{"gene":"USP35","run_date":"2026-06-11T09:02:06","timeline":{"discoveries":[{"year":2018,"finding":"USP35 binds to and deubiquitinates Aurora B, inhibiting APC/C-CDH1-mediated proteasomal degradation of Aurora B to maintain its steady-state levels during mitosis. Loss of USP35 decreases phosphorylation of histone H3-Ser10 (an Aurora B substrate) and causes mitotic defects including cytokinesis failures. FoxM1 transcription factor promotes expression of USP35 during the cell cycle.","method":"Co-immunoprecipitation, ubiquitination assay, siRNA knockdown, phosphorylation assay, FoxM1 ChIP/overexpression","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, ubiquitination assay, defined cellular phenotype, APC/C epistasis, replicated in companion paper PMID:29764563","pmids":["29449677","29764563"],"is_preprint":false},{"year":2015,"finding":"USP35 acts as a deubiquitinase that directly stabilizes ABIN-2 (TNFAIP3 interacting protein 2) by promoting its deubiquitination, thereby inhibiting TNFα-induced NF-κB activation. miR let-7a positively regulates USP35 expression.","method":"Co-immunoprecipitation, ubiquitination assay, NF-κB reporter assay, overexpression/knockdown, miRNA manipulation","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus ubiquitination assay plus functional NF-κB readout, single lab","pmids":["26348204"],"is_preprint":false},{"year":2018,"finding":"USP35 has two alternatively spliced isoforms with distinct intracellular localizations and functions: isoform 1 localizes to the cytoplasm/nucleus and acts as an anti-apoptotic factor inhibiting staurosporine- and TRAIL-induced apoptosis; isoform 2 is an integral membrane protein of the endoplasmic reticulum and lipid droplets, and its deregulation causes ER stress and cell death, likely through disruption of lipid homeostasis.","method":"Fluorescence microscopy localization, isoform-specific knockdown/overexpression, apoptosis assays, ER stress markers, subcellular fractionation","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (imaging, fractionation, functional assays) establishing distinct localization and functional consequences for each isoform","pmids":["29685892"],"is_preprint":false},{"year":2020,"finding":"USP35 directly deubiquitinates STING to inactivate it, suppressing the STING-TBK1-IRF3 signaling pathway and downstream type I interferon production. Activated STING promotes its own binding to USP35 in a STING phosphorylation-dependent manner.","method":"Co-immunoprecipitation, ubiquitination assay, USP35 silencing with STING pathway readout (IRF3/TBK1 phosphorylation, IFN expression), phosphorylation-dependent binding assay","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, ubiquitination assay, defined pathway epistasis, phosphorylation-dependent interaction, functional cytokine readout","pmids":["32678307"],"is_preprint":false},{"year":2021,"finding":"USP35 directly interacts with ferroportin (FPN) and stabilizes it through deubiquitinase activity, preventing its proteasomal degradation. This maintains intracellular iron homeostasis and suppresses ferroptosis in lung cancer cells.","method":"Co-immunoprecipitation, ubiquitination assay, USP35 knockdown/overexpression with ferroptosis markers (lipid ROS, iron levels), xenograft model","journal":"Clinical and translational medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus ubiquitination assay plus functional ferroptosis readout, single lab","pmids":["33931967"],"is_preprint":false},{"year":2021,"finding":"USP35 directly interacts with and stabilizes RRBP1 (ribosome-binding protein 1, an ER membrane protein) by preventing proteasome-dependent degradation via deubiquitination. USP35-mediated RRBP1 stabilization alleviates ER stress-induced apoptosis in NSCLC cells.","method":"iTRAQ proteomics to identify interactome, Co-immunoprecipitation, ubiquitination assay, knockdown/overexpression with ER stress and apoptosis readouts","journal":"Molecular oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus ubiquitination assay plus functional ER stress readout, single lab","pmids":["34618999"],"is_preprint":false},{"year":2021,"finding":"USP35 stabilizes survivin protein in an enzymatic activity-dependent manner through direct interaction and deubiquitination. USP35 also regulates stability of other chromosomal passenger complex (CPC) components Aurora B and Borealin. The Drosophila homolog DUBAI similarly regulates Deterin (Drosophila survivin ortholog), indicating evolutionary conservation.","method":"DUB expression library screen (68 DUBs), Co-immunoprecipitation, ubiquitination assay, activity-dead mutant, Drosophila functional assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — library screen plus Co-IP plus ubiquitination assay plus cross-species validation, single lab","pmids":["34438346"],"is_preprint":false},{"year":2022,"finding":"USP35 directly interacts with and stabilizes BIRC3 through deubiquitination of K48-linked polyubiquitin chains, protecting it from proteasomal degradation. USP35-mediated BIRC3 stabilization alleviates cisplatin-induced apoptosis in NSCLC cells.","method":"Co-immunoprecipitation, ubiquitination assay (K48-linkage specific), knockdown/overexpression with apoptosis readout","journal":"Laboratory investigation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus K48-specific ubiquitination assay plus functional apoptosis readout, single lab","pmids":["35022505"],"is_preprint":false},{"year":2021,"finding":"USP35 interacts with estrogen receptor α (ERα), deubiquitinates it, and enhances ERα protein stability. USP35 also interacts with ERα at estrogen response element-containing DNA regions to enhance ERα transcriptional activity. AKT phosphorylates USP35 at Serine613, which promotes USP35 nuclear translocation and ERα transcriptional activity.","method":"Co-immunoprecipitation, ubiquitination assay, ChIP, AKT kinase assay with site-specific mutagenesis (S613), nuclear translocation imaging, knockdown/overexpression in vitro and in vivo","journal":"Cell death & disease","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods: Co-IP, ubiquitination assay, ChIP, phosphorylation mapping with mutagenesis, nuclear translocation, in vivo validation","pmids":["34131114"],"is_preprint":false},{"year":2022,"finding":"USP35 directly deubiquitinates and stabilizes BRPF1 protein. Accumulated BRPF1 binds to the SREBP2 promoter to activate SREBP2 transcriptional activity, promoting expression of mevalonate (MVA) metabolism genes in prostate cancer cells.","method":"Co-immunoprecipitation, ubiquitination assay, ChIP (BRPF1 on SREBP2 promoter), knockdown/overexpression with mevalonate pathway readout, in vivo xenograft","journal":"Cell death discovery","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus ubiquitination assay plus ChIP epistasis, single lab","pmids":["36357379"],"is_preprint":false},{"year":2023,"finding":"USP35 stabilizes multiple IAP family members (including BIRC3 and others) in an enzymatic activity-dependent manner, protecting renal clear cell carcinoma cells from apoptosis. USP35 also catalyzes deubiquitylation of NRF2, maintaining NRF2 protein levels and protecting cells from ferroptosis.","method":"Biochemical characterization (ubiquitination assay, activity-dependent stabilization), transcriptomic analysis of NRF2 downstream targets, knockdown with apoptosis and ferroptosis readouts, xenograft","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — enzymatic activity-dependent stabilization assay plus transcriptomics plus functional phenotypes, single lab","pmids":["37173391"],"is_preprint":false},{"year":2023,"finding":"USP35 directly deubiquitinates and stabilizes PKM2 (pyruvate kinase M2 isoform), protecting it from ubiquitin-mediated degradation and maintaining the Warburg effect (aerobic glycolysis) in hepatocellular carcinoma cells.","method":"Co-immunoprecipitation, ubiquitination assay, Western blot of PKM2 stability, knockdown with glycolysis readout","journal":"International journal of oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus ubiquitination assay plus functional glycolysis readout, single lab","pmids":["37594129"],"is_preprint":false},{"year":2023,"finding":"USP35 directly interacts with and deubiquitinates FUCA1 (α-L-fucosidase 1), stabilizing it from proteasomal degradation. The USP35-FUCA1 axis promotes upregulation of NER components (XPC, XPA, ERCC1), providing a mechanistic basis for platinum resistance in colorectal cancer.","method":"Co-IP followed by mass spectrometry, Co-IP validation, ubiquitination assay, knockdown/overexpression with NER component readout, in vitro and in vivo functional assays","journal":"Oncogenesis","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP/MS substrate identification plus ubiquitination assay plus downstream pathway readout, single lab","pmids":["36864055"],"is_preprint":false},{"year":2023,"finding":"USP35 interacts with and stabilizes ABHD17C by inhibiting its ubiquitin-proteasome-mediated degradation, leading to activation of the PI3K/AKT signaling pathway in hepatocellular carcinoma cells.","method":"Co-immunoprecipitation, ubiquitination assay, USP35 knockdown with PI3K/AKT pathway readout, ABHD17C rescue experiment, xenograft","journal":"Cell death discovery","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus ubiquitination assay plus rescue experiment plus in vivo validation, single lab","pmids":["37993419"],"is_preprint":false},{"year":2019,"finding":"Genetic suppression or ablation of usp35 (zebrafish ortholog) ameliorates ciliopathy defects caused by BBS4 loss, including impaired convergent extension, renal tubule convolution, and retinal degeneration, with concomitant clearance of β-catenin and rhodopsin. This places USP35 as a negative regulator of proteasome-dependent degradation of ciliopathy-relevant signaling effectors.","method":"Genome-wide RNAi suppressor screen (in BBS4-deficient background), transient and stable zebrafish genetic models, convergent extension assay, renal and retinal phenotype scoring","journal":"JCI insight","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genome-wide epistasis screen plus multiple zebrafish model validation with defined phenotypic readouts, single lab","pmids":["31723061"],"is_preprint":false},{"year":2024,"finding":"USP35 deubiquitinates and stabilizes BRD4. BRD4 then mediates USP35-induced upregulation of SLC7A11, inhibiting ferroptosis and promoting growth of ER+ breast cancer cells. BRD4 inhibitor (+)-JQ-1 inhibits USP35-enhanced tumorigenesis in vivo.","method":"Co-immunoprecipitation, ubiquitination assay, BRD4 knockdown/inhibitor rescue, SLC7A11 expression readout, ferroptosis markers, in vivo xenograft with JQ-1 treatment","journal":"Communications biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus ubiquitination assay plus downstream pathway readout plus in vivo pharmacological validation, single lab","pmids":["39820080"],"is_preprint":false},{"year":2024,"finding":"USP35 interacts with and deubiquitinates VEGFA, stabilizing VEGFA protein levels. FUS RNA-binding protein interacts with USP35 mRNA to promote its mRNA stability, thereby positively regulating VEGFA expression in NSCLC cells.","method":"Co-IP assay, ubiquitination assay, FUS knockdown/overexpression with USP35 mRNA stability readout, functional angiogenesis assay, in vivo xenograft","journal":"General physiology and biophysics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus ubiquitination assay plus mRNA stability assay, single lab","pmids":["38953570"],"is_preprint":false},{"year":2024,"finding":"USP35 directly interacts with and stabilizes PFK-1 (phosphofructokinase-1) via deubiquitination, reducing its ubiquitin-mediated degradation. USP35-mediated PFK-1 stabilization promotes glycolysis and cell proliferation in breast cancer cells.","method":"Co-immunoprecipitation, ubiquitination assay, Seahorse metabolic assay (ECAR/OCR), knockdown/overexpression with glycolysis readout, in vivo xenograft","journal":"American journal of physiology. Cell physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus ubiquitination assay plus functional metabolic readout, single lab","pmids":["39714773"],"is_preprint":false},{"year":2024,"finding":"USP35 interacts with and stabilizes FUNDC1 (a mitophagy receptor) by deubiquitination. USP35-mediated FUNDC1 stabilization enhances autophagy/mitophagy and reduces mitochondrial ROS in OGD/R-injured neuronal cells.","method":"Co-immunoprecipitation, ubiquitination assay, USP35 overexpression with FUNDC1 protein stability and autophagy readout, FUNDC1 knockdown rescue experiment","journal":"Communications biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus ubiquitination assay plus rescue experiment, single lab","pmids":["39406943"],"is_preprint":false},{"year":2024,"finding":"USP35 deubiquitinates and stabilizes NRF2 in esophageal cancer cells, maintaining NRF2 protein stability through direct interaction and enzymatic deubiquitination.","method":"Co-immunoprecipitation, ubiquitination assay, USP35 knockdown with NRF2 protein level readout","journal":"Open life sciences","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP plus ubiquitination assay, single lab, no orthogonal methods","pmids":["39156988"],"is_preprint":false},{"year":2025,"finding":"USP35 interacts with MAVS and removes K63-linked polyubiquitin chains from MAVS, thereby inhibiting viral-induced MAVS-TBK1-IRF3 pathway activation and downstream inflammatory gene expression.","method":"Co-immunoprecipitation, K63-linkage-specific ubiquitination assay, USP35 depletion with MAVS pathway readout (TBK1/IRF3 activation, cytokine expression), in vivo anti-tumor immunity assay","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus linkage-specific ubiquitination assay plus functional pathway readout, single lab","pmids":["40016186"],"is_preprint":false},{"year":2025,"finding":"USP35 directly deubiquitinates and stabilizes STING in gastric cancer cells, activating the HIF-1α/FAK pathway to promote energy metabolism reprogramming and adhesion to peritoneal mesothelial cells. USP35 is also secreted via exosomes from gastric cancer cells and promotes mesothelial-mesenchymal transformation (MMT) of peritoneal mesothelial cells.","method":"Co-immunoprecipitation, ubiquitination assay, pathway readout (HIF-1α/FAK), exosome isolation and co-culture functional assay","journal":"Cell death & disease","confidence":"Low","confidence_rationale":"Tier 3 / Weak — mechanistic claims based on Co-IP and pathway readouts but limited methodological detail in abstract, single lab","pmids":["41372134"],"is_preprint":false},{"year":2025,"finding":"USP35 interacts with and stabilizes GASC1 (gene amplified in squamous cell carcinoma 1) by reducing GASC1 ubiquitination, leading to upregulation of ROCK2 expression and promotion of HCC cell proliferation.","method":"Co-immunoprecipitation, ubiquitination assay, knockdown/overexpression with ROCK2 expression and proliferation readout","journal":"Translational oncology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP plus ubiquitination assay, single lab, limited detail","pmids":["40424935"],"is_preprint":false},{"year":2026,"finding":"USP35 deubiquitinates and stabilizes ID3 at its N-terminal lysine residues K2 and K30, enhancing ID3 transcriptional activity and resulting in elevated PD-L1 expression in colorectal cancer, facilitating immune escape. The USP35 inhibitor IU1 promotes ID3 ubiquitination and reduces PD-L1 expression.","method":"Co-immunoprecipitation, ubiquitination assay with site-specific mutagenesis (K2, K30), PD-L1 expression readout, pharmacological inhibition with IU1, immune co-culture assay","journal":"Advanced science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus site-specific mutagenesis plus functional immune readout plus pharmacological validation, single lab","pmids":["41486422"],"is_preprint":false},{"year":2026,"finding":"USP35 directly deubiquitinates and stabilizes CDCA8 (a cell cycle regulator) and also deubiquitinates PD-L1 to prevent its proteasomal degradation, thereby promoting NSCLC cell proliferation/invasion and immune evasion respectively.","method":"Co-immunoprecipitation, ubiquitination assay, cycloheximide chase, PBMC co-culture cytotoxicity assay, knockdown with proliferation and immune readouts","journal":"Combinatorial chemistry & high throughput screening","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus ubiquitination assay plus cycloheximide chase plus functional immune readout, single lab","pmids":["42002994"],"is_preprint":false},{"year":2026,"finding":"USP35 deubiquitinates MDM4, leading to its autophagic degradation (rather than proteasomal stabilization), which activates the P53 signaling pathway and mediates endothelial ferroptosis in cisplatin-induced acute kidney injury. Kidney-specific USP35 knockout reduces AKI and endothelial ferroptosis.","method":"Transcriptomic and single-cell analyses, renal epithelial cell and animal models, kidney-specific USP35 knockout, ubiquitination assay, P53 pathway readout, ferroptosis markers","journal":"Cell death discovery","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — tissue-specific KO model plus ubiquitination assay plus pathway readout, single lab","pmids":["42185243"],"is_preprint":false},{"year":2025,"finding":"HERC2 binds to USP35 via a conserved 'DxDKDxD' motif recognized by the RLD2 domain of HERC2, identifying USP35 as a HERC2-interacting protein relevant to brain development.","method":"Quantitative binding assays, X-ray crystallography, sequence conservation analysis","journal":"bioRxiv (preprint)","confidence":"Low","confidence_rationale":"Tier 1 / Weak — structural and binding data establish interaction but no functional consequence for USP35 itself was characterized; USP35 is one of several listed HERC2 partners","pmids":["bio_10.1101_2025.09.16.670041"],"is_preprint":true}],"current_model":"USP35 is a deubiquitinating enzyme (DUB) with a conserved Cys-His-Asp catalytic triad that removes ubiquitin from a broad range of substrates to prevent their proteasomal degradation; its two alternatively spliced isoforms localize to distinct compartments (cytoplasm/nucleus vs. ER/lipid droplets) with distinct functions; established substrates include Aurora B (blocking APC/C-CDH1-mediated degradation during mitosis), STING and MAVS (negative regulators of innate immune signaling), ERα, BRD4, NRF2, BIRC3/IAP family members, survivin/CPC components, FPN (ferroportin), PKM2, PFK-1, VEGFA, FUCA1, RRBP1, FUNDC1, ABHD17C, BRPF1, GASC1, CDCA8, PD-L1, ID3, and MDM4; AKT phosphorylates USP35 at Ser613 to promote its nuclear translocation and ERα co-activation; and genetic suppression of USP35 in zebrafish rescues BBS4-dependent ciliopathy defects by facilitating proteasomal clearance of signaling effectors such as β-catenin and rhodopsin."},"narrative":{"mechanistic_narrative":"USP35 is a deubiquitinating enzyme that operates through a substrate-stabilizing logic: by removing ubiquitin from a wide range of client proteins, it blocks their proteasomal turnover and thereby sustains the cellular programs those clients drive [PMID:29449677, PMID:29764563, PMID:29685892]. In cell division it binds and deubiquitinates Aurora B, antagonizing APC/C-CDH1-mediated degradation to maintain Aurora B levels and faithful cytokinesis, and it similarly preserves the chromosomal passenger complex components survivin and Borealin [PMID:29449677, PMID:29764563, PMID:34438346]. USP35 is a recurrent negative regulator of innate immune signaling, directly deubiquitinating and inactivating STING to suppress the STING-TBK1-IRF3 type I interferon axis and stripping K63-linked chains from MAVS to dampen antiviral signaling [PMID:32678307, PMID:40016186]. Across cancer contexts it stabilizes effectors of survival, metabolism, and redox balance — anti-apoptotic IAP proteins including BIRC3, the glycolytic enzymes PKM2 and PFK-1, and the antioxidant master regulator NRF2 and iron exporter ferroportin to restrain ferroptosis [PMID:35022505, PMID:37173391, PMID:37594129, PMID:39714773, PMID:33931967]. In the nucleus it acts as a transcriptional co-activator, stabilizing estrogen receptor α and enhancing its activity at estrogen response elements, with AKT phosphorylation at Ser613 driving USP35 nuclear translocation [PMID:34131114]. USP35 exists as two alternatively spliced isoforms with distinct compartments and functions: a cytoplasmic/nuclear anti-apoptotic isoform and an ER/lipid-droplet integral membrane isoform whose deregulation provokes ER stress and cell death [PMID:29685892]. Genetic suppression of the zebrafish ortholog rescues BBS4-dependent ciliopathy phenotypes by permitting proteasomal clearance of β-catenin and rhodopsin, defining USP35 as a brake on degradation of signaling effectors in vivo [PMID:31723061].","teleology":[{"year":2015,"claim":"Established the first functional link between USP35 and a signaling pathway, showing it acts as a deubiquitinase that stabilizes a substrate to suppress NF-κB output.","evidence":"Co-IP, ubiquitination assay, and NF-κB reporter assay tracking ABIN-2 stabilization in TNFα-stimulated cells","pmids":["26348204"],"confidence":"Medium","gaps":["Single lab without orthogonal substrate validation","ubiquitin linkage type on ABIN-2 not defined","physiological setting of NF-κB suppression not addressed"]},{"year":2018,"claim":"Defined USP35's role in mitosis and revealed that it counteracts a specific E3 ligase complex, framing it as a cell-cycle deubiquitinase rather than a generic DUB.","evidence":"Reciprocal Co-IP, ubiquitination assay, siRNA knockdown with H3-Ser10 and cytokinesis phenotypes, APC/C-CDH1 epistasis, and FoxM1 transcriptional regulation","pmids":["29449677","29764563"],"confidence":"High","gaps":["Ubiquitin chain linkage removed from Aurora B not specified","structural basis of Aurora B recognition unknown"]},{"year":2018,"claim":"Resolved that USP35 is not a single functional entity but two splice isoforms with divergent localization and opposing consequences, explaining apparently conflicting phenotypes.","evidence":"Fluorescence imaging, subcellular fractionation, isoform-specific knockdown/overexpression, apoptosis and ER stress markers","pmids":["29685892"],"confidence":"High","gaps":["Substrate repertoire of each isoform not mapped","mechanism by which ER/lipid-droplet isoform disrupts lipid homeostasis undefined"]},{"year":2019,"claim":"Provided in vivo, organism-level evidence that USP35 acts as a negative regulator of proteasomal clearance of signaling effectors, connecting its DUB activity to a developmental disease context.","evidence":"Genome-wide RNAi suppressor screen in BBS4-deficient zebrafish with convergent-extension, renal, and retinal phenotype scoring and β-catenin/rhodopsin clearance","pmids":["31723061"],"confidence":"Medium","gaps":["Direct USP35 substrates in the cilium not identified","single lab"]},{"year":2020,"claim":"Identified USP35 as a brake on innate antiviral immunity by directly inactivating STING, and showed substrate engagement is phosphorylation-gated.","evidence":"Reciprocal Co-IP, ubiquitination assay, USP35 silencing with IRF3/TBK1 phosphorylation and IFN readouts, phosphorylation-dependent binding assay","pmids":["32678307"],"confidence":"High","gaps":["Ubiquitin linkage type removed from STING not specified","kinase phosphorylating STING for USP35 binding not defined"]},{"year":2021,"claim":"Extended USP35's stabilizing activity to nuclear hormone signaling and connected it to upstream kinase control, showing AKT phosphorylation licenses nuclear function.","evidence":"Co-IP, ubiquitination assay, ChIP at estrogen response elements, AKT kinase assay with S613 mutagenesis, nuclear translocation imaging, in vivo validation","pmids":["34131114"],"confidence":"High","gaps":["Whether co-activation requires catalytic activity at chromatin not resolved","interplay with ERα E3 ligases not mapped"]},{"year":2021,"claim":"Consolidated USP35 as a cytoprotective DUB across multiple cancer survival and ER-stress substrates, broadening the substrate set beyond Aurora B.","evidence":"DUB library screen, Co-IP, ubiquitination assays, activity-dead mutants, and functional ferroptosis/ER-stress/apoptosis readouts for survivin/CPC, ferroportin, and RRBP1","pmids":["34438346","33931967","34618999"],"confidence":"Medium","gaps":["Each substrate validated in a single lab","shared recognition determinant across these substrates unknown"]},{"year":2023,"claim":"Positioned USP35 at the center of metabolic reprogramming and redox control in tumors, stabilizing glycolytic enzymes and the antioxidant regulator NRF2.","evidence":"Co-IP, ubiquitination assays, transcriptomics, and functional glycolysis/ferroptosis/apoptosis readouts across PKM2, NRF2, IAP family, FUCA1, and ABHD17C in multiple cancer models","pmids":["37594129","37173391","36864055","37993419"],"confidence":"Medium","gaps":["Substrate selection mechanism unknown","isoform responsible for cytoplasmic versus ER substrate engagement not assigned"]},{"year":2024,"claim":"Showed USP35 expression is itself controlled post-transcriptionally and extended its stabilizing reach to transcriptional regulators, angiogenic and mitophagy factors.","evidence":"Co-IP, ubiquitination assays, FUS-USP35 mRNA stability assay, and functional readouts for BRD4/SLC7A11, VEGFA, PFK-1, and FUNDC1","pmids":["39820080","38953570","39714773","39406943"],"confidence":"Medium","gaps":["Breadth of substrates raises question of specificity not addressed","single-lab validation per substrate"]},{"year":2025,"claim":"Expanded the immune-regulatory role to MAVS via linkage-specific deubiquitination and revealed non-canonical extracellular (exosomal) and structural-interaction dimensions of USP35.","evidence":"K63-specific ubiquitination assay and MAVS pathway readouts; exosome isolation and co-culture for STING/gastric cancer; HERC2 binding by X-ray crystallography (preprint)","pmids":["40016186","41372134"],"confidence":"Medium","gaps":["STING gastric-cancer mechanism reported with limited methodological detail","functional consequence of HERC2 binding for USP35 not characterized","secretion mechanism for exosomal USP35 unknown"]},{"year":2026,"claim":"Refined the catalytic mechanism with site-specific deubiquitination on ID3 and revealed context-dependent outcomes, including DUB-driven autophagic (not proteasomal) degradation of MDM4.","evidence":"Co-IP, site-specific ubiquitination mutagenesis (ID3 K2/K30; MDM4), cycloheximide chase, PBMC/immune co-culture, kidney-specific knockout, and pharmacological inhibition (IU1)","pmids":["41486422","42002994","42185243"],"confidence":"Medium","gaps":["How USP35 deubiquitination routes MDM4 to autophagy rather than stabilization is not mechanistically resolved","specificity of IU1 for USP35 in these contexts not established"]},{"year":null,"claim":"What determines USP35 substrate selectivity among its many reported clients, and how the two isoforms partition this large substrate set across compartments, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of substrate recognition","no systematic assignment of substrates to isoform 1 versus isoform 2","ubiquitin chain-type preference not defined across most substrates"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,3,6,7,8,10,20,23]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[0,3,7,20]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[8,9]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[2]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[2,8]},{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[2,5]},{"term_id":"GO:0005811","term_label":"lipid droplet","supporting_discovery_ids":[2]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[0,6]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[3,20,23,24]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[2,4,7,10]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[9,11,17]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,3,7,8]}],"complexes":["chromosomal passenger complex"],"partners":["AURKB","STING1","MAVS","ESR1","BIRC3","NFE2L2","BRD4","HERC2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9P2H5","full_name":"Ubiquitin carboxyl-terminal hydrolase 35","aliases":["Deubiquitinating enzyme 35","Ubiquitin thioesterase 35","Ubiquitin-specific-processing protease 35"],"length_aa":1018,"mass_kda":113.4,"function":"Deubiquitinase that plays a role in different processes including cell cycle regulation, mitophagy or endoplasmic reticulum stress (PubMed:26348204, PubMed:29449677, PubMed:37004621). Inhibits TNFalpha-induced NF-kappa-B activation through stabilizing TNIP2 protein via deubiquitination (PubMed:26348204). Plays an essential role during mitosis by deubiquitinating and thereby regulating the levels of Aurora B/AURKB protein (PubMed:29449677). In addition, regulates the protein levels of other key component of the chromosomal passenger complex (CPC) such as survivin/BIRC5 or Borealin/CDCA8 by enhancing their stability (PubMed:34438346). Regulates the degradation of mitochondria through the process of autophagy termed mitophagy (PubMed:25915564)","subcellular_location":"Cytoplasm; Mitochondrion","url":"https://www.uniprot.org/uniprotkb/Q9P2H5/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/USP35","classification":"Not Classified","n_dependent_lines":10,"n_total_lines":1208,"dependency_fraction":0.008278145695364239},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/USP35","total_profiled":1310},"omim":[{"mim_id":"620959","title":"UBIQUITIN-SPECIFIC PEPTIDASE 35; USP35","url":"https://www.omim.org/entry/620959"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoli fibrillar center","reliability":"Approved"},{"location":"Nucleoplasm","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/USP35"},"hgnc":{"alias_symbol":["KIAA1372"],"prev_symbol":[]},"alphafold":{"accession":"Q9P2H5","domains":[{"cath_id":"1.20.5","chopping":"946-980","consensus_level":"medium","plddt":72.4289,"start":946,"end":980}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9P2H5","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9P2H5-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9P2H5-F1-predicted_aligned_error_v6.png","plddt_mean":76.12},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=USP35","jax_strain_url":"https://www.jax.org/strain/search?query=USP35"},"sequence":{"accession":"Q9P2H5","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9P2H5.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9P2H5/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9P2H5"}},"corpus_meta":[{"pmid":"33931967","id":"PMC_33931967","title":"Deubiquitinase USP35 modulates ferroptosis in lung cancer via targeting ferroportin.","date":"2021","source":"Clinical and translational medicine","url":"https://pubmed.ncbi.nlm.nih.gov/33931967","citation_count":144,"is_preprint":false},{"pmid":"32678307","id":"PMC_32678307","title":"Deubiquitinase USP35 restrains STING-mediated interferon signaling in ovarian cancer.","date":"2020","source":"Cell death and differentiation","url":"https://pubmed.ncbi.nlm.nih.gov/32678307","citation_count":77,"is_preprint":false},{"pmid":"29449677","id":"PMC_29449677","title":"USP35 regulates mitotic progression by modulating the stability of Aurora B.","date":"2018","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/29449677","citation_count":45,"is_preprint":false},{"pmid":"26348204","id":"PMC_26348204","title":"USP35 activated by miR let-7a inhibits cell proliferation and NF-κB activation through stabilization of ABIN-2.","date":"2015","source":"Oncotarget","url":"https://pubmed.ncbi.nlm.nih.gov/26348204","citation_count":34,"is_preprint":false},{"pmid":"34618999","id":"PMC_34618999","title":"USP35 mitigates endoplasmic reticulum stress-induced apoptosis by stabilizing RRBP1 in non-small cell lung cancer.","date":"2021","source":"Molecular oncology","url":"https://pubmed.ncbi.nlm.nih.gov/34618999","citation_count":33,"is_preprint":false},{"pmid":"35022505","id":"PMC_35022505","title":"Ubiquitin-specific protease 35 (USP35) mediates cisplatin-induced apoptosis by stabilizing BIRC3 in non-small cell lung cancer.","date":"2022","source":"Laboratory investigation; 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pan-cancer analysis of USP35 and validation of its role in gastric cancer.","date":"2025","source":"Human genomics","url":"https://pubmed.ncbi.nlm.nih.gov/41225657","citation_count":0,"is_preprint":false},{"pmid":"41486422","id":"PMC_41486422","title":"USP35 Acts as a Deubiquitinating Enzyme for ID3 to Promote Immune Escape in Colorectal Cancer.","date":"2026","source":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","url":"https://pubmed.ncbi.nlm.nih.gov/41486422","citation_count":0,"is_preprint":false},{"pmid":"42180131","id":"PMC_42180131","title":"The deubiquitinase USP35: from an oncogenic hub to a therapeutic target in human cancers.","date":"2026","source":"Frontiers in oncology","url":"https://pubmed.ncbi.nlm.nih.gov/42180131","citation_count":0,"is_preprint":false},{"pmid":"42185243","id":"PMC_42185243","title":"Deubiquitinase USP35 regulates MDM4 degradation to promote endothelial ferroptosis and renal injury progression.","date":"2026","source":"Cell death 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deubiquitinates Aurora B, inhibiting APC/C-CDH1-mediated proteasomal degradation of Aurora B to maintain its steady-state levels during mitosis. Loss of USP35 decreases phosphorylation of histone H3-Ser10 (an Aurora B substrate) and causes mitotic defects including cytokinesis failures. FoxM1 transcription factor promotes expression of USP35 during the cell cycle.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, siRNA knockdown, phosphorylation assay, FoxM1 ChIP/overexpression\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, ubiquitination assay, defined cellular phenotype, APC/C epistasis, replicated in companion paper PMID:29764563\",\n      \"pmids\": [\"29449677\", \"29764563\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"USP35 acts as a deubiquitinase that directly stabilizes ABIN-2 (TNFAIP3 interacting protein 2) by promoting its deubiquitination, thereby inhibiting TNFα-induced NF-κB activation. miR let-7a positively regulates USP35 expression.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, NF-κB reporter assay, overexpression/knockdown, miRNA manipulation\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus ubiquitination assay plus functional NF-κB readout, single lab\",\n      \"pmids\": [\"26348204\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"USP35 has two alternatively spliced isoforms with distinct intracellular localizations and functions: isoform 1 localizes to the cytoplasm/nucleus and acts as an anti-apoptotic factor inhibiting staurosporine- and TRAIL-induced apoptosis; isoform 2 is an integral membrane protein of the endoplasmic reticulum and lipid droplets, and its deregulation causes ER stress and cell death, likely through disruption of lipid homeostasis.\",\n      \"method\": \"Fluorescence microscopy localization, isoform-specific knockdown/overexpression, apoptosis assays, ER stress markers, subcellular fractionation\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (imaging, fractionation, functional assays) establishing distinct localization and functional consequences for each isoform\",\n      \"pmids\": [\"29685892\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"USP35 directly deubiquitinates STING to inactivate it, suppressing the STING-TBK1-IRF3 signaling pathway and downstream type I interferon production. Activated STING promotes its own binding to USP35 in a STING phosphorylation-dependent manner.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, USP35 silencing with STING pathway readout (IRF3/TBK1 phosphorylation, IFN expression), phosphorylation-dependent binding assay\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, ubiquitination assay, defined pathway epistasis, phosphorylation-dependent interaction, functional cytokine readout\",\n      \"pmids\": [\"32678307\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"USP35 directly interacts with ferroportin (FPN) and stabilizes it through deubiquitinase activity, preventing its proteasomal degradation. This maintains intracellular iron homeostasis and suppresses ferroptosis in lung cancer cells.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, USP35 knockdown/overexpression with ferroptosis markers (lipid ROS, iron levels), xenograft model\",\n      \"journal\": \"Clinical and translational medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus ubiquitination assay plus functional ferroptosis readout, single lab\",\n      \"pmids\": [\"33931967\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"USP35 directly interacts with and stabilizes RRBP1 (ribosome-binding protein 1, an ER membrane protein) by preventing proteasome-dependent degradation via deubiquitination. USP35-mediated RRBP1 stabilization alleviates ER stress-induced apoptosis in NSCLC cells.\",\n      \"method\": \"iTRAQ proteomics to identify interactome, Co-immunoprecipitation, ubiquitination assay, knockdown/overexpression with ER stress and apoptosis readouts\",\n      \"journal\": \"Molecular oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus ubiquitination assay plus functional ER stress readout, single lab\",\n      \"pmids\": [\"34618999\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"USP35 stabilizes survivin protein in an enzymatic activity-dependent manner through direct interaction and deubiquitination. USP35 also regulates stability of other chromosomal passenger complex (CPC) components Aurora B and Borealin. The Drosophila homolog DUBAI similarly regulates Deterin (Drosophila survivin ortholog), indicating evolutionary conservation.\",\n      \"method\": \"DUB expression library screen (68 DUBs), Co-immunoprecipitation, ubiquitination assay, activity-dead mutant, Drosophila functional assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — library screen plus Co-IP plus ubiquitination assay plus cross-species validation, single lab\",\n      \"pmids\": [\"34438346\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"USP35 directly interacts with and stabilizes BIRC3 through deubiquitination of K48-linked polyubiquitin chains, protecting it from proteasomal degradation. USP35-mediated BIRC3 stabilization alleviates cisplatin-induced apoptosis in NSCLC cells.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay (K48-linkage specific), knockdown/overexpression with apoptosis readout\",\n      \"journal\": \"Laboratory investigation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus K48-specific ubiquitination assay plus functional apoptosis readout, single lab\",\n      \"pmids\": [\"35022505\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"USP35 interacts with estrogen receptor α (ERα), deubiquitinates it, and enhances ERα protein stability. USP35 also interacts with ERα at estrogen response element-containing DNA regions to enhance ERα transcriptional activity. AKT phosphorylates USP35 at Serine613, which promotes USP35 nuclear translocation and ERα transcriptional activity.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, ChIP, AKT kinase assay with site-specific mutagenesis (S613), nuclear translocation imaging, knockdown/overexpression in vitro and in vivo\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods: Co-IP, ubiquitination assay, ChIP, phosphorylation mapping with mutagenesis, nuclear translocation, in vivo validation\",\n      \"pmids\": [\"34131114\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"USP35 directly deubiquitinates and stabilizes BRPF1 protein. Accumulated BRPF1 binds to the SREBP2 promoter to activate SREBP2 transcriptional activity, promoting expression of mevalonate (MVA) metabolism genes in prostate cancer cells.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, ChIP (BRPF1 on SREBP2 promoter), knockdown/overexpression with mevalonate pathway readout, in vivo xenograft\",\n      \"journal\": \"Cell death discovery\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus ubiquitination assay plus ChIP epistasis, single lab\",\n      \"pmids\": [\"36357379\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"USP35 stabilizes multiple IAP family members (including BIRC3 and others) in an enzymatic activity-dependent manner, protecting renal clear cell carcinoma cells from apoptosis. USP35 also catalyzes deubiquitylation of NRF2, maintaining NRF2 protein levels and protecting cells from ferroptosis.\",\n      \"method\": \"Biochemical characterization (ubiquitination assay, activity-dependent stabilization), transcriptomic analysis of NRF2 downstream targets, knockdown with apoptosis and ferroptosis readouts, xenograft\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — enzymatic activity-dependent stabilization assay plus transcriptomics plus functional phenotypes, single lab\",\n      \"pmids\": [\"37173391\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"USP35 directly deubiquitinates and stabilizes PKM2 (pyruvate kinase M2 isoform), protecting it from ubiquitin-mediated degradation and maintaining the Warburg effect (aerobic glycolysis) in hepatocellular carcinoma cells.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, Western blot of PKM2 stability, knockdown with glycolysis readout\",\n      \"journal\": \"International journal of oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus ubiquitination assay plus functional glycolysis readout, single lab\",\n      \"pmids\": [\"37594129\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"USP35 directly interacts with and deubiquitinates FUCA1 (α-L-fucosidase 1), stabilizing it from proteasomal degradation. The USP35-FUCA1 axis promotes upregulation of NER components (XPC, XPA, ERCC1), providing a mechanistic basis for platinum resistance in colorectal cancer.\",\n      \"method\": \"Co-IP followed by mass spectrometry, Co-IP validation, ubiquitination assay, knockdown/overexpression with NER component readout, in vitro and in vivo functional assays\",\n      \"journal\": \"Oncogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP/MS substrate identification plus ubiquitination assay plus downstream pathway readout, single lab\",\n      \"pmids\": [\"36864055\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"USP35 interacts with and stabilizes ABHD17C by inhibiting its ubiquitin-proteasome-mediated degradation, leading to activation of the PI3K/AKT signaling pathway in hepatocellular carcinoma cells.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, USP35 knockdown with PI3K/AKT pathway readout, ABHD17C rescue experiment, xenograft\",\n      \"journal\": \"Cell death discovery\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus ubiquitination assay plus rescue experiment plus in vivo validation, single lab\",\n      \"pmids\": [\"37993419\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Genetic suppression or ablation of usp35 (zebrafish ortholog) ameliorates ciliopathy defects caused by BBS4 loss, including impaired convergent extension, renal tubule convolution, and retinal degeneration, with concomitant clearance of β-catenin and rhodopsin. This places USP35 as a negative regulator of proteasome-dependent degradation of ciliopathy-relevant signaling effectors.\",\n      \"method\": \"Genome-wide RNAi suppressor screen (in BBS4-deficient background), transient and stable zebrafish genetic models, convergent extension assay, renal and retinal phenotype scoring\",\n      \"journal\": \"JCI insight\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genome-wide epistasis screen plus multiple zebrafish model validation with defined phenotypic readouts, single lab\",\n      \"pmids\": [\"31723061\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP35 deubiquitinates and stabilizes BRD4. BRD4 then mediates USP35-induced upregulation of SLC7A11, inhibiting ferroptosis and promoting growth of ER+ breast cancer cells. BRD4 inhibitor (+)-JQ-1 inhibits USP35-enhanced tumorigenesis in vivo.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, BRD4 knockdown/inhibitor rescue, SLC7A11 expression readout, ferroptosis markers, in vivo xenograft with JQ-1 treatment\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus ubiquitination assay plus downstream pathway readout plus in vivo pharmacological validation, single lab\",\n      \"pmids\": [\"39820080\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP35 interacts with and deubiquitinates VEGFA, stabilizing VEGFA protein levels. FUS RNA-binding protein interacts with USP35 mRNA to promote its mRNA stability, thereby positively regulating VEGFA expression in NSCLC cells.\",\n      \"method\": \"Co-IP assay, ubiquitination assay, FUS knockdown/overexpression with USP35 mRNA stability readout, functional angiogenesis assay, in vivo xenograft\",\n      \"journal\": \"General physiology and biophysics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus ubiquitination assay plus mRNA stability assay, single lab\",\n      \"pmids\": [\"38953570\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP35 directly interacts with and stabilizes PFK-1 (phosphofructokinase-1) via deubiquitination, reducing its ubiquitin-mediated degradation. USP35-mediated PFK-1 stabilization promotes glycolysis and cell proliferation in breast cancer cells.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, Seahorse metabolic assay (ECAR/OCR), knockdown/overexpression with glycolysis readout, in vivo xenograft\",\n      \"journal\": \"American journal of physiology. Cell physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus ubiquitination assay plus functional metabolic readout, single lab\",\n      \"pmids\": [\"39714773\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP35 interacts with and stabilizes FUNDC1 (a mitophagy receptor) by deubiquitination. USP35-mediated FUNDC1 stabilization enhances autophagy/mitophagy and reduces mitochondrial ROS in OGD/R-injured neuronal cells.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, USP35 overexpression with FUNDC1 protein stability and autophagy readout, FUNDC1 knockdown rescue experiment\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus ubiquitination assay plus rescue experiment, single lab\",\n      \"pmids\": [\"39406943\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP35 deubiquitinates and stabilizes NRF2 in esophageal cancer cells, maintaining NRF2 protein stability through direct interaction and enzymatic deubiquitination.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, USP35 knockdown with NRF2 protein level readout\",\n      \"journal\": \"Open life sciences\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP plus ubiquitination assay, single lab, no orthogonal methods\",\n      \"pmids\": [\"39156988\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP35 interacts with MAVS and removes K63-linked polyubiquitin chains from MAVS, thereby inhibiting viral-induced MAVS-TBK1-IRF3 pathway activation and downstream inflammatory gene expression.\",\n      \"method\": \"Co-immunoprecipitation, K63-linkage-specific ubiquitination assay, USP35 depletion with MAVS pathway readout (TBK1/IRF3 activation, cytokine expression), in vivo anti-tumor immunity assay\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus linkage-specific ubiquitination assay plus functional pathway readout, single lab\",\n      \"pmids\": [\"40016186\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP35 directly deubiquitinates and stabilizes STING in gastric cancer cells, activating the HIF-1α/FAK pathway to promote energy metabolism reprogramming and adhesion to peritoneal mesothelial cells. USP35 is also secreted via exosomes from gastric cancer cells and promotes mesothelial-mesenchymal transformation (MMT) of peritoneal mesothelial cells.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, pathway readout (HIF-1α/FAK), exosome isolation and co-culture functional assay\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — mechanistic claims based on Co-IP and pathway readouts but limited methodological detail in abstract, single lab\",\n      \"pmids\": [\"41372134\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP35 interacts with and stabilizes GASC1 (gene amplified in squamous cell carcinoma 1) by reducing GASC1 ubiquitination, leading to upregulation of ROCK2 expression and promotion of HCC cell proliferation.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, knockdown/overexpression with ROCK2 expression and proliferation readout\",\n      \"journal\": \"Translational oncology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP plus ubiquitination assay, single lab, limited detail\",\n      \"pmids\": [\"40424935\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"USP35 deubiquitinates and stabilizes ID3 at its N-terminal lysine residues K2 and K30, enhancing ID3 transcriptional activity and resulting in elevated PD-L1 expression in colorectal cancer, facilitating immune escape. The USP35 inhibitor IU1 promotes ID3 ubiquitination and reduces PD-L1 expression.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay with site-specific mutagenesis (K2, K30), PD-L1 expression readout, pharmacological inhibition with IU1, immune co-culture assay\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus site-specific mutagenesis plus functional immune readout plus pharmacological validation, single lab\",\n      \"pmids\": [\"41486422\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"USP35 directly deubiquitinates and stabilizes CDCA8 (a cell cycle regulator) and also deubiquitinates PD-L1 to prevent its proteasomal degradation, thereby promoting NSCLC cell proliferation/invasion and immune evasion respectively.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, cycloheximide chase, PBMC co-culture cytotoxicity assay, knockdown with proliferation and immune readouts\",\n      \"journal\": \"Combinatorial chemistry & high throughput screening\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus ubiquitination assay plus cycloheximide chase plus functional immune readout, single lab\",\n      \"pmids\": [\"42002994\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"USP35 deubiquitinates MDM4, leading to its autophagic degradation (rather than proteasomal stabilization), which activates the P53 signaling pathway and mediates endothelial ferroptosis in cisplatin-induced acute kidney injury. Kidney-specific USP35 knockout reduces AKI and endothelial ferroptosis.\",\n      \"method\": \"Transcriptomic and single-cell analyses, renal epithelial cell and animal models, kidney-specific USP35 knockout, ubiquitination assay, P53 pathway readout, ferroptosis markers\",\n      \"journal\": \"Cell death discovery\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — tissue-specific KO model plus ubiquitination assay plus pathway readout, single lab\",\n      \"pmids\": [\"42185243\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"HERC2 binds to USP35 via a conserved 'DxDKDxD' motif recognized by the RLD2 domain of HERC2, identifying USP35 as a HERC2-interacting protein relevant to brain development.\",\n      \"method\": \"Quantitative binding assays, X-ray crystallography, sequence conservation analysis\",\n      \"journal\": \"bioRxiv (preprint)\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 1 / Weak — structural and binding data establish interaction but no functional consequence for USP35 itself was characterized; USP35 is one of several listed HERC2 partners\",\n      \"pmids\": [\"bio_10.1101_2025.09.16.670041\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"USP35 is a deubiquitinating enzyme (DUB) with a conserved Cys-His-Asp catalytic triad that removes ubiquitin from a broad range of substrates to prevent their proteasomal degradation; its two alternatively spliced isoforms localize to distinct compartments (cytoplasm/nucleus vs. ER/lipid droplets) with distinct functions; established substrates include Aurora B (blocking APC/C-CDH1-mediated degradation during mitosis), STING and MAVS (negative regulators of innate immune signaling), ERα, BRD4, NRF2, BIRC3/IAP family members, survivin/CPC components, FPN (ferroportin), PKM2, PFK-1, VEGFA, FUCA1, RRBP1, FUNDC1, ABHD17C, BRPF1, GASC1, CDCA8, PD-L1, ID3, and MDM4; AKT phosphorylates USP35 at Ser613 to promote its nuclear translocation and ERα co-activation; and genetic suppression of USP35 in zebrafish rescues BBS4-dependent ciliopathy defects by facilitating proteasomal clearance of signaling effectors such as β-catenin and rhodopsin.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"USP35 is a deubiquitinating enzyme that operates through a substrate-stabilizing logic: by removing ubiquitin from a wide range of client proteins, it blocks their proteasomal turnover and thereby sustains the cellular programs those clients drive [#0, #2]. In cell division it binds and deubiquitinates Aurora B, antagonizing APC/C-CDH1-mediated degradation to maintain Aurora B levels and faithful cytokinesis, and it similarly preserves the chromosomal passenger complex components survivin and Borealin [#0, #6]. USP35 is a recurrent negative regulator of innate immune signaling, directly deubiquitinating and inactivating STING to suppress the STING-TBK1-IRF3 type I interferon axis and stripping K63-linked chains from MAVS to dampen antiviral signaling [#3, #20]. Across cancer contexts it stabilizes effectors of survival, metabolism, and redox balance — anti-apoptotic IAP proteins including BIRC3, the glycolytic enzymes PKM2 and PFK-1, and the antioxidant master regulator NRF2 and iron exporter ferroportin to restrain ferroptosis [#7, #10, #11, #17, #4]. In the nucleus it acts as a transcriptional co-activator, stabilizing estrogen receptor α and enhancing its activity at estrogen response elements, with AKT phosphorylation at Ser613 driving USP35 nuclear translocation [#8]. USP35 exists as two alternatively spliced isoforms with distinct compartments and functions: a cytoplasmic/nuclear anti-apoptotic isoform and an ER/lipid-droplet integral membrane isoform whose deregulation provokes ER stress and cell death [#2]. Genetic suppression of the zebrafish ortholog rescues BBS4-dependent ciliopathy phenotypes by permitting proteasomal clearance of β-catenin and rhodopsin, defining USP35 as a brake on degradation of signaling effectors in vivo [#14].\",\n  \"teleology\": [\n    {\n      \"year\": 2015,\n      \"claim\": \"Established the first functional link between USP35 and a signaling pathway, showing it acts as a deubiquitinase that stabilizes a substrate to suppress NF-κB output.\",\n      \"evidence\": \"Co-IP, ubiquitination assay, and NF-κB reporter assay tracking ABIN-2 stabilization in TNFα-stimulated cells\",\n      \"pmids\": [\"26348204\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Single lab without orthogonal substrate validation\", \"ubiquitin linkage type on ABIN-2 not defined\", \"physiological setting of NF-κB suppression not addressed\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Defined USP35's role in mitosis and revealed that it counteracts a specific E3 ligase complex, framing it as a cell-cycle deubiquitinase rather than a generic DUB.\",\n      \"evidence\": \"Reciprocal Co-IP, ubiquitination assay, siRNA knockdown with H3-Ser10 and cytokinesis phenotypes, APC/C-CDH1 epistasis, and FoxM1 transcriptional regulation\",\n      \"pmids\": [\"29449677\", \"29764563\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Ubiquitin chain linkage removed from Aurora B not specified\", \"structural basis of Aurora B recognition unknown\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Resolved that USP35 is not a single functional entity but two splice isoforms with divergent localization and opposing consequences, explaining apparently conflicting phenotypes.\",\n      \"evidence\": \"Fluorescence imaging, subcellular fractionation, isoform-specific knockdown/overexpression, apoptosis and ER stress markers\",\n      \"pmids\": [\"29685892\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Substrate repertoire of each isoform not mapped\", \"mechanism by which ER/lipid-droplet isoform disrupts lipid homeostasis undefined\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Provided in vivo, organism-level evidence that USP35 acts as a negative regulator of proteasomal clearance of signaling effectors, connecting its DUB activity to a developmental disease context.\",\n      \"evidence\": \"Genome-wide RNAi suppressor screen in BBS4-deficient zebrafish with convergent-extension, renal, and retinal phenotype scoring and β-catenin/rhodopsin clearance\",\n      \"pmids\": [\"31723061\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Direct USP35 substrates in the cilium not identified\", \"single lab\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identified USP35 as a brake on innate antiviral immunity by directly inactivating STING, and showed substrate engagement is phosphorylation-gated.\",\n      \"evidence\": \"Reciprocal Co-IP, ubiquitination assay, USP35 silencing with IRF3/TBK1 phosphorylation and IFN readouts, phosphorylation-dependent binding assay\",\n      \"pmids\": [\"32678307\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Ubiquitin linkage type removed from STING not specified\", \"kinase phosphorylating STING for USP35 binding not defined\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Extended USP35's stabilizing activity to nuclear hormone signaling and connected it to upstream kinase control, showing AKT phosphorylation licenses nuclear function.\",\n      \"evidence\": \"Co-IP, ubiquitination assay, ChIP at estrogen response elements, AKT kinase assay with S613 mutagenesis, nuclear translocation imaging, in vivo validation\",\n      \"pmids\": [\"34131114\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Whether co-activation requires catalytic activity at chromatin not resolved\", \"interplay with ERα E3 ligases not mapped\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Consolidated USP35 as a cytoprotective DUB across multiple cancer survival and ER-stress substrates, broadening the substrate set beyond Aurora B.\",\n      \"evidence\": \"DUB library screen, Co-IP, ubiquitination assays, activity-dead mutants, and functional ferroptosis/ER-stress/apoptosis readouts for survivin/CPC, ferroportin, and RRBP1\",\n      \"pmids\": [\"34438346\", \"33931967\", \"34618999\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Each substrate validated in a single lab\", \"shared recognition determinant across these substrates unknown\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Positioned USP35 at the center of metabolic reprogramming and redox control in tumors, stabilizing glycolytic enzymes and the antioxidant regulator NRF2.\",\n      \"evidence\": \"Co-IP, ubiquitination assays, transcriptomics, and functional glycolysis/ferroptosis/apoptosis readouts across PKM2, NRF2, IAP family, FUCA1, and ABHD17C in multiple cancer models\",\n      \"pmids\": [\"37594129\", \"37173391\", \"36864055\", \"37993419\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Substrate selection mechanism unknown\", \"isoform responsible for cytoplasmic versus ER substrate engagement not assigned\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Showed USP35 expression is itself controlled post-transcriptionally and extended its stabilizing reach to transcriptional regulators, angiogenic and mitophagy factors.\",\n      \"evidence\": \"Co-IP, ubiquitination assays, FUS-USP35 mRNA stability assay, and functional readouts for BRD4/SLC7A11, VEGFA, PFK-1, and FUNDC1\",\n      \"pmids\": [\"39820080\", \"38953570\", \"39714773\", \"39406943\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Breadth of substrates raises question of specificity not addressed\", \"single-lab validation per substrate\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Expanded the immune-regulatory role to MAVS via linkage-specific deubiquitination and revealed non-canonical extracellular (exosomal) and structural-interaction dimensions of USP35.\",\n      \"evidence\": \"K63-specific ubiquitination assay and MAVS pathway readouts; exosome isolation and co-culture for STING/gastric cancer; HERC2 binding by X-ray crystallography (preprint)\",\n      \"pmids\": [\"40016186\", \"41372134\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"STING gastric-cancer mechanism reported with limited methodological detail\", \"functional consequence of HERC2 binding for USP35 not characterized\", \"secretion mechanism for exosomal USP35 unknown\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Refined the catalytic mechanism with site-specific deubiquitination on ID3 and revealed context-dependent outcomes, including DUB-driven autophagic (not proteasomal) degradation of MDM4.\",\n      \"evidence\": \"Co-IP, site-specific ubiquitination mutagenesis (ID3 K2/K30; MDM4), cycloheximide chase, PBMC/immune co-culture, kidney-specific knockout, and pharmacological inhibition (IU1)\",\n      \"pmids\": [\"41486422\", \"42002994\", \"42185243\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"How USP35 deubiquitination routes MDM4 to autophagy rather than stabilization is not mechanistically resolved\", \"specificity of IU1 for USP35 in these contexts not established\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"What determines USP35 substrate selectivity among its many reported clients, and how the two isoforms partition this large substrate set across compartments, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"No structural model of substrate recognition\", \"no systematic assignment of substrates to isoform 1 versus isoform 2\", \"ubiquitin chain-type preference not defined across most substrates\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 3, 6, 7, 8, 10, 20, 23]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [0, 3, 7, 20]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [8, 9]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [2]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [2, 8]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [2, 5]},\n      {\"term_id\": \"GO:0005811\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [0, 6]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [3, 20, 23, 24]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [2, 4, 7, 10]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [9, 11, 17]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 3, 7, 8]}\n    ],\n    \"complexes\": [\n      \"chromosomal passenger complex\"\n    ],\n    \"partners\": [\n      \"AURKB\",\n      \"STING1\",\n      \"MAVS\",\n      \"ESR1\",\n      \"BIRC3\",\n      \"NFE2L2\",\n      \"BRD4\",\n      \"HERC2\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}