{"gene":"USP28","run_date":"2026-06-11T09:02:06","timeline":{"discoveries":[{"year":2007,"finding":"USP28 binds to MYC through an interaction with FBW7alpha (nucleoplasmic isoform), stabilizing MYC in the nucleus but not in the nucleolus where FBW7gamma mediates MYC degradation. USP28 is required for MYC stability in human tumor cells.","method":"shRNA screen, Co-IP, subcellular fractionation, knockdown/rescue experiments","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, shRNA screen, subcellular localization, replicated across multiple labs subsequently","pmids":["17558397"],"is_preprint":false},{"year":2006,"finding":"USP28 is required to stabilize Chk2 and 53BP1 in response to DNA damage (double-strand breaks). USP28 was discovered through analysis of 53BP1 complexes and is required for DNA-damage-induced apoptosis through the Chk2-p53-PUMA pathway. USP28 also stabilizes Mdc1, Claspin, and TopBP1 in the DNA-damage checkpoint pathway.","method":"Co-IP (53BP1 complex analysis), cell line functional assays, RNAi knockdown, apoptosis assays","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP discovery, multiple substrates identified, pathway epistasis established, replicated in subsequent studies","pmids":["16901786"],"is_preprint":false},{"year":2007,"finding":"Usp28 dissociates from Fbw7alpha in response to UV irradiation, providing a mechanism by which Fbw7-mediated degradation of Myc is enhanced upon DNA damage. Myc decline after DNA damage is mediated by proteasomal degradation via the Fbw7 ubiquitin ligase.","method":"Co-IP, UV irradiation, protein stability assays, proteasome inhibitor experiments","journal":"Cell cycle (Georgetown, Tex.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP showing dissociation, single lab, two orthogonal methods","pmids":["17873522"],"is_preprint":false},{"year":2011,"finding":"USP28 antagonizes GSK-3β/Fbw7-dependent HIF-1α degradation. Fbw7 and USP28 reciprocally regulate cell migration and angiogenesis in an HIF-1α-dependent manner.","method":"GSK-3β and Fbw7-deficient cell experiments, overexpression/knockdown, migration and angiogenesis assays","journal":"Blood","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic loss-of-function, multiple cell-based readouts, single lab","pmids":["22144179"],"is_preprint":false},{"year":2013,"finding":"USP28 is a deubiquitinase of LSD1. USP28 directly interacts with LSD1 and stabilizes it via deubiquitination, counteracting its proteasome-mediated degradation. Knockdown of USP28 results in LSD1 destabilization and suppression of cancer stem cell-like characteristics rescued by ectopic LSD1 expression.","method":"siRNA screen against all human deubiquitinases, Co-IP, deubiquitination assay, rescue experiments, in vivo xenograft","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — unbiased DUB screen, Co-IP, in vitro deubiquitination, rescue with LSD1 re-expression, single lab with multiple orthogonal methods","pmids":["24075993"],"is_preprint":false},{"year":2014,"finding":"USP28 antagonizes c-MYC, c-JUN, and NOTCH1 ubiquitin-dependent degradation in the intestine. Usp28 deletion in mice reduced intestinal proliferation and impaired secretory lineage cell differentiation. USP28 and c-MYC form a positive feedback loop where USP28 is a c-MYC target gene.","method":"Murine genetic models (Usp28 knockout), intestinal tumorigenesis models, protein stability assays, mouse lifespan analysis","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo genetic models, multiple substrates validated, c-MYC feedback loop established, replicated across labs","pmids":["24960159"],"is_preprint":false},{"year":2014,"finding":"USP28 preferentially antagonizes autocatalytic (self-)ubiquitination of Fbw7, stabilizing Fbw7. Monoallelic Usp28 deletion maintains stable Fbw7 and drives Fbw7 substrate degradation, while complete knockout triggers Fbw7 degradation and accumulation of Fbw7 substrates. Both complete loss and overexpression of Usp28 promote Ras-driven oncogenic transformation.","method":"Usp28 knockout mouse models (monoallelic and biallelic), embryonic fibroblast assays, Ras transformation assay, protein stability measurements","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo genetic models (mono- and biallelic KO), multiple tissues, functional transformation assay, dose-dependent effects demonstrated","pmids":["25437563"],"is_preprint":false},{"year":2014,"finding":"USP28 is recruited to double-strand breaks in a manner that requires the tandem BRCT domains of 53BP1. However, USP28 depletion caused only minor DDR defects, and mice lacking USP28 showed normal longevity, immunological development, and radiation responses, indicating USP28 is not a critical factor in double-strand break metabolism.","method":"Recruitment assay to DSBs, BRCT domain mutant analysis, USP28-depleted cells, Usp28 knockout mice with radiation challenge","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — domain-specific recruitment established, in vivo knockout mouse model with radiation response, negative result well-controlled","pmids":["24687851"],"is_preprint":false},{"year":2014,"finding":"SUMO modification of the N-terminal domain of USP28 negatively regulates its deubiquitinating activity. Truncation of the N-terminal domain does not impair deubiquitinating activity on diubiquitin or polyubiquitin chain substrates. USP28 shows chain preference for Lys11-, Lys48-, and Lys63-linked diubiquitin linkages.","method":"In vitro deubiquitination assays, SUMO modification assays, N-terminal truncation mutants, linkage-specificity assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro biochemical assays with mutant analysis, chain preference directly measured, single lab","pmids":["25359778"],"is_preprint":false},{"year":2015,"finding":"Usp28 inactivation in the intestine rescues hyperproliferation and impaired goblet/Paneth cell differentiation of Fbw7-deficient mice. Usp28 deficiency corrects accumulation of SCF(Fbw7) substrate proteins (NICD1, c-Jun, c-Myc) independently of Fbw7 presence. Usp28 interacts with unphosphorylated phosphodegron motifs (the same motif recognized by Fbw7 when phosphorylated), offering a mechanistic explanation for identical substrate selection.","method":"Intestinal-specific Usp28/Fbw7 double knockout mice, protein stability assays, phosphodegron interaction assays","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo genetic epistasis (double KO), phosphodegron interaction mechanism established, multiple substrates validated","pmids":["25716680"],"is_preprint":false},{"year":2016,"finding":"USP28 and 53BP1 form a module acting upstream of p53 to arrest cell growth after centrosome loss or prolonged mitosis. USP28 and 53BP1 stabilize p53 after centrosome loss, independently of their role in the DNA damage response. This USP28-53BP1-p53-p21 signaling axis also arrests growth after prolonged prometaphase.","method":"Genome-wide CRISPR/Cas9 knockout screen (centrinone resistance), genetic deletion of TP53BP1 and USP28, p53 protein level assays, cell cycle analysis","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — independent CRISPR screens in three different labs (PMIDs 27432897, 27432896, 27371829), epistasis established, replicated across labs","pmids":["27432897","27432896","27371829"],"is_preprint":false},{"year":2016,"finding":"53BP1 mediates p53 activation independently of its DNA repair activity but requiring USP28. USP28 can directly deubiquitinate p53 in vitro and ectopically stabilize p53 in vivo. 53BP1 and USP28 function in parallel to the spindle assembly checkpoint to select against disturbed or delayed mitosis.","method":"CRISPR loss-of-function screen, in vitro deubiquitination assay of p53, in vivo p53 stabilization assay, genetic epistasis","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — direct in vitro deubiquitination of p53 demonstrated, CRISPR screen, in vivo validation, replicated across three concurrent labs","pmids":["27371829"],"is_preprint":false},{"year":2018,"finding":"USP28 functions through a feedback loop to destabilize RAF family members; loss of USP28 stabilizes BRAF, enhances downstream MAPK activation, and promotes resistance to RAF inhibitor therapy. USP28 is deleted in a proportion of melanoma patients.","method":"Cell culture and in vivo models, BRAF stability assays, USP28 knockdown/knockout, MAPK pathway activation measurement","journal":"The Journal of experimental medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with defined cellular phenotype, in vivo model, single lab","pmids":["29880484"],"is_preprint":false},{"year":2018,"finding":"LIN28A protein undergoes ubiquitination and USP28 interacts with and stabilizes LIN28A by reversing its proteasomal degradation via deubiquitinating activity.","method":"Co-IP, deubiquitination assay, protein stability (cycloheximide chase), knockdown/overexpression","journal":"Biochimica et biophysica acta. Molecular basis of disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination assay, stability assay, single lab with multiple methods","pmids":["30543854"],"is_preprint":false},{"year":2019,"finding":"USP28 forms a constitutively active dimer through a dimerizing insertion that spatially separates independently active catalytic domains. Unlike USP25, USP28 does not form an autoinhibited tetramer. Oligomeric states of USP28 confirmed in cells and modulating oligomerization affects substrate stabilization in accordance with in vitro activity.","method":"Crystal structure (structural determination), biochemical activity assays, cell-based substrate stabilization assays, in vitro oligomerization experiments","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure, in vitro reconstitution, cell-based validation, independently replicated in concurrent companion paper (PMID 30926243)","pmids":["30926242","30926243"],"is_preprint":false},{"year":2019,"finding":"USP28 is a constitutively active dimer, while USP25 forms an autoinhibited tetramer; structures of the catalytic domains of both proteins determined. USP28 dimerization is intrinsic to its constitutive activity. Cancer-associated USP25 mutations lead to activation in vitro and in vivo.","method":"Crystal structure of catalytic domains, biochemical activity assays, in vitro and in vivo oligomerization studies","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structures of both proteins, in vitro reconstitution, cell-based validation, replicated in companion paper","pmids":["30926243"],"is_preprint":false},{"year":2019,"finding":"SENP1-mediated deSUMOylation of USP28 activates USP28 under hypoxia. SUMOylation of USP28 under normoxia limits its deubiquitinating activity toward HIF-1α. Hypoxia activates USP28 through SENP1-mediated deSUMOylation, creating a positive feedback loop to further accumulate HIF-1α.","method":"Co-IP, GST pulldown, in vitro deubiquitination assay, in vivo deSUMOylation assay, luciferase reporter assay, SUMOylation-deficient mutant","journal":"Cancer cell international","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, GST pulldown, in vitro deubiquitination, SUMO mutant analysis; single lab","pmids":["30622440"],"is_preprint":false},{"year":2019,"finding":"USP28 knockdown caused increased ubiquitination of histone H2A at K119, reduced expression of p53, p21 and p16INK4a, and increased cell proliferation, establishing USP28 as a deubiquitinase for histone H2A.","method":"RNAi screen of DUBs, USP28 knockdown, H2A ubiquitination assay, cell proliferation assay","journal":"Experimental cell research","confidence":"Low","confidence_rationale":"Tier 3 / Weak — RNAi screen hit, single Co-IP/knockdown experiment, no direct in vitro deubiquitination of H2A demonstrated, single lab","pmids":["30910399"],"is_preprint":false},{"year":2020,"finding":"Nuclear caspase-8 cleaves and inactivates USP28, preventing USP28 from deubiquitinating and stabilizing wild-type p53. This cleavage results in de facto p53 protein loss, switching cell fate from apoptosis toward mitosis, allowing tumor cells to override the p53-dependent G2/M checkpoint.","method":"Nuclear caspase-8 localization studies, Co-IP, USP28 cleavage assay, p53 stability assays, genetic depletion with cell cycle/apoptosis readouts","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — caspase-8 cleavage of USP28 demonstrated biochemically, mechanistic pathway established, p53 deubiquitination linked to substrate stability, single lab with multiple orthogonal methods","pmids":["31982308"],"is_preprint":false},{"year":2020,"finding":"The 53BP1-USP28 mitotic surveillance pathway is active in developing brain. Depletion of centrosome proteins in neural progenitor cells prolongs mitosis and increases TP53-mediated apoptosis. Inactivation of the mitotic surveillance pathway (53BP1 or USP28 deletion) restored NPC proliferation and brain size without correcting upstream centrosome defects or extended mitosis.","method":"Mouse genetic models (53BP1 and USP28 knockout), centrosome protein depletion in NPCs, apoptosis assays, brain size measurements","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo genetic epistasis in brain development, multiple genetic combinations, pathway placement confirmed","pmids":["33226141"],"is_preprint":false},{"year":2020,"finding":"USP28 stabilizes ΔNp63 and maintains elevated ΔNp63 levels in squamous cell carcinoma by counteracting its proteasome-mediated degradation. Impaired USP28 activity (genetic or pharmacological) abrogates transcriptional identity and suppresses growth of human SCC cells. CRISPR/Cas9 in vivo mouse models show endogenous USP28 is required for both induction and maintenance of lung SCC.","method":"CRISPR/Cas9 in vivo mouse models, siRNA, pharmacological inhibition, protein stability assays, gene expression analysis","journal":"EMBO molecular medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo CRISPR mouse models, pharmacological and genetic validation, multiple cancer types, replicated in subsequent studies","pmids":["32128997"],"is_preprint":false},{"year":2021,"finding":"ATR phosphorylates USP28 in response to cisplatin treatment, increasing its enzymatic activity. USP28 is recruited to sites of DNA damage in cisplatin-treated cells, and this phosphorylation positively regulates DNA damage repair in SCC by stabilizing ΔNp63.","method":"ATR inhibitor studies, phosphorylation assays, USP28 recruitment to DNA damage sites, enzymatic activity assays, knockdown/inhibitor experiments","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ATR-USP28 phosphorylation and activity link established, recruitment to damage sites shown, single lab","pmids":["34611298"],"is_preprint":false},{"year":2021,"finding":"USP28 deubiquitinates and stabilizes FOXM1, a critical mediator of Wnt/β-catenin signaling. USP28-mediated stabilization of FOXM1 promotes nucleus β-catenin trans-activation and activation of the Wnt/β-catenin pathway in pancreatic cancer.","method":"Co-IP, deubiquitination assay, protein stability assay, rescue experiments with FOXM1 re-expression, in vivo xenograft","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, deubiquitination assay, rescue experiment confirming causal role of FOXM1; single lab","pmids":["34584067"],"is_preprint":false},{"year":2021,"finding":"Genetic inactivation of Usp28 induced regression of established murine LSCC lung tumors. A small-molecule USP28 inhibitor decreased c-MYC, c-JUN, and ΔNp63 protein levels and caused regression of autochthonous murine LSCC and human LSCC xenografts, phenocopying genetic deletion.","method":"Genetic mouse tumor model, small-molecule inhibitor, tumor regression analysis, protein stability assays","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo genetic and pharmacological validation, autochthonous tumor model plus xenograft, phenocopy by inhibitor confirming on-target mechanism","pmids":["34636321"],"is_preprint":false},{"year":2021,"finding":"USP28 interacts with and deubiquitinates UCK1, antagonizing KLHL2-mediated polyubiquitination of UCK1 at K81. ATM-mediated phosphorylation of USP28 results in its disassociation from KLHL2 and UCK1 destabilization.","method":"Mass spectrometry, Co-IP, ubiquitination assays (identifying K81 site), ATM phosphorylation studies, AML cell line and murine AML model","journal":"Theranostics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS identification, Co-IP, site-specific ubiquitination, ATM-mediated regulation; single lab with multiple methods","pmids":["31938050"],"is_preprint":false},{"year":2022,"finding":"USP28 interacts with PIM1 and PIM2 kinases, and this interaction increases in response to hypoxia, correlating with reduced PIM ubiquitination and increased protein stability. USP28 overexpression increased PIM protein stability; USP28 knockdown significantly increased ubiquitination of PIM1 and PIM2.","method":"Co-IP, ubiquitination assay, overexpression/knockdown, in vivo xenograft","journal":"Cells","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP, ubiquitination assay, in vivo data; single lab","pmids":["35326457"],"is_preprint":false},{"year":2022,"finding":"USP28 interacts with NuMA1 and affects centrosome clustering in tetraploid cells. Loss of USP28 reduces checkpoint activation in tetraploid cells, facilitating their proliferation.","method":"Mass spectrometry, immunoprecipitation, RNAi screen, cell viability assays","journal":"Cellular oncology (Dordrecht, Netherlands)","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single IP-MS finding, NuMA1 interaction not deeply validated mechanistically, single lab","pmids":["34962618"],"is_preprint":false},{"year":2023,"finding":"USP28 directly binds to mature SREBP2, leading to its deubiquitination and stabilization. USP28 silencing reduces expression of mevalonate pathway enzymes and lowers metabolic flux. USP28 deletion rendered cancer cells highly sensitive to MVP inhibition by statins, rescued by geranyl-geranyl pyrophosphate.","method":"Co-IP, deubiquitination assay, metabolic flux analysis, CRISPR/Cas9 deletion, tissue microarray, mouse tumor model","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, deubiquitination, metabolic rescue, CRISPR in vivo model; single lab with multiple orthogonal approaches","pmids":["37202505"],"is_preprint":false},{"year":2023,"finding":"USP28 dimerization limits USP28 activity and restricts recruitment of the elongation factor PAF1c by MYC. Monomeric USP28 stabilizes MYC and promotes PAF1c recruitment, leading to ectopic DNA synthesis and replication-associated DNA damage. 53BP1 selectively binds USP28 dimers and stimulates dimerization; genotoxic stress diminishes 53BP1-USP28 interaction and promotes USP28 dimer disassembly.","method":"Dimerization mutants, ChIP, DNA replication assays, DNA damage assays, Co-IP with 53BP1","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — dimerization mutant analysis, ChIP, DNA synthesis assays; single lab","pmids":["38227944"],"is_preprint":false},{"year":2023,"finding":"The HNF-1β transcription factor directly regulates USP28 expression as a target gene. USP28 in turn stabilizes Claspin (by deubiquitination), which promotes Chk1 activation and G2/M cell cycle progression in response to DNA damage in ovarian clear cell carcinoma.","method":"siRNA knockdown, immunoprecipitation (Claspin ubiquitination), RNAi-mediated gene silencing, Chk1 phosphorylation assay","journal":"Oncotarget","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP for Claspin stabilization, HNF-1β regulation of USP28 by siRNA only, single lab","pmids":["29707125"],"is_preprint":false},{"year":2023,"finding":"USP28 directly interacts with PPARα, deubiquitinating and stabilizing PPARα (at Lys152), which promotes Mfn2 transcription to impede mitochondrial morphofunctional defects. This USP28-PPARα-Mfn2 axis was cardioprotective in diabetic hearts.","method":"Co-IP/mass spectrometry, protein pull-down, ChIP sequencing, ChIP assays, site-specific ubiquitination mapping, cardiac-specific USP28 KO mice, AAV9-USP28 overexpression","journal":"Circulation","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — Co-IP/MS, protein pull-down, ChIP-seq, site-specific modification (K152), in vivo KO and OE models; single lab but multiple orthogonal methods","pmids":["37994595"],"is_preprint":false},{"year":2024,"finding":"DYRK2 phosphorylates USP28, promoting its ubiquitination and proteasomal degradation. Conversely, USP28 deubiquitinates DYRK2, stabilizing it and enhancing its kinase activity. The DYRK2 region 521-541 (particularly T525) plays a crucial role in USP28-mediated DYRK2 stabilization. This reciprocal regulation modulates p53 signaling and apoptotic responses to DNA damage.","method":"Phosphorylation assay, ubiquitination assay, Co-IP, kinase activity assay, site-directed mutants, genetic depletion with apoptosis readout","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — bidirectional regulation shown with phosphorylation and deubiquitination assays, domain mapping; single lab","pmids":["40858801"],"is_preprint":false},{"year":2024,"finding":"USP28 deubiquitinates and stabilizes TRIM21; cardiomyocyte USP28 deubiquitinating TRIM21 negatively regulates Nrf2 antioxidant response, increasing oxidative stress and promoting cardiac hypertrophy. Identified by Co-IP/mass spectrometry screen.","method":"Co-IP/mass spectrometry substrate screen, Co-IP validation, USP28 cardiomyocyte-specific KO mice (Ang II and TAC models), deubiquitination assay, USP28 inhibitor (Otilonium Bromide)","journal":"Theranostics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP/MS substrate identification, in vivo KO model, pharmacological validation; single lab","pmids":["39431010"],"is_preprint":false},{"year":2024,"finding":"USP28 interacts with and extends the half-life of MAST1 protein via its deubiquitinating activity. CRISPR/Cas9 knockout of USP28 blocked MAST1-driven cisplatin resistance.","method":"CRISPR/Cas9 genome-wide screen, Co-IP, protein half-life assay (cycloheximide chase), deubiquitination assay, xenograft model","journal":"Cellular and molecular life sciences : CMLS","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genome-wide CRISPR screen hit, Co-IP, deubiquitination, in vivo xenograft; single lab","pmids":["38498222"],"is_preprint":false},{"year":2024,"finding":"USP28 inhibits K48-linked polyubiquitination of STAT3 and reverses its protein degradation in ADPKD. USP28 also directly interacts with and stabilizes c-Myc, both processes synergistically enhancing renal cystogenesis.","method":"Co-IP, ubiquitination assay (K48-specific), protein stability assay, pharmacological inhibition, in vivo cystogenesis model","journal":"Biochemical pharmacology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — K48-specific ubiquitination, Co-IP, in vivo model; single lab","pmids":["36442624"],"is_preprint":false},{"year":2024,"finding":"The C-terminal domain of USP28 mediates PLK1-dependent binding to 53BP1, a dimerization-driven interaction necessary for mitotic stress memory, p53 stabilization, and cell cycle arrest. Cancer-associated missense mutations in this domain disrupt 53BP1 binding, impair nuclear localization, and destabilize USP28, compromising p53 stabilization. USP28 stabilizes p53 through deubiquitination.","method":"Cancer mutation analysis, domain interaction mapping, nuclear localization assays, deubiquitination assay (p53 substrate), Co-IP, cell cycle assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — direct deubiquitination of p53 demonstrated, domain mapping of 53BP1 interaction, PLK1-dependent binding mechanism, isoform characterization; single lab with multiple orthogonal methods","pmids":["41365927"],"is_preprint":false},{"year":2024,"finding":"USP28 interacts with KRT1 and exerts deubiquitination on KRT1, maintaining KRT1 stability. USP28 knockdown results in decreased IFITM3 expression.","method":"IP-MS analysis, Co-IP, immunofluorescence, ubiquitination assay, xenograft model","journal":"Experimental cell research","confidence":"Low","confidence_rationale":"Tier 3 / Weak — Co-IP/IP-MS identification, limited mechanistic follow-up on KRT1-IFITM3 link, single lab","pmids":["40222446"],"is_preprint":false},{"year":2024,"finding":"USP28 stabilizes NOTCH1 intracellular domain (NICD) through direct interaction, independently of FBXW7 and the NICD-PEST domain, enhancing NOTCH1 signaling in CLL. USP28 is deleted in 90% of del(11q) CLL patients, resulting in decreased expression and dysregulation of NOTCH1 target genes.","method":"Co-IP, RNA-Seq of USP28 WT/KO cells, RBPJ ChIP, pharmacological inhibition (AZ1), primary CLL cells","journal":"Leukemia","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP showing FBXW7-independent interaction, RNA-Seq/ChIP for target genes, primary patient cells; single lab","pmids":["40456839"],"is_preprint":false},{"year":2024,"finding":"Crystal structure of USP28 in complex with inhibitors (AZ1, Vismodegib, FT206) reveals a common inhibitor-binding pocket. A key glutamate residue (E366 in USP28/E373 in USP25) plays a central structural role for pocket stability, inhibition, and activity.","method":"Crystal structure determination, mutagenesis of E366, inhibitor binding assays","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structures with multiple inhibitors, mutagenesis validation of key residue; single lab","pmids":["38816515"],"is_preprint":false},{"year":2025,"finding":"DTX3L (E3 ubiquitin ligase) ubiquitinates USP28, leading to its proteasomal degradation, while USP28 counteracts by deubiquitinating both itself (autoubiquitination reversal) and DTX3L. This cross-regulation fine-tunes DSB repair in NHEJ, HR, SSA, and MMEJ pathways.","method":"Co-IP, ubiquitination assay, DSB repair pathway assays (NHEJ, HR, SSA, MMEJ), knockdown rescue experiments","journal":"iScience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutual ubiquitination/deubiquitination demonstrated, multiple DSB repair pathways tested, epistasis; single lab","pmids":["40703443"],"is_preprint":false},{"year":2025,"finding":"USP28 deubiquitinates SIRT1, stabilizing it and promoting osimertinib resistance in NSCLC. Co-IP confirmed the USP28-SIRT1 interaction; SIRT1 knockdown attenuated USP28 overexpression effects.","method":"Co-IP, SIRT1 protein stability assay, knockdown/overexpression rescue experiments, in vivo xenograft","journal":"The Kaohsiung journal of medical sciences","confidence":"Low","confidence_rationale":"Tier 3 / Weak — Co-IP, stability assay, rescue experiment; single lab, limited mechanistic depth","pmids":["40855785"],"is_preprint":false},{"year":2025,"finding":"USP28 deubiquitinates and stabilizes SOX9 protein, and this is counteracted by FBXW7-mediated ubiquitination during olaparib treatment. USP28-stabilized SOX9 binds to promoters of DDR genes (SMARCA4, UIMC1, SLX4), promoting PARP inhibitor resistance in ovarian cancer.","method":"Co-IP, deubiquitination assay, ChIP-Seq, USP28 inhibitor (AZ1), FBXW7 interaction studies","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, deubiquitination assay, ChIP-Seq, inhibitor validation; single lab with multiple methods","pmids":["40240356"],"is_preprint":false},{"year":2025,"finding":"USP28 directly binds to PPARγ, preventing its ubiquitination and degradation, maintaining PPARγ signaling. In the absence of USP28, PPARγ is downregulated and its signaling pathway is inhibited, enhancing cellular defenses against excess fat; USP28 inactivation significantly reduced MASH severity in mice.","method":"Co-IP, ubiquitination assay, liver-specific USP28 KO mice (MCD diet and WD-CCl4 models), pharmacological inhibition","journal":"Molecular therapy : the journal of the American Society of Gene Therapy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination assay, in vivo KO and pharmacological validation; single lab","pmids":["39905730"],"is_preprint":false},{"year":2025,"finding":"USP28 inhibitor CAS-010 disrupts the 53BP1-USP28 interaction, and USP28 catalytic activity contributes to maintaining this interaction. USP28 negatively regulates p53 transactivation in wild-type settings through its deubiquitinase activity.","method":"Crystal structure of USP28 with inhibitor, 53BP1-USP28 interaction assay, p53 transactivation assay, catalytic mutant analysis","journal":"Cell chemical biology","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — crystal structure, catalytic activity linked to 53BP1 interaction; single lab","pmids":["40902594"],"is_preprint":false},{"year":2025,"finding":"USP28 interacts with HEY1 and deubiquitinates its K87 residue, stabilizing HEY1 and enhancing cancer stem-like properties of liver cancer cells. IKKβ phosphorylates HEY1 at S40, facilitating its interaction with USP28.","method":"Co-IP, site-specific deubiquitination (K87), IKKβ phosphorylation assay, in vivo tumor model, anti-PD-1 combination assay","journal":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, site-specific ubiquitination mapping (K87), phosphorylation-dependent interaction, in vivo model; single lab","pmids":["42189121"],"is_preprint":false},{"year":2025,"finding":"USP28 directly interacts with BCR-ABL and promotes the expression of IFITM3 in CML cells, contributing to imatinib resistance. Combined inhibition of USP28 and BCR-ABL reduced IFITM3 expression and tumor progression.","method":"Co-IP (USP28-BCR-ABL interaction), overexpression, small molecule inhibitor combination, xenograft model","journal":"Biomedicine & pharmacotherapy","confidence":"Low","confidence_rationale":"Tier 3 / Weak — Co-IP, limited mechanistic follow-up on how USP28-BCR-ABL interaction leads to IFITM3; single lab","pmids":["38394852"],"is_preprint":false},{"year":2001,"finding":"USP28 was identified as a new ubiquitin-specific protease gene at chromosome 11q23 with preferential expression in heart and muscle. USP28 and USP25 form a new subfamily of deubiquitinating enzymes. Both genes have alternatively spliced exons that could generate protein isoforms with distinct tissue-specific activity.","method":"Database homology search, cDNA library screening, RT-PCR, isoform characterization","journal":"Genome biology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — original gene characterization paper, tissue-specific isoform identification, replicated in subsequent work","pmids":["11597335"],"is_preprint":false}],"current_model":"USP28 is a dimeric deubiquitinase (USP family) that stabilizes multiple oncoproteins—including c-MYC, c-JUN, NOTCH1, ΔNp63, LSD1, HIF-1α, and FOXM1—by removing ubiquitin chains and counteracting SCF(FBW7)-mediated proteasomal degradation, while also forming a 53BP1-dependent complex that deubiquitinates and stabilizes p53 to enforce cell cycle arrest after centrosome loss or prolonged mitosis; its activity is regulated by SUMOylation (inhibitory), SENP1-mediated deSUMOylation (activating), ATR phosphorylation (activating in DNA damage), caspase-8 cleavage (inactivating in cancer), DYRK2-mediated phosphorylation and ubiquitination (destabilizing), and DTX3L-mediated ubiquitination (destabilizing), and its constitutive activity depends on dimerization which is modulated by 53BP1 binding."},"narrative":{"mechanistic_narrative":"USP28 is a constitutively active, dimeric ubiquitin-specific protease that controls cell proliferation, oncoprotein abundance, and a p53-dependent mitotic surveillance checkpoint by reversing proteasomal degradation of selected substrates [PMID:24960159, PMID:30926242, PMID:30926243]. Its founding role is the protection of oncoproteins from SCF(FBW7)-mediated turnover: USP28 binds the nucleoplasmic FBW7alpha isoform and stabilizes c-MYC, and dissociates from FBW7 upon DNA damage to permit MYC decline [PMID:17558397, PMID:17873522], and in vivo it antagonizes FBW7-directed degradation of c-MYC, c-JUN, and NOTCH1 in intestinal proliferation and lineage differentiation [PMID:24960159]. Mechanistically, USP28 recognizes the same phosphodegron motifs targeted by FBW7 (in their unphosphorylated state) and also stabilizes FBW7 itself by reversing its autoubiquitination, so that both loss and excess of USP28 perturb FBW7 substrate balance and promote oncogenic transformation [PMID:25716680, PMID:25437563]. In a second, FBW7-independent arm, USP28 forms a 53BP1-dependent module that directly deubiquitinates and stabilizes p53 to arrest growth after centrosome loss or prolonged mitosis, a mitotic surveillance pathway operating in parallel to the spindle assembly checkpoint and active in the developing brain [PMID:27432897, PMID:27432896, PMID:27371829, PMID:33226141]. The 53BP1 interaction is mediated by the USP28 C-terminal domain in a PLK1- and dimerization-dependent manner; cancer-associated missense mutations in this domain disrupt 53BP1 binding, impair nuclear localization, and destabilize USP28 [PMID:41365927]. USP28 activity is set by its oligomeric state—its dimerizing insertion spatially separates two independently active catalytic domains, distinguishing it from the autoinhibited tetramer of its paralog USP25—and dimerization both limits MYC-driven transcriptional elongation and is selectively bound and stabilized by 53BP1 [PMID:30926242, PMID:30926243, PMID:38227944]. This activity is further tuned by post-translational regulation: N-terminal SUMOylation is inhibitory and SENP1-mediated deSUMOylation is activating under hypoxia [PMID:25359778, PMID:30622440], ATR phosphorylation increases activity during DNA damage [PMID:34611298], nuclear caspase-8 cleaves and inactivates USP28 to abolish p53 stabilization [PMID:31982308], and DYRK2 phosphorylation and DTX3L ubiquitination drive its degradation in reciprocal regulatory loops [PMID:40858801, PMID:40703443]. USP28 is required for induction and maintenance of squamous cell carcinoma through stabilization of ΔNp63, and genetic or small-molecule inhibition of USP28 lowers c-MYC, c-JUN, and ΔNp63 and causes regression of lung tumors, establishing it as a tractable cancer target [PMID:32128997, PMID:34636321]. Beyond these themes, a broad set of substrates extends USP28's deubiquitinase activity across additional oncogenic, metabolic, and stress pathways [PMID:24075993, PMID:34584067, PMID:37202505, PMID:37994595, PMID:39431010].","teleology":[{"year":2001,"claim":"Establishing USP28 as a distinct deubiquitinase gene defined the molecular class and the existence of tissue-specific isoforms before any function was known.","evidence":"homology search and cDNA cloning at 11q23 with isoform characterization","pmids":["11597335"],"confidence":"Medium","gaps":["No catalytic activity or substrate demonstrated","Functional significance of tissue-specific isoforms not tested"]},{"year":2006,"claim":"Discovery of USP28 within 53BP1 complexes answered what links it to the DNA damage response, showing it stabilizes checkpoint factors and enables damage-induced apoptosis.","evidence":"Co-IP of 53BP1 complexes, RNAi knockdown, apoptosis assays in cell lines","pmids":["16901786"],"confidence":"High","gaps":["Direct deubiquitination of named checkpoint substrates not shown in vitro","Mechanism of 53BP1 recruitment not yet defined"]},{"year":2007,"claim":"Identification of the USP28-FBW7alpha interaction answered how MYC is protected from degradation and tied the protease to oncoprotein stability in a subcellularly compartmentalized manner.","evidence":"shRNA screen, reciprocal Co-IP, subcellular fractionation, knockdown/rescue in tumor cells","pmids":["17558397","17873522"],"confidence":"High","gaps":["Whether USP28 acts directly on MYC ubiquitin chains versus via FBW7 not fully resolved","Structural basis of FBW7alpha selectivity unknown"]},{"year":2014,"claim":"In vivo deletion and double-knockout genetics defined USP28 as an FBW7 antagonist that recognizes the same phosphodegron motifs and also stabilizes FBW7 itself, explaining its dual oncogenic/tumor-suppressive behavior.","evidence":"Usp28 knockout and Usp28/Fbw7 double-knockout mice, Ras transformation assays, phosphodegron interaction assays","pmids":["24960159","25437563","25716680"],"confidence":"High","gaps":["Determinants distinguishing FBW7-dependent from FBW7-independent substrates not defined","Quantitative basis of the dose-dependent transformation phenotype unclear"]},{"year":2014,"claim":"Biochemistry of USP28 regulation and chain specificity answered how its activity is restrained, showing inhibitory N-terminal SUMOylation and preference for K11/K48/K63 chains.","evidence":"in vitro deubiquitination assays, SUMO modification, N-terminal truncation and linkage-specificity assays","pmids":["25359778"],"confidence":"High","gaps":["Physiological substrates whose chains are SUMO-regulated not mapped","SUMO sites and writer/eraser enzymes not identified in this study"]},{"year":2016,"claim":"Convergent CRISPR screens placed USP28 in a 53BP1-p53 mitotic surveillance pathway, answering how cells arrest after centrosome loss or prolonged mitosis independently of DNA repair.","evidence":"genome-wide CRISPR centrinone-resistance screens in multiple labs, in vitro p53 deubiquitination, genetic epistasis","pmids":["27432897","27432896","27371829"],"confidence":"High","gaps":["How mitotic delay is sensed and transmitted to USP28 not defined","Relationship between this pathway and FBW7-substrate functions unresolved"]},{"year":2019,"claim":"Crystal structures answered the basis of USP28's constitutive activity, showing it forms an active dimer rather than the autoinhibited tetramer of USP25, and that oligomeric state tunes substrate stabilization.","evidence":"crystal structures of catalytic domains, in vitro activity and oligomerization assays, cell-based substrate stabilization, companion papers","pmids":["30926242","30926243"],"confidence":"High","gaps":["How cellular signals shift oligomeric state was not addressed here","Structure of full-length protein with regulatory domains not determined"]},{"year":2019,"claim":"Demonstrating SENP1-mediated deSUMOylation under hypoxia answered how USP28 activity is switched on to stabilize HIF-1alpha, defining a positive feedback loop.","evidence":"Co-IP, GST pulldown, in vitro deubiquitination, SUMO-deficient mutants, luciferase reporters","pmids":["30622440"],"confidence":"Medium","gaps":["In vivo relevance of the SENP1-USP28-HIF1a loop not tested","Single lab"]},{"year":2020,"claim":"Identification of ΔNp63 as a substrate and caspase-8 as an inactivating protease answered how USP28 maintains squamous carcinoma identity and how its tumor-suppressive p53 arm can be silenced in cancer.","evidence":"in vivo CRISPR SCC mouse models, pharmacological/genetic inhibition, nuclear caspase-8 cleavage and p53 stability assays","pmids":["32128997","31982308"],"confidence":"High","gaps":["Trigger and regulation of nuclear caspase-8 activity not defined","Whether caspase-8 cleavage affects FBW7-substrate arm not tested"]},{"year":2021,"claim":"DNA-damage signaling to USP28 was clarified by showing ATR phosphorylation activates it and links damage repair to ΔNp63 stability in SCC, and pharmacological inhibition validated USP28 as a druggable target.","evidence":"ATR inhibitor and phosphorylation assays, recruitment to damage sites; small-molecule USP28 inhibitor causing tumor regression that phenocopies genetic deletion","pmids":["34611298","34636321"],"confidence":"High","gaps":["ATR phosphosites on USP28 not all mapped","Selectivity of the inhibitor versus USP25 not fully resolved in this context"]},{"year":2023,"claim":"Dimerization mutant and ChIP analyses answered how USP28 oligomeric state couples to transcription, showing monomeric USP28 stabilizes MYC and drives PAF1c recruitment and replication stress, while 53BP1 selectively binds and stabilizes dimers.","evidence":"dimerization mutants, ChIP, DNA synthesis and damage assays, Co-IP with 53BP1","pmids":["38227944"],"confidence":"Medium","gaps":["What physiologically drives monomer-dimer switching in cells","Single lab"]},{"year":2024,"claim":"Mapping the C-terminal, PLK1-dependent 53BP1-binding domain and characterizing cancer mutations answered how the mitotic surveillance complex assembles and how mutations cripple p53 stabilization.","evidence":"cancer mutation analysis, domain interaction mapping, nuclear localization and p53 deubiquitination assays, Co-IP, cell cycle assays","pmids":["41365927"],"confidence":"High","gaps":["How PLK1 phosphorylation is timed within mitosis not detailed","Effect of mutations on FBW7-substrate functions not assessed"]},{"year":2024,"claim":"Inhibitor co-crystal structures answered the structural basis of pharmacological USP28 inhibition, identifying a common binding pocket and a key glutamate critical for activity.","evidence":"crystal structures with AZ1, Vismodegib, FT206; E366 mutagenesis and inhibitor binding assays","pmids":["38816515"],"confidence":"High","gaps":["USP25/USP28 selectivity of the pocket not fully resolved","Allosteric coupling to dimerization not addressed"]},{"year":2025,"claim":"Identification of DTX3L and DYRK2 as enzymes that degrade USP28, with reciprocal USP28 stabilization of each, answered how USP28 abundance is fine-tuned and feeds back on DSB repair and p53 signaling.","evidence":"Co-IP, ubiquitination/phosphorylation assays, DSB repair pathway assays, kinase activity and domain mapping","pmids":["40703443","40858801"],"confidence":"Medium","gaps":["Hierarchy among caspase-8, DYRK2, and DTX3L control of USP28 not defined","Single labs"]},{"year":2025,"claim":"Chemical-biology disruption of the 53BP1-USP28 interface answered whether USP28 catalytic activity sustains the complex, showing catalytic activity maintains 53BP1 binding and that USP28 negatively regulates p53 transactivation in wild-type settings.","evidence":"crystal structure with CAS-010 inhibitor, 53BP1 interaction and p53 transactivation assays, catalytic-mutant analysis","pmids":["40902594"],"confidence":"Medium","gaps":["Reconciliation with prior models where USP28 stabilizes p53 not fully resolved","Single lab"]},{"year":null,"claim":"It remains unclear what governs the choice between USP28's FBW7-substrate stabilization arm and its 53BP1-p53 surveillance arm, and how its many post-translational regulators are integrated to set substrate selectivity in a given cell state.","evidence":"no single study in the timeline resolves the determinants of arm selection","pmids":[],"confidence":"Low","gaps":["No unified model linking oligomeric state, PTMs, and substrate choice","Limited cross-substrate prioritization data","Many substrate claims rest on single-lab Co-IP studies"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[4,11,14,35]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[8,14]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,6,9]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,35]},{"term_id":"GO:0005654","term_label":"nucleoplasm","supporting_discovery_ids":[0]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[10,11,19]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[1,7,39]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,5,6]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[20,23]}],"complexes":["53BP1-USP28 mitotic surveillance complex"],"partners":["TP53BP1","FBXW7","TP53","MYC","DTX3L","DYRK2","CASP8","SENP1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q96RU2","full_name":"Ubiquitin carboxyl-terminal hydrolase 28","aliases":["Deubiquitinating enzyme 28","Ubiquitin thioesterase 28","Ubiquitin-specific-processing protease 28"],"length_aa":1077,"mass_kda":122.5,"function":"Deubiquitinase involved in DNA damage response checkpoint and MYC proto-oncogene stability. Involved in DNA damage induced apoptosis by specifically deubiquitinating proteins of the DNA damage pathway such as CLSPN. Also involved in G2 DNA damage checkpoint, by deubiquitinating CLSPN, and preventing its degradation by the anaphase promoting complex/cyclosome (APC/C). In contrast, it does not deubiquitinate PLK1. Specifically deubiquitinates MYC in the nucleoplasm, leading to prevent MYC degradation by the proteasome: acts by specifically interacting with isoform 1 of FBXW7 (FBW7alpha) in the nucleoplasm and counteracting ubiquitination of MYC by the SCF(FBW7) complex. In contrast, it does not interact with isoform 4 of FBXW7 (FBW7gamma) in the nucleolus, allowing MYC degradation and explaining the selective MYC degradation in the nucleolus. Deubiquitinates ZNF304, hence preventing ZNF304 degradation by the proteasome and leading to the activated KRAS-mediated promoter hypermethylation and transcriptional silencing of tumor suppressor genes (TSGs) in a subset of colorectal cancers (CRC) cells (PubMed:24623306)","subcellular_location":"Nucleus, nucleoplasm","url":"https://www.uniprot.org/uniprotkb/Q96RU2/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/USP28","classification":"Not Classified","n_dependent_lines":3,"n_total_lines":1208,"dependency_fraction":0.0024834437086092716},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/USP28","total_profiled":1310},"omim":[{"mim_id":"610748","title":"UBIQUITIN-SPECIFIC PROTEASE 28; USP28","url":"https://www.omim.org/entry/610748"},{"mim_id":"604736","title":"UBIQUITIN-SPECIFIC PROTEASE 25; USP25","url":"https://www.omim.org/entry/604736"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Nuclear bodies","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"skeletal muscle","ntpm":53.2}],"url":"https://www.proteinatlas.org/search/USP28"},"hgnc":{"alias_symbol":["KIAA1515"],"prev_symbol":[]},"alphafold":{"accession":"Q96RU2","domains":[{"cath_id":"-","chopping":"746-881","consensus_level":"medium","plddt":81.0901,"start":746,"end":881},{"cath_id":"-","chopping":"901-1077","consensus_level":"medium","plddt":81.1167,"start":901,"end":1077}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96RU2","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q96RU2-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q96RU2-F1-predicted_aligned_error_v6.png","plddt_mean":73.06},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=USP28","jax_strain_url":"https://www.jax.org/strain/search?query=USP28"},"sequence":{"accession":"Q96RU2","fasta_url":"https://rest.uniprot.org/uniprotkb/Q96RU2.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q96RU2/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96RU2"}},"corpus_meta":[{"pmid":"17558397","id":"PMC_17558397","title":"The ubiquitin-specific protease USP28 is required for MYC stability.","date":"2007","source":"Nature cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/17558397","citation_count":398,"is_preprint":false},{"pmid":"16901786","id":"PMC_16901786","title":"A role for the deubiquitinating enzyme USP28 in control of the DNA-damage response.","date":"2006","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/16901786","citation_count":292,"is_preprint":false},{"pmid":"33664871","id":"PMC_33664871","title":"Exosomal miR-500a-5p derived from cancer-associated fibroblasts promotes breast cancer cell proliferation and metastasis through targeting USP28.","date":"2021","source":"Theranostics","url":"https://pubmed.ncbi.nlm.nih.gov/33664871","citation_count":195,"is_preprint":false},{"pmid":"27432897","id":"PMC_27432897","title":"53BP1 and USP28 mediate p53 activation and G1 arrest after centrosome loss or extended mitotic duration.","date":"2016","source":"The Journal of cell 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A Unifying Paradigm for Squamous Cell Carcinoma.","date":"2021","source":"Cells","url":"https://pubmed.ncbi.nlm.nih.gov/34685632","citation_count":38,"is_preprint":false},{"pmid":"32578360","id":"PMC_32578360","title":"USP28 and USP25 are downregulated by Vismodegib in vitro and in colorectal cancer cell lines.","date":"2020","source":"The FEBS journal","url":"https://pubmed.ncbi.nlm.nih.gov/32578360","citation_count":32,"is_preprint":false},{"pmid":"34611298","id":"PMC_34611298","title":"Inhibition of USP28 overcomes Cisplatin-resistance of squamous tumors by suppression of the Fanconi anemia pathway.","date":"2021","source":"Cell death and differentiation","url":"https://pubmed.ncbi.nlm.nih.gov/34611298","citation_count":31,"is_preprint":false},{"pmid":"30206969","id":"PMC_30206969","title":"Knockdown of USP28 enhances the radiosensitivity of esophageal cancer cells via the c-Myc/hypoxia-inducible factor-1 alpha pathway.","date":"2018","source":"Journal of cellular biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/30206969","citation_count":29,"is_preprint":false},{"pmid":"25359778","id":"PMC_25359778","title":"Regulation of USP28 deubiquitinating activity by SUMO conjugation.","date":"2014","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/25359778","citation_count":29,"is_preprint":false},{"pmid":"37202505","id":"PMC_37202505","title":"USP28 controls SREBP2 and the mevalonate pathway to drive tumour growth in squamous cancer.","date":"2023","source":"Cell death and differentiation","url":"https://pubmed.ncbi.nlm.nih.gov/37202505","citation_count":28,"is_preprint":false},{"pmid":"29707125","id":"PMC_29707125","title":"The HNF-1β-USP28-Claspin pathway upregulates DNA damage-induced Chk1 activation in ovarian clear cell carcinoma.","date":"2018","source":"Oncotarget","url":"https://pubmed.ncbi.nlm.nih.gov/29707125","citation_count":27,"is_preprint":false},{"pmid":"29545478","id":"PMC_29545478","title":"USP28 Deficiency Promotes Breast and Liver Carcinogenesis as well as Tumor Angiogenesis in a HIF-independent Manner.","date":"2018","source":"Molecular cancer research : MCR","url":"https://pubmed.ncbi.nlm.nih.gov/29545478","citation_count":27,"is_preprint":false},{"pmid":"30485491","id":"PMC_30485491","title":"USP28 contributes to the proliferation and metastasis of gastric cancer.","date":"2018","source":"Journal of cellular biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/30485491","citation_count":26,"is_preprint":false},{"pmid":"30543854","id":"PMC_30543854","title":"The stability and oncogenic function of LIN28A are regulated by USP28.","date":"2018","source":"Biochimica et biophysica acta. 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(nucleoplasmic isoform), stabilizing MYC in the nucleus but not in the nucleolus where FBW7gamma mediates MYC degradation. USP28 is required for MYC stability in human tumor cells.\",\n      \"method\": \"shRNA screen, Co-IP, subcellular fractionation, knockdown/rescue experiments\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, shRNA screen, subcellular localization, replicated across multiple labs subsequently\",\n      \"pmids\": [\"17558397\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"USP28 is required to stabilize Chk2 and 53BP1 in response to DNA damage (double-strand breaks). USP28 was discovered through analysis of 53BP1 complexes and is required for DNA-damage-induced apoptosis through the Chk2-p53-PUMA pathway. USP28 also stabilizes Mdc1, Claspin, and TopBP1 in the DNA-damage checkpoint pathway.\",\n      \"method\": \"Co-IP (53BP1 complex analysis), cell line functional assays, RNAi knockdown, apoptosis assays\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP discovery, multiple substrates identified, pathway epistasis established, replicated in subsequent studies\",\n      \"pmids\": [\"16901786\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Usp28 dissociates from Fbw7alpha in response to UV irradiation, providing a mechanism by which Fbw7-mediated degradation of Myc is enhanced upon DNA damage. Myc decline after DNA damage is mediated by proteasomal degradation via the Fbw7 ubiquitin ligase.\",\n      \"method\": \"Co-IP, UV irradiation, protein stability assays, proteasome inhibitor experiments\",\n      \"journal\": \"Cell cycle (Georgetown, Tex.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP showing dissociation, single lab, two orthogonal methods\",\n      \"pmids\": [\"17873522\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"USP28 antagonizes GSK-3β/Fbw7-dependent HIF-1α degradation. Fbw7 and USP28 reciprocally regulate cell migration and angiogenesis in an HIF-1α-dependent manner.\",\n      \"method\": \"GSK-3β and Fbw7-deficient cell experiments, overexpression/knockdown, migration and angiogenesis assays\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic loss-of-function, multiple cell-based readouts, single lab\",\n      \"pmids\": [\"22144179\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"USP28 is a deubiquitinase of LSD1. USP28 directly interacts with LSD1 and stabilizes it via deubiquitination, counteracting its proteasome-mediated degradation. Knockdown of USP28 results in LSD1 destabilization and suppression of cancer stem cell-like characteristics rescued by ectopic LSD1 expression.\",\n      \"method\": \"siRNA screen against all human deubiquitinases, Co-IP, deubiquitination assay, rescue experiments, in vivo xenograft\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — unbiased DUB screen, Co-IP, in vitro deubiquitination, rescue with LSD1 re-expression, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"24075993\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"USP28 antagonizes c-MYC, c-JUN, and NOTCH1 ubiquitin-dependent degradation in the intestine. Usp28 deletion in mice reduced intestinal proliferation and impaired secretory lineage cell differentiation. USP28 and c-MYC form a positive feedback loop where USP28 is a c-MYC target gene.\",\n      \"method\": \"Murine genetic models (Usp28 knockout), intestinal tumorigenesis models, protein stability assays, mouse lifespan analysis\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo genetic models, multiple substrates validated, c-MYC feedback loop established, replicated across labs\",\n      \"pmids\": [\"24960159\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"USP28 preferentially antagonizes autocatalytic (self-)ubiquitination of Fbw7, stabilizing Fbw7. Monoallelic Usp28 deletion maintains stable Fbw7 and drives Fbw7 substrate degradation, while complete knockout triggers Fbw7 degradation and accumulation of Fbw7 substrates. Both complete loss and overexpression of Usp28 promote Ras-driven oncogenic transformation.\",\n      \"method\": \"Usp28 knockout mouse models (monoallelic and biallelic), embryonic fibroblast assays, Ras transformation assay, protein stability measurements\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo genetic models (mono- and biallelic KO), multiple tissues, functional transformation assay, dose-dependent effects demonstrated\",\n      \"pmids\": [\"25437563\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"USP28 is recruited to double-strand breaks in a manner that requires the tandem BRCT domains of 53BP1. However, USP28 depletion caused only minor DDR defects, and mice lacking USP28 showed normal longevity, immunological development, and radiation responses, indicating USP28 is not a critical factor in double-strand break metabolism.\",\n      \"method\": \"Recruitment assay to DSBs, BRCT domain mutant analysis, USP28-depleted cells, Usp28 knockout mice with radiation challenge\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — domain-specific recruitment established, in vivo knockout mouse model with radiation response, negative result well-controlled\",\n      \"pmids\": [\"24687851\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"SUMO modification of the N-terminal domain of USP28 negatively regulates its deubiquitinating activity. Truncation of the N-terminal domain does not impair deubiquitinating activity on diubiquitin or polyubiquitin chain substrates. USP28 shows chain preference for Lys11-, Lys48-, and Lys63-linked diubiquitin linkages.\",\n      \"method\": \"In vitro deubiquitination assays, SUMO modification assays, N-terminal truncation mutants, linkage-specificity assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro biochemical assays with mutant analysis, chain preference directly measured, single lab\",\n      \"pmids\": [\"25359778\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Usp28 inactivation in the intestine rescues hyperproliferation and impaired goblet/Paneth cell differentiation of Fbw7-deficient mice. Usp28 deficiency corrects accumulation of SCF(Fbw7) substrate proteins (NICD1, c-Jun, c-Myc) independently of Fbw7 presence. Usp28 interacts with unphosphorylated phosphodegron motifs (the same motif recognized by Fbw7 when phosphorylated), offering a mechanistic explanation for identical substrate selection.\",\n      \"method\": \"Intestinal-specific Usp28/Fbw7 double knockout mice, protein stability assays, phosphodegron interaction assays\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo genetic epistasis (double KO), phosphodegron interaction mechanism established, multiple substrates validated\",\n      \"pmids\": [\"25716680\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"USP28 and 53BP1 form a module acting upstream of p53 to arrest cell growth after centrosome loss or prolonged mitosis. USP28 and 53BP1 stabilize p53 after centrosome loss, independently of their role in the DNA damage response. This USP28-53BP1-p53-p21 signaling axis also arrests growth after prolonged prometaphase.\",\n      \"method\": \"Genome-wide CRISPR/Cas9 knockout screen (centrinone resistance), genetic deletion of TP53BP1 and USP28, p53 protein level assays, cell cycle analysis\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — independent CRISPR screens in three different labs (PMIDs 27432897, 27432896, 27371829), epistasis established, replicated across labs\",\n      \"pmids\": [\"27432897\", \"27432896\", \"27371829\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"53BP1 mediates p53 activation independently of its DNA repair activity but requiring USP28. USP28 can directly deubiquitinate p53 in vitro and ectopically stabilize p53 in vivo. 53BP1 and USP28 function in parallel to the spindle assembly checkpoint to select against disturbed or delayed mitosis.\",\n      \"method\": \"CRISPR loss-of-function screen, in vitro deubiquitination assay of p53, in vivo p53 stabilization assay, genetic epistasis\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — direct in vitro deubiquitination of p53 demonstrated, CRISPR screen, in vivo validation, replicated across three concurrent labs\",\n      \"pmids\": [\"27371829\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"USP28 functions through a feedback loop to destabilize RAF family members; loss of USP28 stabilizes BRAF, enhances downstream MAPK activation, and promotes resistance to RAF inhibitor therapy. USP28 is deleted in a proportion of melanoma patients.\",\n      \"method\": \"Cell culture and in vivo models, BRAF stability assays, USP28 knockdown/knockout, MAPK pathway activation measurement\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with defined cellular phenotype, in vivo model, single lab\",\n      \"pmids\": [\"29880484\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"LIN28A protein undergoes ubiquitination and USP28 interacts with and stabilizes LIN28A by reversing its proteasomal degradation via deubiquitinating activity.\",\n      \"method\": \"Co-IP, deubiquitination assay, protein stability (cycloheximide chase), knockdown/overexpression\",\n      \"journal\": \"Biochimica et biophysica acta. Molecular basis of disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination assay, stability assay, single lab with multiple methods\",\n      \"pmids\": [\"30543854\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"USP28 forms a constitutively active dimer through a dimerizing insertion that spatially separates independently active catalytic domains. Unlike USP25, USP28 does not form an autoinhibited tetramer. Oligomeric states of USP28 confirmed in cells and modulating oligomerization affects substrate stabilization in accordance with in vitro activity.\",\n      \"method\": \"Crystal structure (structural determination), biochemical activity assays, cell-based substrate stabilization assays, in vitro oligomerization experiments\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure, in vitro reconstitution, cell-based validation, independently replicated in concurrent companion paper (PMID 30926243)\",\n      \"pmids\": [\"30926242\", \"30926243\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"USP28 is a constitutively active dimer, while USP25 forms an autoinhibited tetramer; structures of the catalytic domains of both proteins determined. USP28 dimerization is intrinsic to its constitutive activity. Cancer-associated USP25 mutations lead to activation in vitro and in vivo.\",\n      \"method\": \"Crystal structure of catalytic domains, biochemical activity assays, in vitro and in vivo oligomerization studies\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structures of both proteins, in vitro reconstitution, cell-based validation, replicated in companion paper\",\n      \"pmids\": [\"30926243\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"SENP1-mediated deSUMOylation of USP28 activates USP28 under hypoxia. SUMOylation of USP28 under normoxia limits its deubiquitinating activity toward HIF-1α. Hypoxia activates USP28 through SENP1-mediated deSUMOylation, creating a positive feedback loop to further accumulate HIF-1α.\",\n      \"method\": \"Co-IP, GST pulldown, in vitro deubiquitination assay, in vivo deSUMOylation assay, luciferase reporter assay, SUMOylation-deficient mutant\",\n      \"journal\": \"Cancer cell international\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, GST pulldown, in vitro deubiquitination, SUMO mutant analysis; single lab\",\n      \"pmids\": [\"30622440\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"USP28 knockdown caused increased ubiquitination of histone H2A at K119, reduced expression of p53, p21 and p16INK4a, and increased cell proliferation, establishing USP28 as a deubiquitinase for histone H2A.\",\n      \"method\": \"RNAi screen of DUBs, USP28 knockdown, H2A ubiquitination assay, cell proliferation assay\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — RNAi screen hit, single Co-IP/knockdown experiment, no direct in vitro deubiquitination of H2A demonstrated, single lab\",\n      \"pmids\": [\"30910399\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Nuclear caspase-8 cleaves and inactivates USP28, preventing USP28 from deubiquitinating and stabilizing wild-type p53. This cleavage results in de facto p53 protein loss, switching cell fate from apoptosis toward mitosis, allowing tumor cells to override the p53-dependent G2/M checkpoint.\",\n      \"method\": \"Nuclear caspase-8 localization studies, Co-IP, USP28 cleavage assay, p53 stability assays, genetic depletion with cell cycle/apoptosis readouts\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — caspase-8 cleavage of USP28 demonstrated biochemically, mechanistic pathway established, p53 deubiquitination linked to substrate stability, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"31982308\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"The 53BP1-USP28 mitotic surveillance pathway is active in developing brain. Depletion of centrosome proteins in neural progenitor cells prolongs mitosis and increases TP53-mediated apoptosis. Inactivation of the mitotic surveillance pathway (53BP1 or USP28 deletion) restored NPC proliferation and brain size without correcting upstream centrosome defects or extended mitosis.\",\n      \"method\": \"Mouse genetic models (53BP1 and USP28 knockout), centrosome protein depletion in NPCs, apoptosis assays, brain size measurements\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo genetic epistasis in brain development, multiple genetic combinations, pathway placement confirmed\",\n      \"pmids\": [\"33226141\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"USP28 stabilizes ΔNp63 and maintains elevated ΔNp63 levels in squamous cell carcinoma by counteracting its proteasome-mediated degradation. Impaired USP28 activity (genetic or pharmacological) abrogates transcriptional identity and suppresses growth of human SCC cells. CRISPR/Cas9 in vivo mouse models show endogenous USP28 is required for both induction and maintenance of lung SCC.\",\n      \"method\": \"CRISPR/Cas9 in vivo mouse models, siRNA, pharmacological inhibition, protein stability assays, gene expression analysis\",\n      \"journal\": \"EMBO molecular medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo CRISPR mouse models, pharmacological and genetic validation, multiple cancer types, replicated in subsequent studies\",\n      \"pmids\": [\"32128997\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ATR phosphorylates USP28 in response to cisplatin treatment, increasing its enzymatic activity. USP28 is recruited to sites of DNA damage in cisplatin-treated cells, and this phosphorylation positively regulates DNA damage repair in SCC by stabilizing ΔNp63.\",\n      \"method\": \"ATR inhibitor studies, phosphorylation assays, USP28 recruitment to DNA damage sites, enzymatic activity assays, knockdown/inhibitor experiments\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ATR-USP28 phosphorylation and activity link established, recruitment to damage sites shown, single lab\",\n      \"pmids\": [\"34611298\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"USP28 deubiquitinates and stabilizes FOXM1, a critical mediator of Wnt/β-catenin signaling. USP28-mediated stabilization of FOXM1 promotes nucleus β-catenin trans-activation and activation of the Wnt/β-catenin pathway in pancreatic cancer.\",\n      \"method\": \"Co-IP, deubiquitination assay, protein stability assay, rescue experiments with FOXM1 re-expression, in vivo xenograft\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, deubiquitination assay, rescue experiment confirming causal role of FOXM1; single lab\",\n      \"pmids\": [\"34584067\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Genetic inactivation of Usp28 induced regression of established murine LSCC lung tumors. A small-molecule USP28 inhibitor decreased c-MYC, c-JUN, and ΔNp63 protein levels and caused regression of autochthonous murine LSCC and human LSCC xenografts, phenocopying genetic deletion.\",\n      \"method\": \"Genetic mouse tumor model, small-molecule inhibitor, tumor regression analysis, protein stability assays\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo genetic and pharmacological validation, autochthonous tumor model plus xenograft, phenocopy by inhibitor confirming on-target mechanism\",\n      \"pmids\": [\"34636321\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"USP28 interacts with and deubiquitinates UCK1, antagonizing KLHL2-mediated polyubiquitination of UCK1 at K81. ATM-mediated phosphorylation of USP28 results in its disassociation from KLHL2 and UCK1 destabilization.\",\n      \"method\": \"Mass spectrometry, Co-IP, ubiquitination assays (identifying K81 site), ATM phosphorylation studies, AML cell line and murine AML model\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS identification, Co-IP, site-specific ubiquitination, ATM-mediated regulation; single lab with multiple methods\",\n      \"pmids\": [\"31938050\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"USP28 interacts with PIM1 and PIM2 kinases, and this interaction increases in response to hypoxia, correlating with reduced PIM ubiquitination and increased protein stability. USP28 overexpression increased PIM protein stability; USP28 knockdown significantly increased ubiquitination of PIM1 and PIM2.\",\n      \"method\": \"Co-IP, ubiquitination assay, overexpression/knockdown, in vivo xenograft\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP, ubiquitination assay, in vivo data; single lab\",\n      \"pmids\": [\"35326457\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"USP28 interacts with NuMA1 and affects centrosome clustering in tetraploid cells. Loss of USP28 reduces checkpoint activation in tetraploid cells, facilitating their proliferation.\",\n      \"method\": \"Mass spectrometry, immunoprecipitation, RNAi screen, cell viability assays\",\n      \"journal\": \"Cellular oncology (Dordrecht, Netherlands)\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single IP-MS finding, NuMA1 interaction not deeply validated mechanistically, single lab\",\n      \"pmids\": [\"34962618\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"USP28 directly binds to mature SREBP2, leading to its deubiquitination and stabilization. USP28 silencing reduces expression of mevalonate pathway enzymes and lowers metabolic flux. USP28 deletion rendered cancer cells highly sensitive to MVP inhibition by statins, rescued by geranyl-geranyl pyrophosphate.\",\n      \"method\": \"Co-IP, deubiquitination assay, metabolic flux analysis, CRISPR/Cas9 deletion, tissue microarray, mouse tumor model\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, deubiquitination, metabolic rescue, CRISPR in vivo model; single lab with multiple orthogonal approaches\",\n      \"pmids\": [\"37202505\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"USP28 dimerization limits USP28 activity and restricts recruitment of the elongation factor PAF1c by MYC. Monomeric USP28 stabilizes MYC and promotes PAF1c recruitment, leading to ectopic DNA synthesis and replication-associated DNA damage. 53BP1 selectively binds USP28 dimers and stimulates dimerization; genotoxic stress diminishes 53BP1-USP28 interaction and promotes USP28 dimer disassembly.\",\n      \"method\": \"Dimerization mutants, ChIP, DNA replication assays, DNA damage assays, Co-IP with 53BP1\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — dimerization mutant analysis, ChIP, DNA synthesis assays; single lab\",\n      \"pmids\": [\"38227944\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"The HNF-1β transcription factor directly regulates USP28 expression as a target gene. USP28 in turn stabilizes Claspin (by deubiquitination), which promotes Chk1 activation and G2/M cell cycle progression in response to DNA damage in ovarian clear cell carcinoma.\",\n      \"method\": \"siRNA knockdown, immunoprecipitation (Claspin ubiquitination), RNAi-mediated gene silencing, Chk1 phosphorylation assay\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP for Claspin stabilization, HNF-1β regulation of USP28 by siRNA only, single lab\",\n      \"pmids\": [\"29707125\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"USP28 directly interacts with PPARα, deubiquitinating and stabilizing PPARα (at Lys152), which promotes Mfn2 transcription to impede mitochondrial morphofunctional defects. This USP28-PPARα-Mfn2 axis was cardioprotective in diabetic hearts.\",\n      \"method\": \"Co-IP/mass spectrometry, protein pull-down, ChIP sequencing, ChIP assays, site-specific ubiquitination mapping, cardiac-specific USP28 KO mice, AAV9-USP28 overexpression\",\n      \"journal\": \"Circulation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — Co-IP/MS, protein pull-down, ChIP-seq, site-specific modification (K152), in vivo KO and OE models; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"37994595\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"DYRK2 phosphorylates USP28, promoting its ubiquitination and proteasomal degradation. Conversely, USP28 deubiquitinates DYRK2, stabilizing it and enhancing its kinase activity. The DYRK2 region 521-541 (particularly T525) plays a crucial role in USP28-mediated DYRK2 stabilization. This reciprocal regulation modulates p53 signaling and apoptotic responses to DNA damage.\",\n      \"method\": \"Phosphorylation assay, ubiquitination assay, Co-IP, kinase activity assay, site-directed mutants, genetic depletion with apoptosis readout\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — bidirectional regulation shown with phosphorylation and deubiquitination assays, domain mapping; single lab\",\n      \"pmids\": [\"40858801\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP28 deubiquitinates and stabilizes TRIM21; cardiomyocyte USP28 deubiquitinating TRIM21 negatively regulates Nrf2 antioxidant response, increasing oxidative stress and promoting cardiac hypertrophy. Identified by Co-IP/mass spectrometry screen.\",\n      \"method\": \"Co-IP/mass spectrometry substrate screen, Co-IP validation, USP28 cardiomyocyte-specific KO mice (Ang II and TAC models), deubiquitination assay, USP28 inhibitor (Otilonium Bromide)\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP/MS substrate identification, in vivo KO model, pharmacological validation; single lab\",\n      \"pmids\": [\"39431010\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP28 interacts with and extends the half-life of MAST1 protein via its deubiquitinating activity. CRISPR/Cas9 knockout of USP28 blocked MAST1-driven cisplatin resistance.\",\n      \"method\": \"CRISPR/Cas9 genome-wide screen, Co-IP, protein half-life assay (cycloheximide chase), deubiquitination assay, xenograft model\",\n      \"journal\": \"Cellular and molecular life sciences : CMLS\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genome-wide CRISPR screen hit, Co-IP, deubiquitination, in vivo xenograft; single lab\",\n      \"pmids\": [\"38498222\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP28 inhibits K48-linked polyubiquitination of STAT3 and reverses its protein degradation in ADPKD. USP28 also directly interacts with and stabilizes c-Myc, both processes synergistically enhancing renal cystogenesis.\",\n      \"method\": \"Co-IP, ubiquitination assay (K48-specific), protein stability assay, pharmacological inhibition, in vivo cystogenesis model\",\n      \"journal\": \"Biochemical pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — K48-specific ubiquitination, Co-IP, in vivo model; single lab\",\n      \"pmids\": [\"36442624\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"The C-terminal domain of USP28 mediates PLK1-dependent binding to 53BP1, a dimerization-driven interaction necessary for mitotic stress memory, p53 stabilization, and cell cycle arrest. Cancer-associated missense mutations in this domain disrupt 53BP1 binding, impair nuclear localization, and destabilize USP28, compromising p53 stabilization. USP28 stabilizes p53 through deubiquitination.\",\n      \"method\": \"Cancer mutation analysis, domain interaction mapping, nuclear localization assays, deubiquitination assay (p53 substrate), Co-IP, cell cycle assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — direct deubiquitination of p53 demonstrated, domain mapping of 53BP1 interaction, PLK1-dependent binding mechanism, isoform characterization; single lab with multiple orthogonal methods\",\n      \"pmids\": [\"41365927\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP28 interacts with KRT1 and exerts deubiquitination on KRT1, maintaining KRT1 stability. USP28 knockdown results in decreased IFITM3 expression.\",\n      \"method\": \"IP-MS analysis, Co-IP, immunofluorescence, ubiquitination assay, xenograft model\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — Co-IP/IP-MS identification, limited mechanistic follow-up on KRT1-IFITM3 link, single lab\",\n      \"pmids\": [\"40222446\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP28 stabilizes NOTCH1 intracellular domain (NICD) through direct interaction, independently of FBXW7 and the NICD-PEST domain, enhancing NOTCH1 signaling in CLL. USP28 is deleted in 90% of del(11q) CLL patients, resulting in decreased expression and dysregulation of NOTCH1 target genes.\",\n      \"method\": \"Co-IP, RNA-Seq of USP28 WT/KO cells, RBPJ ChIP, pharmacological inhibition (AZ1), primary CLL cells\",\n      \"journal\": \"Leukemia\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP showing FBXW7-independent interaction, RNA-Seq/ChIP for target genes, primary patient cells; single lab\",\n      \"pmids\": [\"40456839\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Crystal structure of USP28 in complex with inhibitors (AZ1, Vismodegib, FT206) reveals a common inhibitor-binding pocket. A key glutamate residue (E366 in USP28/E373 in USP25) plays a central structural role for pocket stability, inhibition, and activity.\",\n      \"method\": \"Crystal structure determination, mutagenesis of E366, inhibitor binding assays\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structures with multiple inhibitors, mutagenesis validation of key residue; single lab\",\n      \"pmids\": [\"38816515\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"DTX3L (E3 ubiquitin ligase) ubiquitinates USP28, leading to its proteasomal degradation, while USP28 counteracts by deubiquitinating both itself (autoubiquitination reversal) and DTX3L. This cross-regulation fine-tunes DSB repair in NHEJ, HR, SSA, and MMEJ pathways.\",\n      \"method\": \"Co-IP, ubiquitination assay, DSB repair pathway assays (NHEJ, HR, SSA, MMEJ), knockdown rescue experiments\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutual ubiquitination/deubiquitination demonstrated, multiple DSB repair pathways tested, epistasis; single lab\",\n      \"pmids\": [\"40703443\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP28 deubiquitinates SIRT1, stabilizing it and promoting osimertinib resistance in NSCLC. Co-IP confirmed the USP28-SIRT1 interaction; SIRT1 knockdown attenuated USP28 overexpression effects.\",\n      \"method\": \"Co-IP, SIRT1 protein stability assay, knockdown/overexpression rescue experiments, in vivo xenograft\",\n      \"journal\": \"The Kaohsiung journal of medical sciences\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — Co-IP, stability assay, rescue experiment; single lab, limited mechanistic depth\",\n      \"pmids\": [\"40855785\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP28 deubiquitinates and stabilizes SOX9 protein, and this is counteracted by FBXW7-mediated ubiquitination during olaparib treatment. USP28-stabilized SOX9 binds to promoters of DDR genes (SMARCA4, UIMC1, SLX4), promoting PARP inhibitor resistance in ovarian cancer.\",\n      \"method\": \"Co-IP, deubiquitination assay, ChIP-Seq, USP28 inhibitor (AZ1), FBXW7 interaction studies\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, deubiquitination assay, ChIP-Seq, inhibitor validation; single lab with multiple methods\",\n      \"pmids\": [\"40240356\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP28 directly binds to PPARγ, preventing its ubiquitination and degradation, maintaining PPARγ signaling. In the absence of USP28, PPARγ is downregulated and its signaling pathway is inhibited, enhancing cellular defenses against excess fat; USP28 inactivation significantly reduced MASH severity in mice.\",\n      \"method\": \"Co-IP, ubiquitination assay, liver-specific USP28 KO mice (MCD diet and WD-CCl4 models), pharmacological inhibition\",\n      \"journal\": \"Molecular therapy : the journal of the American Society of Gene Therapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination assay, in vivo KO and pharmacological validation; single lab\",\n      \"pmids\": [\"39905730\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP28 inhibitor CAS-010 disrupts the 53BP1-USP28 interaction, and USP28 catalytic activity contributes to maintaining this interaction. USP28 negatively regulates p53 transactivation in wild-type settings through its deubiquitinase activity.\",\n      \"method\": \"Crystal structure of USP28 with inhibitor, 53BP1-USP28 interaction assay, p53 transactivation assay, catalytic mutant analysis\",\n      \"journal\": \"Cell chemical biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — crystal structure, catalytic activity linked to 53BP1 interaction; single lab\",\n      \"pmids\": [\"40902594\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP28 interacts with HEY1 and deubiquitinates its K87 residue, stabilizing HEY1 and enhancing cancer stem-like properties of liver cancer cells. IKKβ phosphorylates HEY1 at S40, facilitating its interaction with USP28.\",\n      \"method\": \"Co-IP, site-specific deubiquitination (K87), IKKβ phosphorylation assay, in vivo tumor model, anti-PD-1 combination assay\",\n      \"journal\": \"Advanced science (Weinheim, Baden-Wurttemberg, Germany)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, site-specific ubiquitination mapping (K87), phosphorylation-dependent interaction, in vivo model; single lab\",\n      \"pmids\": [\"42189121\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP28 directly interacts with BCR-ABL and promotes the expression of IFITM3 in CML cells, contributing to imatinib resistance. Combined inhibition of USP28 and BCR-ABL reduced IFITM3 expression and tumor progression.\",\n      \"method\": \"Co-IP (USP28-BCR-ABL interaction), overexpression, small molecule inhibitor combination, xenograft model\",\n      \"journal\": \"Biomedicine & pharmacotherapy\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — Co-IP, limited mechanistic follow-up on how USP28-BCR-ABL interaction leads to IFITM3; single lab\",\n      \"pmids\": [\"38394852\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"USP28 was identified as a new ubiquitin-specific protease gene at chromosome 11q23 with preferential expression in heart and muscle. USP28 and USP25 form a new subfamily of deubiquitinating enzymes. Both genes have alternatively spliced exons that could generate protein isoforms with distinct tissue-specific activity.\",\n      \"method\": \"Database homology search, cDNA library screening, RT-PCR, isoform characterization\",\n      \"journal\": \"Genome biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — original gene characterization paper, tissue-specific isoform identification, replicated in subsequent work\",\n      \"pmids\": [\"11597335\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"USP28 is a dimeric deubiquitinase (USP family) that stabilizes multiple oncoproteins—including c-MYC, c-JUN, NOTCH1, ΔNp63, LSD1, HIF-1α, and FOXM1—by removing ubiquitin chains and counteracting SCF(FBW7)-mediated proteasomal degradation, while also forming a 53BP1-dependent complex that deubiquitinates and stabilizes p53 to enforce cell cycle arrest after centrosome loss or prolonged mitosis; its activity is regulated by SUMOylation (inhibitory), SENP1-mediated deSUMOylation (activating), ATR phosphorylation (activating in DNA damage), caspase-8 cleavage (inactivating in cancer), DYRK2-mediated phosphorylation and ubiquitination (destabilizing), and DTX3L-mediated ubiquitination (destabilizing), and its constitutive activity depends on dimerization which is modulated by 53BP1 binding.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"USP28 is a constitutively active, dimeric ubiquitin-specific protease that controls cell proliferation, oncoprotein abundance, and a p53-dependent mitotic surveillance checkpoint by reversing proteasomal degradation of selected substrates [#5, #14]. Its founding role is the protection of oncoproteins from SCF(FBW7)-mediated turnover: USP28 binds the nucleoplasmic FBW7alpha isoform and stabilizes c-MYC, and dissociates from FBW7 upon DNA damage to permit MYC decline [#0, #2], and in vivo it antagonizes FBW7-directed degradation of c-MYC, c-JUN, and NOTCH1 in intestinal proliferation and lineage differentiation [#5]. Mechanistically, USP28 recognizes the same phosphodegron motifs targeted by FBW7 (in their unphosphorylated state) and also stabilizes FBW7 itself by reversing its autoubiquitination, so that both loss and excess of USP28 perturb FBW7 substrate balance and promote oncogenic transformation [#9, #6]. In a second, FBW7-independent arm, USP28 forms a 53BP1-dependent module that directly deubiquitinates and stabilizes p53 to arrest growth after centrosome loss or prolonged mitosis, a mitotic surveillance pathway operating in parallel to the spindle assembly checkpoint and active in the developing brain [#10, #11, #19]. The 53BP1 interaction is mediated by the USP28 C-terminal domain in a PLK1- and dimerization-dependent manner; cancer-associated missense mutations in this domain disrupt 53BP1 binding, impair nuclear localization, and destabilize USP28 [#35]. USP28 activity is set by its oligomeric state\\u2014its dimerizing insertion spatially separates two independently active catalytic domains, distinguishing it from the autoinhibited tetramer of its paralog USP25\\u2014and dimerization both limits MYC-driven transcriptional elongation and is selectively bound and stabilized by 53BP1 [#14, #15, #28]. This activity is further tuned by post-translational regulation: N-terminal SUMOylation is inhibitory and SENP1-mediated deSUMOylation is activating under hypoxia [#8, #16], ATR phosphorylation increases activity during DNA damage [#21], nuclear caspase-8 cleaves and inactivates USP28 to abolish p53 stabilization [#18], and DYRK2 phosphorylation and DTX3L ubiquitination drive its degradation in reciprocal regulatory loops [#31, #39]. USP28 is required for induction and maintenance of squamous cell carcinoma through stabilization of \\u0394Np63, and genetic or small-molecule inhibition of USP28 lowers c-MYC, c-JUN, and \\u0394Np63 and causes regression of lung tumors, establishing it as a tractable cancer target [#20, #23]. Beyond these themes, a broad set of substrates extends USP28's deubiquitinase activity across additional oncogenic, metabolic, and stress pathways [#4, #22, #27, #30, #32].\"\n  ,\n  \"teleology\": [\n    {\n      \"year\": 2001,\n      \"claim\": \"Establishing USP28 as a distinct deubiquitinase gene defined the molecular class and the existence of tissue-specific isoforms before any function was known.\",\n      \"evidence\": \"homology search and cDNA cloning at 11q23 with isoform characterization\",\n      \"pmids\": [\"11597335\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No catalytic activity or substrate demonstrated\", \"Functional significance of tissue-specific isoforms not tested\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Discovery of USP28 within 53BP1 complexes answered what links it to the DNA damage response, showing it stabilizes checkpoint factors and enables damage-induced apoptosis.\",\n      \"evidence\": \"Co-IP of 53BP1 complexes, RNAi knockdown, apoptosis assays in cell lines\",\n      \"pmids\": [\"16901786\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct deubiquitination of named checkpoint substrates not shown in vitro\", \"Mechanism of 53BP1 recruitment not yet defined\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Identification of the USP28-FBW7alpha interaction answered how MYC is protected from degradation and tied the protease to oncoprotein stability in a subcellularly compartmentalized manner.\",\n      \"evidence\": \"shRNA screen, reciprocal Co-IP, subcellular fractionation, knockdown/rescue in tumor cells\",\n      \"pmids\": [\"17558397\", \"17873522\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether USP28 acts directly on MYC ubiquitin chains versus via FBW7 not fully resolved\", \"Structural basis of FBW7alpha selectivity unknown\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"In vivo deletion and double-knockout genetics defined USP28 as an FBW7 antagonist that recognizes the same phosphodegron motifs and also stabilizes FBW7 itself, explaining its dual oncogenic/tumor-suppressive behavior.\",\n      \"evidence\": \"Usp28 knockout and Usp28/Fbw7 double-knockout mice, Ras transformation assays, phosphodegron interaction assays\",\n      \"pmids\": [\"24960159\", \"25437563\", \"25716680\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Determinants distinguishing FBW7-dependent from FBW7-independent substrates not defined\", \"Quantitative basis of the dose-dependent transformation phenotype unclear\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Biochemistry of USP28 regulation and chain specificity answered how its activity is restrained, showing inhibitory N-terminal SUMOylation and preference for K11/K48/K63 chains.\",\n      \"evidence\": \"in vitro deubiquitination assays, SUMO modification, N-terminal truncation and linkage-specificity assays\",\n      \"pmids\": [\"25359778\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological substrates whose chains are SUMO-regulated not mapped\", \"SUMO sites and writer/eraser enzymes not identified in this study\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Convergent CRISPR screens placed USP28 in a 53BP1-p53 mitotic surveillance pathway, answering how cells arrest after centrosome loss or prolonged mitosis independently of DNA repair.\",\n      \"evidence\": \"genome-wide CRISPR centrinone-resistance screens in multiple labs, in vitro p53 deubiquitination, genetic epistasis\",\n      \"pmids\": [\"27432897\", \"27432896\", \"27371829\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How mitotic delay is sensed and transmitted to USP28 not defined\", \"Relationship between this pathway and FBW7-substrate functions unresolved\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Crystal structures answered the basis of USP28's constitutive activity, showing it forms an active dimer rather than the autoinhibited tetramer of USP25, and that oligomeric state tunes substrate stabilization.\",\n      \"evidence\": \"crystal structures of catalytic domains, in vitro activity and oligomerization assays, cell-based substrate stabilization, companion papers\",\n      \"pmids\": [\"30926242\", \"30926243\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How cellular signals shift oligomeric state was not addressed here\", \"Structure of full-length protein with regulatory domains not determined\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Demonstrating SENP1-mediated deSUMOylation under hypoxia answered how USP28 activity is switched on to stabilize HIF-1alpha, defining a positive feedback loop.\",\n      \"evidence\": \"Co-IP, GST pulldown, in vitro deubiquitination, SUMO-deficient mutants, luciferase reporters\",\n      \"pmids\": [\"30622440\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo relevance of the SENP1-USP28-HIF1a loop not tested\", \"Single lab\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identification of \\u0394Np63 as a substrate and caspase-8 as an inactivating protease answered how USP28 maintains squamous carcinoma identity and how its tumor-suppressive p53 arm can be silenced in cancer.\",\n      \"evidence\": \"in vivo CRISPR SCC mouse models, pharmacological/genetic inhibition, nuclear caspase-8 cleavage and p53 stability assays\",\n      \"pmids\": [\"32128997\", \"31982308\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Trigger and regulation of nuclear caspase-8 activity not defined\", \"Whether caspase-8 cleavage affects FBW7-substrate arm not tested\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"DNA-damage signaling to USP28 was clarified by showing ATR phosphorylation activates it and links damage repair to \\u0394Np63 stability in SCC, and pharmacological inhibition validated USP28 as a druggable target.\",\n      \"evidence\": \"ATR inhibitor and phosphorylation assays, recruitment to damage sites; small-molecule USP28 inhibitor causing tumor regression that phenocopies genetic deletion\",\n      \"pmids\": [\"34611298\", \"34636321\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"ATR phosphosites on USP28 not all mapped\", \"Selectivity of the inhibitor versus USP25 not fully resolved in this context\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Dimerization mutant and ChIP analyses answered how USP28 oligomeric state couples to transcription, showing monomeric USP28 stabilizes MYC and drives PAF1c recruitment and replication stress, while 53BP1 selectively binds and stabilizes dimers.\",\n      \"evidence\": \"dimerization mutants, ChIP, DNA synthesis and damage assays, Co-IP with 53BP1\",\n      \"pmids\": [\"38227944\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"What physiologically drives monomer-dimer switching in cells\", \"Single lab\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Mapping the C-terminal, PLK1-dependent 53BP1-binding domain and characterizing cancer mutations answered how the mitotic surveillance complex assembles and how mutations cripple p53 stabilization.\",\n      \"evidence\": \"cancer mutation analysis, domain interaction mapping, nuclear localization and p53 deubiquitination assays, Co-IP, cell cycle assays\",\n      \"pmids\": [\"41365927\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How PLK1 phosphorylation is timed within mitosis not detailed\", \"Effect of mutations on FBW7-substrate functions not assessed\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Inhibitor co-crystal structures answered the structural basis of pharmacological USP28 inhibition, identifying a common binding pocket and a key glutamate critical for activity.\",\n      \"evidence\": \"crystal structures with AZ1, Vismodegib, FT206; E366 mutagenesis and inhibitor binding assays\",\n      \"pmids\": [\"38816515\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"USP25/USP28 selectivity of the pocket not fully resolved\", \"Allosteric coupling to dimerization not addressed\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identification of DTX3L and DYRK2 as enzymes that degrade USP28, with reciprocal USP28 stabilization of each, answered how USP28 abundance is fine-tuned and feeds back on DSB repair and p53 signaling.\",\n      \"evidence\": \"Co-IP, ubiquitination/phosphorylation assays, DSB repair pathway assays, kinase activity and domain mapping\",\n      \"pmids\": [\"40703443\", \"40858801\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Hierarchy among caspase-8, DYRK2, and DTX3L control of USP28 not defined\", \"Single labs\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Chemical-biology disruption of the 53BP1-USP28 interface answered whether USP28 catalytic activity sustains the complex, showing catalytic activity maintains 53BP1 binding and that USP28 negatively regulates p53 transactivation in wild-type settings.\",\n      \"evidence\": \"crystal structure with CAS-010 inhibitor, 53BP1 interaction and p53 transactivation assays, catalytic-mutant analysis\",\n      \"pmids\": [\"40902594\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Reconciliation with prior models where USP28 stabilizes p53 not fully resolved\", \"Single lab\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unclear what governs the choice between USP28's FBW7-substrate stabilization arm and its 53BP1-p53 surveillance arm, and how its many post-translational regulators are integrated to set substrate selectivity in a given cell state.\",\n      \"evidence\": \"no single study in the timeline resolves the determinants of arm selection\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No unified model linking oligomeric state, PTMs, and substrate choice\", \"Limited cross-substrate prioritization data\", \"Many substrate claims rest on single-lab Co-IP studies\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [4, 11, 14, 35]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [8, 14]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 6, 9]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 35]},\n      {\"term_id\": \"GO:0005654\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [10, 11, 19]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [1, 7, 39]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 5, 6]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [20, 23]}\n    ],\n    \"complexes\": [\"53BP1-USP28 mitotic surveillance complex\"],\n    \"partners\": [\"TP53BP1\", \"FBXW7\", \"TP53\", \"MYC\", \"DTX3L\", \"DYRK2\", \"CASP8\", \"SENP1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":9,"faith_total":9,"faith_pct":100.0}}