophagy; additionally, it functions through both catalytic-dependent and catalytic-independent mechanisms in vivo, as demonstrated by the dispensability of its DUB activity for cell growth (but not spermatogenesis) in Drosophila."},"narrative":{"mechanistic_narrative":"USP36 is a nucleolar deubiquitinating enzyme that coordinates ribosome biogenesis, rRNA processing, and the stability of numerous nucleolar and signaling proteins [PMID:19208757, PMID:29273634]. It is recruited to the nucleolus through a C-terminal basic motif that is bound by nucleophosmin/B23, and there it removes polyubiquitin from substrates such as fibrillarin and nucleophosmin to protect them from proteasomal degradation, sustaining rRNA transcription/processing and cell proliferation [PMID:19208757, PMID:19679658]. A defining activity is stabilization of the oncoprotein c-Myc: USP36 deubiquitinates c-Myc in the nucleolus, antagonizing SCF(Fbw7)-mediated turnover, and is itself a c-Myc target gene, forming a positive feedback loop [PMID:25775507]. Beyond deubiquitination, USP36 is a non-canonical SUMO E3 ligase that interacts with SUMO2/Ubc9 and SUMOylates snoRNP and processing factors (Nop58, Nhp2, Nop56, DKC1, EXOSC10 at K583, Las1L at K565, DGCR8) to promote their RNA binding and rRNA/miRNA processing, often without altering substrate abundance [PMID:33852194, PMID:36912080, PMID:36950067, PMID:39356143]. USP36 also processes ribosomal precursors directly: it cleaves the FUBI-eS30 fusion to drive late cytoplasmic 40S maturation, a dual ubiquitin/FUBI hydrolase activity explained by crystal structures of USP36 bound to ubiquitin and FUBI [PMID:34318747, PMID:37443395]. Through deubiquitination it stabilizes a broad set of substrates linking it to DNA replication stress and repair (PrimPol via K29 chains, ALKBH5), histone H2B/H2A regulation, and multiple oncogenic and stress pathways (YAP, ERα, AR, survivin/cIAP1, RBM28-p53), with catalytic activity confirmed by the C131A inactive mutant in several contexts [PMID:33237263, PMID:36239338, PMID:29274341, PMID:36470870, PMID:39215346, PMID:38876304, PMID:39343961, PMID:38307305]. Usp36 knockout is embryonic-lethal in mice, and in Drosophila its deubiquitinating activity is dispensable for viability and cell growth but essential for spermatogenesis, establishing both catalytic-dependent and catalytic-independent functions [PMID:29273634, PMID:40646716].","teleology":[{"year":2005,"claim":"Established USP36 as a bona fide deubiquitinase, providing the biochemical foundation for all subsequent substrate work.","evidence":"RT-PCR cloning, ubiquitin-cleavage activity assay, and domain analysis identifying a PEST motif and self-ubiquitination","pmids":["15809067"],"confidence":"Medium","gaps":["No substrates identified","No cellular localization or pathway context","Single in vitro study"]},{"year":2009,"claim":"Defined USP36 as a nucleolar enzyme whose recruitment depends on nucleophosmin/B23 binding a C-terminal basic motif, answering where USP36 acts and how it is targeted.","evidence":"NLS mapping, reciprocal Co-IP, RNAi, in vitro deubiquitylation, and rRNA/proliferation readouts in human cells","pmids":["19208757","19679658"],"confidence":"High","gaps":["Substrate repertoire beyond fibrillarin/nucleophosmin unknown at this stage","No structural basis for catalysis"]},{"year":2011,"claim":"Extended USP36 substrate range beyond the nucleolus by showing it stabilizes mitochondrial SOD2, hinting at functions outside ribosome biogenesis.","evidence":"2D-gel/MALDI-TOF MS, yeast two-hybrid, Co-IP, and ubiquitination/half-life assays","pmids":["21268071"],"confidence":"Medium","gaps":["Mechanism reconciling nucleolar localization with mitochondrial substrate unclear","Single lab"]},{"year":2012,"claim":"Connected USP36 to selective autophagy, showing its loss triggers p62/SQSTM1-dependent clearance of ubiquitinated cargo independent of TOR signaling.","evidence":"Drosophila loss-of-function genetics with RNAi confirmation in human cells and epistasis with p62","pmids":["22622177"],"confidence":"High","gaps":["Direct substrates driving aggregate accumulation not defined","Link between nucleolar role and autophagy unclear"]},{"year":2015,"claim":"Identified c-Myc as a key USP36 substrate and a c-Myc/USP36 positive feedback loop, establishing USP36 as an oncogenic stabilizer that counteracts SCF(Fbw7).","evidence":"In vitro deubiquitination, cellular Co-IP, stability assays, and Fbw7 isoform-specific interaction mapping","pmids":["25775507"],"confidence":"High","gaps":["Whether deubiquitination occurs only in the nucleolar compartment unresolved","In vivo tumor relevance not tested here"]},{"year":2017,"claim":"Demonstrated USP36 is essential in vivo and stabilizes the rRNA helicase DHX33, linking USP36 to early embryonic viability and translation.","evidence":"Usp36 knockout mouse (morula-to-blastocyst lethality), EM, Northern blot, OPP protein synthesis, ubiquitination assays","pmids":["29273634","29274341"],"confidence":"High","gaps":["Catalytic dependence of essentiality not dissected","H2B deubiquitination role separate from rRNA functions not integrated"]},{"year":2020,"claim":"Revealed a DNA replication stress function by showing USP36 removes K29-linked chains from PrimPol and is itself stabilized after stress.","evidence":"Co-IP, MS, in vivo/in vitro ubiquitination assays with linkage specificity, and drug sensitivity assays","pmids":["33237263"],"confidence":"High","gaps":["Upstream signal regulating USP36 stabilization not defined","Nucleolar vs nucleoplasmic site of PrimPol action unclear"]},{"year":2021,"claim":"Uncovered a second enzymatic mode—non-canonical SUMO E3 ligase activity—and direct FUBI-eS30 processing, redefining USP36 as a dual-activity ribosome biogenesis factor.","evidence":"In vitro SUMOylation reconstitution on snoRNP factors, snoRNA-binding and rRNA processing assays, plus in vitro FUBI cleavage with purified enzyme and CRISPRi/RNAi","pmids":["33852194","34318747"],"confidence":"High","gaps":["How DUB, SUMO ligase, and protease activities are coordinated unknown","Structural basis for FUBI/ubiquitin discrimination not yet shown"]},{"year":2023,"claim":"Consolidated USP36 as a master nucleolar SUMO ligase across rRNA and miRNA processing machinery and provided structural explanation for its dual ubiquitin/FUBI specificity.","evidence":"Site-specific SUMOylation of EXOSC10 (K583) and DGCR8 with RNA-binding rescue, ALKBH5 deubiquitination in glioma models, and crystal structures of USP36 with ubiquitin and FUBI","pmids":["36912080","36950067","36239338","37443395"],"confidence":"High","gaps":["Determinants selecting between SUMOylation versus deubiquitination of a given substrate unclear","Physiological hierarchy of substrates undefined"]},{"year":2024,"claim":"Expanded the catalytic-dependent substrate network into cancer and disease signaling, with C131A mutants confirming catalytic requirement and Las1L K565 SUMOylation linking USP36 to ITS2 processing.","evidence":"siRNA DUB screens, Co-IP, linkage-specific ubiquitination assays, C131A catalytic mutants, in vivo xenograft and cardiomyopathy mouse models, and site-specific SUMOylation rescue","pmids":["39215346","38307305","39356143","38876304","39343961"],"confidence":"High","gaps":["Tissue-specific selection among many candidate substrates unresolved","Whether disease phenotypes depend on DUB vs SUMO activity often untested"]},{"year":2025,"claim":"Genetically separated catalytic from non-catalytic functions and identified post-translational regulation of USP36 itself.","evidence":"Drosophila CRISPR knock-in of catalytically inactive dUSP36 (viable but sterile) and metabolite-driven methylmalonylation of USP36 at K499 inhibiting its activity","pmids":["40646716","41398045"],"confidence":"High","gaps":["Identity of catalytic-independent functions at the molecular level unknown","Generality of metabolic regulation across cell types untested"]},{"year":null,"claim":"How USP36 integrates its three activities (deubiquitination, SUMO ligation, FUBI/ubiquitin protease) and selects among its large substrate set in a given physiological or disease context remains unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["No unifying model for substrate/activity selection","Mammalian catalytic-independent functions uncharacterized","Mechanism coupling nucleolar localization to non-nucleolar substrates unclear"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,5,7,12,19,23,25]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[2,14,20]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[13,18,22,27]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[13,18,27]}],"localization":[{"term_id":"GO:0005730","term_label":"nucleolus","supporting_discovery_ids":[0,1,5,13]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,8]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[0,7,13,14,22]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[0,14,27]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,5,12,19]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[4,11]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[12]}],"complexes":["Drosha-DGCR8 microprocessor (associated)"],"partners":["NPM1","FBL","MYC","DHX33","PRIMPOL","EXOSC10","DGCR8","LAS1L"],"other_free_text":[]}},"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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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":27420,"output_tokens":8563,"usd":0.105353,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":18211,"output_tokens":4188,"usd":0.097878,"stage2_stop_reason":"end_turn"},"total_usd":0.203231,"stage1_batch_id":"msgbatch_01UT1AahFPCAyn2uVweS7A9z","stage2_batch_id":"msgbatch_01HuKWtKNT4Pqj1bc8eEP5RV","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 region and deubiquitinates nucleolar proteins nucleophosmin/B23 and fibrillarin, stabilizing them against ubiquitylation-mediated proteasomal degradation. RNAi-mediated depletion reduces rRNA transcription and processing, nucleolar morphology, and cell proliferation.\",\n      \"method\": \"Dominant-negative inhibition, RNAi knockdown, co-immunoprecipitation, in vitro deubiquitylation assay, subcellular fractionation/imaging\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods including dominant-negative, RNAi, in vitro deubiquitylation, and functional readouts in a single study\",\n      \"pmids\": [\"19208757\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Nucleophosmin/B23 recruits USP36 to nucleoli by interacting with a specific basic amino acid motif (RGKEKKIKKFKREKRR) in USP36's C-terminal region. Knockdown of nucleophosmin/B23 reduces nucleolar USP36 levels and elevates fibrillarin ubiquitylation.\",\n      \"method\": \"Nucleolar localization signal mapping, Co-IP, RNAi knockdown, ubiquitylation assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, domain mapping, and functional consequence shown in single study with multiple orthogonal methods\",\n      \"pmids\": [\"19679658\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"USP36 possesses deubiquitinase activity (cleaves ubiquitin from substrates), contains a PEST motif, and is itself polyubiquitinated.\",\n      \"method\": \"RT-PCR cloning, ubiquitin-cleavage activity assay, immunoprecipitation, structural domain analysis\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — in vitro DUB activity assay and Co-IP, single lab, single study\",\n      \"pmids\": [\"15809067\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"USP36 interacts with mitochondrial antioxidant enzyme SOD2, reduces its polyubiquitination, and stabilizes it against proteasomal degradation, extending its half-life.\",\n      \"method\": \"2D-gel electrophoresis, MALDI-TOF/MS identification, co-immunoprecipitation, yeast two-hybrid, ubiquitination assay\",\n      \"journal\": \"Journal of cellular biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP plus yeast two-hybrid plus in vivo ubiquitination assay, single lab\",\n      \"pmids\": [\"21268071\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"USP36 (dUsp36) loss of function in Drosophila and human cells activates p62/SQSTM1-dependent selective autophagy of ubiquitinated cargo; dUSP36 loss leads to nuclear accumulation of ubiquitinated proteins (including histone H2B) and cytoplasmic ubiquitinated aggregates eliminated by autophagy. dUSP36 is not part of the TOR signaling pathway.\",\n      \"method\": \"Drosophila loss-of-function genetics, RNAi in human cells, p62/SQSTM1 requirement epistasis, fluorescence imaging of ubiquitin aggregates\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis in Drosophila plus RNAi in human cells, conservation demonstrated, negative TOR epistasis established\",\n      \"pmids\": [\"22622177\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"USP36 is a nucleolar deubiquitinase for c-Myc: it interacts with and deubiquitinates c-Myc in cells and in vitro, stabilizing it against SCF(Fbw7)-mediated degradation in the nucleolus. USP36 interacts with nucleolar Fbw7γ but not nucleoplasmic Fbw7α, yet abolishes c-Myc degradation by both. USP36 is also a c-Myc target gene, forming a positive feedback loop.\",\n      \"method\": \"Co-immunoprecipitation, in vitro deubiquitination assay, knockdown, c-Myc stability assay, isoform-specific interaction mapping\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro deubiquitination plus cellular Co-IP plus epistasis with Fbw7 isoforms, multiple orthogonal approaches\",\n      \"pmids\": [\"25775507\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"USP36 binds Nedd4-2 and regulates its association with TrkA neurotrophin receptor indirectly (USP36 does not directly deubiquitinate TrkA). Depletion of USP36 increases TrkA–Nedd4-2 complex formation, enhancing TrkA ubiquitination; USP36 overexpression disrupts this complex. USP36 also interferes with Nedd4-2-dependent regulation of Kv7.2/3 channels.\",\n      \"method\": \"siRNA screen, Co-IP, overexpression/depletion, TrkA ubiquitination assay, PC12 differentiation assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, functional rescue, multiple substrate contexts, single lab\",\n      \"pmids\": [\"27445338\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"USP36 deubiquitinates and stabilizes the DEAH-box RNA helicase DHX33 in the nucleolus. Usp36 knockout in mice is lethal at the morula-to-blastocyst transition. USP36 depletion impairs rRNA synthesis, protein translation, cell proliferation, and induces apoptosis and cell cycle arrest in human cancer cells.\",\n      \"method\": \"Usp36 knockout mouse model, electron microscopy, Northern blot, O-propargyl-puromycin protein synthesis assay, ubiquitination assay, Co-IP\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vivo mouse KO model, multiple orthogonal methods (EM, Northern blot, protein synthesis), mechanistic link to DHX33 via ubiquitination assay\",\n      \"pmids\": [\"29273634\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"USP36 is a histone H2B deubiquitinase: it interacts with H2B and deubiquitinates monoubiquitinated H2B (H2Bub1) in cells and in vitro. USP36 depletion increases H2Bub1 at the p21 locus gene body, inducing p21 expression and inhibiting cell proliferation.\",\n      \"method\": \"Co-IP, in vitro deubiquitination assay, overexpression, knockdown, ChIP at p21 locus\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro DUB assay plus ChIP functional linkage, single lab\",\n      \"pmids\": [\"29274341\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"USP36 controls cellular localization of CHD7; in neuroblastoma, lncRNAs CASC15/NBAT1 modulate USP36 localization, which in turn regulates CHD7 stability and downstream SOX9 expression.\",\n      \"method\": \"Loss-of-function experiments, co-immunoprecipitation, immunoblotting, localization studies\",\n      \"journal\": \"Cancer cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, cellular imaging, functional knockdown with mechanistic readout, single lab\",\n      \"pmids\": [\"29533783\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"USP36 deubiquitinates and stabilizes PME-1 (protein phosphatase methylesterase 1), thereby promoting ERK and Akt signaling pathways.\",\n      \"method\": \"Co-IP, USP36 knockdown/overexpression, ubiquitination assay, western blot for ERK/Akt activation\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — Co-IP and ubiquitination assay, single lab, single study\",\n      \"pmids\": [\"29577269\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"USP36 knockdown impairs Parkin-dependent mitophagy by reducing mRNA and protein levels of Beclin-1 and ATG14L (without altering nucleolar USP36 localization during mitophagy); restoration of ATG14L rescues mitophagy in USP36-silenced cells.\",\n      \"method\": \"High-content imaging siRNA screen, knockdown, immunofluorescence, western blot, ATG14L overexpression rescue\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA screen plus rescue experiment plus mechanistic pathway linking, single lab\",\n      \"pmids\": [\"31550441\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"USP36 interacts with PrimPol, deubiquitinates K29-linked polyubiquitin chains on PrimPol, and increases PrimPol protein stability in response to DNA replication stress. USP36 is itself deubiquitinated following replication stress, facilitating its upregulation.\",\n      \"method\": \"Co-IP, mass spectrometry, in vivo and in vitro ubiquitination assays, replication stress assays, cisplatin/olaparib sensitivity\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — Co-IP plus MS plus in vitro ubiquitination assay plus specific linkage identification (K29), multiple orthogonal methods\",\n      \"pmids\": [\"33237263\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"USP36 promotes nucleolar SUMOylation: it interacts with SUMO2 and Ubc9, directly mediates SUMOylation in cells and in vitro, and specifically SUMOylates snoRNP components Nop58, Nhp2, Nop56, and DKC1, promoting their binding to snoRNAs. USP36 knockdown markedly impairs rRNA processing and translation.\",\n      \"method\": \"Overexpression, knockdown, genetic deletion, Co-IP, in vitro SUMOylation assay, snoRNA binding assay, rRNA processing assay\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro SUMOylation reconstitution plus cellular studies plus rRNA processing functional readout, multiple substrates confirmed\",\n      \"pmids\": [\"33852194\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"USP36 cleaves the ribosomal fusion protein FUBI-eS30: purified USP36 cuts FUBI-eS30 in vitro, and USP36 depletion by RNAi or CRISPRi impairs FUBI-eS30 processing and late steps of cytoplasmic 40S maturation including 18S rRNA maturation and recycling of biogenesis factors.\",\n      \"method\": \"Differential affinity purification, RNAi, CRISPRi, in vitro processing assay with purified USP36, rRNA processing analysis\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with purified enzyme plus genetic depletion, orthogonal methods confirming processing role\",\n      \"pmids\": [\"34318747\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"USP36 deubiquitinates and stabilizes DOCK4 (a guanine nucleotide exchange factor); increased USP36-DOCK4 stabilization activates Wnt/β-catenin signaling and promotes epithelial-to-mesenchymal transition in diabetic renal tubular epithelial cells.\",\n      \"method\": \"Co-IP, deubiquitination assay, knockdown/overexpression, Wnt pathway reporter\",\n      \"journal\": \"Frontiers in cell and developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — Co-IP plus deubiquitination assay, single lab, single study\",\n      \"pmids\": [\"33968925\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"USP36 deubiquitinates and stabilizes YAP by blocking K48-linked polyubiquitination, thereby promoting Hippo/YAP signaling in esophageal squamous carcinoma.\",\n      \"method\": \"siRNA DUB screening, Co-IP, ubiquitination assay, knockdown/overexpression, western blot\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — Co-IP and ubiquitination assay, single lab, single study\",\n      \"pmids\": [\"36470870\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"USP36 activates Snail2 deubiquitination in glioblastoma, downstream of PRL1 oncogene; PRL1 promotes EMT and invasion by activating USP36-mediated Snail2 stabilization.\",\n      \"method\": \"Co-IP, ubiquitination assay, knockdown/overexpression, in vitro and in vivo tumorigenesis assays\",\n      \"journal\": \"Frontiers in oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — Co-IP plus ubiquitination assay, single lab, single study\",\n      \"pmids\": [\"35111679\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"USP36 interacts with EXOSC10 (nucleolar RNA exosome subunit) and mediates its SUMOylation at K583. K583 SUMOylation is required for EXOSC10 binding to pre-rRNAs; K583R mutant fails to rescue rRNA processing and cell growth defects upon EXOSC10 knockdown. USP36 does not significantly regulate EXOSC10 protein levels.\",\n      \"method\": \"Co-IP, in vitro SUMOylation assay, mutant rescue, rRNA processing assay, RNA binding assay\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro SUMOylation plus site-specific mutagenesis plus rescue experiments, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"36912080\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"USP36 deubiquitinates and stabilizes ALKBH5, regulating ALKBH5-mediated gene expression in glioblastoma. Depletion of USP36 impairs glioma stem cell self-renewal, proliferation, and sensitizes cells to temozolomide.\",\n      \"method\": \"DUB siRNA library screen, mass spectrometry, Co-IP, in vivo and in vitro ubiquitination assays, neurosphere formation, intracranial tumor growth\",\n      \"journal\": \"Neuro-oncology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — MS-identified interaction, Co-IP, in vitro ubiquitination, in vivo tumor model, multiple orthogonal methods\",\n      \"pmids\": [\"36239338\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Crystal structures of USP36 in complex with ubiquitin and Fubi reveal the substrate recognition mechanism for dual ubiquitin/Fubi cleavage activity. USP16 was also identified as having dual ubiquitin/Fubi cleavage activity by chemoproteomics; both USP16 and USP36 participate in Fubi-S30 maturation.\",\n      \"method\": \"Crystal structure determination, chemoproteomics, Fubi C-terminal hydrolase activity assay, substrate specificity analysis\",\n      \"journal\": \"Nature chemical biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structures of USP36 with both substrates, biochemical activity measurements, mechanistic explanation of specificity\",\n      \"pmids\": [\"37443395\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Ribotoxic stress activates JNK, which in turn activates USP36, leading to stabilization of Snail1 in the nucleolus. Nucleolar Snail1 facilitates ribosome biogenesis and tumor cell survival, and mediates solid tumor resistance to homoharringtonine (HHT).\",\n      \"method\": \"Co-IP, knockdown, JNK inhibition, HHT treatment, ribosome biogenesis assay, combination therapy in vivo\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus epistasis (JNK-USP36-Snail1) plus in vivo rescue, single lab\",\n      \"pmids\": [\"37833415\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"USP36 associates with the Drosha-DGCR8 microprocessor complex and mediates DGCR8 SUMOylation by SUMO2. This SUMOylation promotes DGCR8 binding to pri-miRNAs without affecting DGCR8 levels or the Drosha-DGCR8 complex. USP36 knockdown attenuates pri-miRNA processing and reduces mature miRNA levels.\",\n      \"method\": \"Co-IP, in vitro/in vivo SUMOylation assay, SUMO site mapping, RNA immunoprecipitation, knockdown, cell proliferation assay\",\n      \"journal\": \"Cancer research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro SUMOylation, RNA binding assay, functional rescue with SUMOylation mutant, multiple orthogonal approaches\",\n      \"pmids\": [\"36950067\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP36 binds PARP1 and mediates its deubiquitination, increasing PARP1 protein stability in cardiomyocytes exposed to doxorubicin. The catalytically inactive C131A mutant of USP36 fails to stabilize PARP1, confirming catalytic dependence. Cardiac knockdown of USP36 (rAAV9-shUSP36) preserved cardiac function in a doxorubicin-induced cardiomyopathy mouse model.\",\n      \"method\": \"Co-IP, ubiquitination assay, catalytic mutant (C131A), rAAV9 in vivo knockdown, cardiac function assessment\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — catalytic mutant validation plus in vivo mouse model, multiple orthogonal methods\",\n      \"pmids\": [\"38307305\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP36 deubiquitinates survivin (removing K48-linked chains) and cIAP1 (removing K11-linked chains), stabilizing both anti-apoptotic proteins. USP36 disrupts XIAP-SMAC complex formation and promotes RIP1 ubiquitination, thereby inhibiting both intrinsic and extrinsic apoptosis in colorectal cancer cells.\",\n      \"method\": \"siRNA gene silencing, Co-IP, ubiquitin linkage-specific assays, apoptosis assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus linkage-specific ubiquitination assays, functional apoptosis readouts, single lab\",\n      \"pmids\": [\"38876304\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP36 deubiquitinates ERα, inhibiting K48-linked polyubiquitination and stabilizing ERα protein; the catalytically inactive C131A mutant fails to promote breast cancer progression through ERα signaling. USP36 knockdown destabilizes the tamoxifen-resistant ERα mutant (Y537S).\",\n      \"method\": \"DUB siRNA library screen, Co-IP, ubiquitination assay, catalytic mutant (C131A), xenograft model, RNA sequencing\",\n      \"journal\": \"Journal of experimental & clinical cancer research : CR\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — siRNA screen, catalytic mutant validation, in vivo xenograft, multiple orthogonal approaches\",\n      \"pmids\": [\"39215346\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP36 deubiquitinates and stabilizes RBM28 at K162; upregulated RBM28 binds p53 and suppresses its transcriptional activity, inactivating the p53 signaling pathway in colorectal cancer.\",\n      \"method\": \"Co-IP, ubiquitination assay (site-specific K162), knockdown/overexpression, p53 transcription reporter\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with site-specific mutagenesis plus functional epistasis, single lab\",\n      \"pmids\": [\"39343961\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP36 SUMOylates Las1L at K565; K565 SUMOylation is essential for ITS2 pre-rRNA processing but not for Las1L protein levels or Las1L-Nol9 complex formation. USP36 also deubiquitinates Las1L and Nol9, regulating their stability.\",\n      \"method\": \"Co-IP, in vitro SUMOylation assay, K565R mutant rescue, ITS2 processing assay, ubiquitination assay\",\n      \"journal\": \"Cancer research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro SUMOylation, site-specific mutagenesis with functional rescue, multiple substrates, orthogonal DUB assay\",\n      \"pmids\": [\"39356143\"],\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 degradation, impairing osteogenic differentiation. Overexpression of WDR5 rescues the differentiation defects in USP36-deficient osteoblasts.\",\n      \"method\": \"Co-IP, ubiquitination assay, knockdown, Alizarin red staining, WDR5 rescue overexpression\",\n      \"journal\": \"Journal of orthopaedic surgery and research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — Co-IP plus ubiquitination assay plus rescue, single lab, single study\",\n      \"pmids\": [\"39152465\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"A germline USP36 variant (rs3744797, K814N) stabilizes MLLT3 via deubiquitination in the nucleolus, activating HIF1α and Snai downstream signaling and conferring resistance to EGFR-TKIs in non-small cell lung cancer.\",\n      \"method\": \"m6A-genome-wide-variant analysis, Co-IP, ubiquitination assay, in vitro/in vivo functional assays, patient cohort\",\n      \"journal\": \"Clinical cancer research : an official journal of the American Association for Cancer Research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus ubiquitination assay plus in vivo model, single lab, single study\",\n      \"pmids\": [\"38261467\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"MMA (methylmalonic acid) induces methylmalonylation of USP36 at K499, inhibiting USP36-mediated deubiquitination and SUMOylation of SUFU, thereby promoting GLI1 expression and Hedgehog pathway activation in clear-cell renal cell carcinoma macrophages.\",\n      \"method\": \"Metabolomic profiling, site-specific modification analysis, ubiquitination and SUMOylation assays, in vitro and in vivo functional experiments\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple biochemical assays plus in vivo validation, novel PTM on USP36 itself, single lab\",\n      \"pmids\": [\"41398045\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In Drosophila, catalytically inactive dUSP36 (CRISPR knock-in) allows survival to adulthood with minor growth defects, demonstrating that the deubiquitinating activity is dispensable for cell growth but essential for spermatogenesis, revealing both catalytic-dependent and catalytic-independent mechanisms.\",\n      \"method\": \"CRISPR/Cas9 catalytic mutation knock-in, null mutant comparison, viability and fertility assays\",\n      \"journal\": \"Genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — CRISPR knock-in of catalytic mutant with null comparison in whole organism, rigorous genetic dissection of catalytic vs. non-catalytic functions\",\n      \"pmids\": [\"40646716\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP36 stabilizes APEX1 by removing K48-linked ubiquitin chains, suppressing ferroptosis in melanoma cells. USP36 knockdown sensitizes xenograft tumors to ferroptosis, and APEX1 knockdown abolishes USP36's anti-ferroptotic effect.\",\n      \"method\": \"Co-IP, ubiquitination assay (K48-specific), overexpression/knockdown, erastin treatment, xenograft model\",\n      \"journal\": \"Clinical and experimental medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — Co-IP plus K48-specific ubiquitination assay plus rescue, single lab, single study\",\n      \"pmids\": [\"41649582\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP36 functions as a SUMO ligase for GNL3 (nucleolar GTP-binding protein), while SENP3 deSUMOylates GNL3. GNL3 SUMOylation is required for interaction with the BLM-DNA2 complex and DNA end resection in homologous recombination repair.\",\n      \"method\": \"Co-IP, in vitro SUMOylation assay, CRISPR/RNAi knockdown, DNA end resection assay, epistasis with SENP3\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro SUMOylation plus functional epistasis, preprint not yet peer-reviewed\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP36 stabilizes KIF2C by removing K48-linked ubiquitin chains, suppressing ferroptosis in breast cancer cells. USP36-deficient xenograft tumors show reduced proliferation and increased ferroptosis.\",\n      \"method\": \"Co-IP, K48-specific ubiquitination assay, knockdown/overexpression, erastin ferroptosis assay, xenograft model\",\n      \"journal\": \"Biochemical pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — Co-IP plus ubiquitination assay, in vivo xenograft, single lab, single study\",\n      \"pmids\": [\"40744233\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP36 deubiquitinates and stabilizes AR (androgen receptor) in prostate cancer cells under oxidative stress (H2O2); USP36–AR interaction is induced by H2O2 and knockdown of USP36 abolishes H2O2-induced activation of the AR-PSA pathway.\",\n      \"method\": \"TurboID proximity labeling plus mass spectrometry, Co-IP, ubiquitination assay, dual-luciferase reporter, knockdown\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — proximity labeling MS for discovery, Co-IP for validation, functional pathway reporter, single lab\",\n      \"pmids\": [\"41298501\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"FBL (fibrillarin) acts as a carrier regulating the balance between BMI1-mediated H2A monoubiquitination and USP36-mediated H2Aub deubiquitination in nucleolar lysosome-like structures during glucose starvation; USP36, FBL, Midnolin, and BMI1 form a complex responsible for H2Aub degradation.\",\n      \"method\": \"Protein interaction screening, Co-IP, knockdown, cell cycle and viability assays\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — Co-IP with complex formation claim, preprint not peer-reviewed, single lab\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"USP36 is a nucleolar deubiquitinating enzyme (and SUMO ligase) that localizes to the nucleolus via a C-terminal basic NLS recruited by nucleophosmin/B23, where it stabilizes diverse substrates (c-Myc, nucleophosmin/B23, fibrillarin, DHX33, SOD2, PrimPol, ALKBH5, and others) by removing polyubiquitin chains, while simultaneously acting as a non-canonical SUMO E3 ligase for multiple nucleolar proteins (Nop58, Nhp2, EXOSC10, DGCR8, Las1L, GNL3), thereby coordinating rRNA processing, ribosome biogenesis, miRNA biogenesis, DNA repair, and selective autophagy; additionally, it functions through both catalytic-dependent and catalytic-independent mechanisms in vivo, as demonstrated by the dispensability of its DUB activity for cell growth (but not spermatogenesis) in Drosophila.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"USP36 is a nucleolar deubiquitinating enzyme that coordinates ribosome biogenesis, rRNA processing, and the stability of numerous nucleolar and signaling proteins [#0, #7]. It is recruited to the nucleolus through a C-terminal basic motif that is bound by nucleophosmin/B23, and there it removes polyubiquitin from substrates such as fibrillarin and nucleophosmin to protect them from proteasomal degradation, sustaining rRNA transcription/processing and cell proliferation [#0, #1]. A defining activity is stabilization of the oncoprotein c-Myc: USP36 deubiquitinates c-Myc in the nucleolus, antagonizing SCF(Fbw7)-mediated turnover, and is itself a c-Myc target gene, forming a positive feedback loop [#5]. Beyond deubiquitination, USP36 is a non-canonical SUMO E3 ligase that interacts with SUMO2/Ubc9 and SUMOylates snoRNP and processing factors (Nop58, Nhp2, Nop56, DKC1, EXOSC10 at K583, Las1L at K565, DGCR8) to promote their RNA binding and rRNA/miRNA processing, often without altering substrate abundance [#13, #18, #22, #27]. USP36 also processes ribosomal precursors directly: it cleaves the FUBI-eS30 fusion to drive late cytoplasmic 40S maturation, a dual ubiquitin/FUBI hydrolase activity explained by crystal structures of USP36 bound to ubiquitin and FUBI [#14, #20]. Through deubiquitination it stabilizes a broad set of substrates linking it to DNA replication stress and repair (PrimPol via K29 chains, ALKBH5), histone H2B/H2A regulation, and multiple oncogenic and stress pathways (YAP, ERα, AR, survivin/cIAP1, RBM28-p53), with catalytic activity confirmed by the C131A inactive mutant in several contexts [#12, #19, #8, #16, #25, #24, #26, #23]. Usp36 knockout is embryonic-lethal in mice, and in Drosophila its deubiquitinating activity is dispensable for viability and cell growth but essential for spermatogenesis, establishing both catalytic-dependent and catalytic-independent functions [#7, #31].\",\n  \"teleology\": [\n    {\n      \"year\": 2005,\n      \"claim\": \"Established USP36 as a bona fide deubiquitinase, providing the biochemical foundation for all subsequent substrate work.\",\n      \"evidence\": \"RT-PCR cloning, ubiquitin-cleavage activity assay, and domain analysis identifying a PEST motif and self-ubiquitination\",\n      \"pmids\": [\"15809067\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No substrates identified\", \"No cellular localization or pathway context\", \"Single in vitro study\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Defined USP36 as a nucleolar enzyme whose recruitment depends on nucleophosmin/B23 binding a C-terminal basic motif, answering where USP36 acts and how it is targeted.\",\n      \"evidence\": \"NLS mapping, reciprocal Co-IP, RNAi, in vitro deubiquitylation, and rRNA/proliferation readouts in human cells\",\n      \"pmids\": [\"19208757\", \"19679658\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Substrate repertoire beyond fibrillarin/nucleophosmin unknown at this stage\", \"No structural basis for catalysis\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Extended USP36 substrate range beyond the nucleolus by showing it stabilizes mitochondrial SOD2, hinting at functions outside ribosome biogenesis.\",\n      \"evidence\": \"2D-gel/MALDI-TOF MS, yeast two-hybrid, Co-IP, and ubiquitination/half-life assays\",\n      \"pmids\": [\"21268071\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism reconciling nucleolar localization with mitochondrial substrate unclear\", \"Single lab\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Connected USP36 to selective autophagy, showing its loss triggers p62/SQSTM1-dependent clearance of ubiquitinated cargo independent of TOR signaling.\",\n      \"evidence\": \"Drosophila loss-of-function genetics with RNAi confirmation in human cells and epistasis with p62\",\n      \"pmids\": [\"22622177\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct substrates driving aggregate accumulation not defined\", \"Link between nucleolar role and autophagy unclear\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Identified c-Myc as a key USP36 substrate and a c-Myc/USP36 positive feedback loop, establishing USP36 as an oncogenic stabilizer that counteracts SCF(Fbw7).\",\n      \"evidence\": \"In vitro deubiquitination, cellular Co-IP, stability assays, and Fbw7 isoform-specific interaction mapping\",\n      \"pmids\": [\"25775507\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether deubiquitination occurs only in the nucleolar compartment unresolved\", \"In vivo tumor relevance not tested here\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Demonstrated USP36 is essential in vivo and stabilizes the rRNA helicase DHX33, linking USP36 to early embryonic viability and translation.\",\n      \"evidence\": \"Usp36 knockout mouse (morula-to-blastocyst lethality), EM, Northern blot, OPP protein synthesis, ubiquitination assays\",\n      \"pmids\": [\"29273634\", \"29274341\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Catalytic dependence of essentiality not dissected\", \"H2B deubiquitination role separate from rRNA functions not integrated\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Revealed a DNA replication stress function by showing USP36 removes K29-linked chains from PrimPol and is itself stabilized after stress.\",\n      \"evidence\": \"Co-IP, MS, in vivo/in vitro ubiquitination assays with linkage specificity, and drug sensitivity assays\",\n      \"pmids\": [\"33237263\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Upstream signal regulating USP36 stabilization not defined\", \"Nucleolar vs nucleoplasmic site of PrimPol action unclear\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Uncovered a second enzymatic mode—non-canonical SUMO E3 ligase activity—and direct FUBI-eS30 processing, redefining USP36 as a dual-activity ribosome biogenesis factor.\",\n      \"evidence\": \"In vitro SUMOylation reconstitution on snoRNP factors, snoRNA-binding and rRNA processing assays, plus in vitro FUBI cleavage with purified enzyme and CRISPRi/RNAi\",\n      \"pmids\": [\"33852194\", \"34318747\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How DUB, SUMO ligase, and protease activities are coordinated unknown\", \"Structural basis for FUBI/ubiquitin discrimination not yet shown\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Consolidated USP36 as a master nucleolar SUMO ligase across rRNA and miRNA processing machinery and provided structural explanation for its dual ubiquitin/FUBI specificity.\",\n      \"evidence\": \"Site-specific SUMOylation of EXOSC10 (K583) and DGCR8 with RNA-binding rescue, ALKBH5 deubiquitination in glioma models, and crystal structures of USP36 with ubiquitin and FUBI\",\n      \"pmids\": [\"36912080\", \"36950067\", \"36239338\", \"37443395\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Determinants selecting between SUMOylation versus deubiquitination of a given substrate unclear\", \"Physiological hierarchy of substrates undefined\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Expanded the catalytic-dependent substrate network into cancer and disease signaling, with C131A mutants confirming catalytic requirement and Las1L K565 SUMOylation linking USP36 to ITS2 processing.\",\n      \"evidence\": \"siRNA DUB screens, Co-IP, linkage-specific ubiquitination assays, C131A catalytic mutants, in vivo xenograft and cardiomyopathy mouse models, and site-specific SUMOylation rescue\",\n      \"pmids\": [\"39215346\", \"38307305\", \"39356143\", \"38876304\", \"39343961\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Tissue-specific selection among many candidate substrates unresolved\", \"Whether disease phenotypes depend on DUB vs SUMO activity often untested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Genetically separated catalytic from non-catalytic functions and identified post-translational regulation of USP36 itself.\",\n      \"evidence\": \"Drosophila CRISPR knock-in of catalytically inactive dUSP36 (viable but sterile) and metabolite-driven methylmalonylation of USP36 at K499 inhibiting its activity\",\n      \"pmids\": [\"40646716\", \"41398045\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of catalytic-independent functions at the molecular level unknown\", \"Generality of metabolic regulation across cell types untested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How USP36 integrates its three activities (deubiquitination, SUMO ligation, FUBI/ubiquitin protease) and selects among its large substrate set in a given physiological or disease context remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No unifying model for substrate/activity selection\", \"Mammalian catalytic-independent functions uncharacterized\", \"Mechanism coupling nucleolar localization to non-nucleolar substrates unclear\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 5, 7, 12, 19, 23, 25]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [2, 14, 20]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [13, 18, 22, 27]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [13, 18, 27]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005730\", \"supporting_discovery_ids\": [0, 1, 5, 13]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [0, 7, 13, 14, 22]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [0, 14, 27]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 5, 12, 19]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [4, 11]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [12]}\n    ],\n    \"complexes\": [\"Drosha-DGCR8 microprocessor (associated)\"],\n    \"partners\": [\"NPM1\", \"FBL\", \"MYC\", \"DHX33\", \"PrimPol\", \"EXOSC10\", \"DGCR8\", \"Las1L\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}