Journal: bioRxiv

Article Title: E3 ubiquitin ligase HUWE1 mediates K6-linked polyubiquitylation and stabilization of Nrf2 in an HBx-dependent manner, thereby inhibiting hepatitis B virus replication

doi: 10.64898/2026.04.20.719611

Figure Lengend Snippet: (A) HepG2 cells were co-transfected with pCAG-FLAG-Nrf2 and either pRK5-HA-Ub or pRK5-HA-Ub-K6 and with either pUC19-HBV-C-AT_JPN, carrying a 1.3-mer overlength HBV, or pUC19-HBV-C-AT_JPN(ΔHBx), carrying a 1.3-mer overlength HBV lacking HBx gene expression. At 48 h posttransfection, cells were harvested and lysed under denaturing conditions. Cell lysates were immunoprecipitated with anti-FLAG M2 affinity beads, followed by immunoblotting with anti-HA PAb (top panel) or anti-FLAG MAb (second panel). Input samples were immunoblotted with anti-HA PAb (third panel), anti-FLAG MAb (fourth panel), anti-HBx PAb (fifth panel), anti-HBc MAb (sixth panel), and anti-GAPDH MAb (seventh panel). GAPDH served as a loading control. (B) HepG2 cells were co-transfected with pCAG-FLAG-Nrf2, pEF1A-HBx-Myc-His 6 , pCAG-HUWE1, and either pRK5-HA-Ub or pRK5-HA-Ub-K6. At 48 h posttransfection, cells were harvested. Cell lysates were subjected to immunoprecipitation with anti-FLAG beads, followed by immunoblotting with anti-HA PAb (top panel) or anti-Nrf2 MAb (second panel). Input samples were immunoblotted with anti-HA PAb (third panel), anti-Nrf2 MAb (fourth panel), anti-HUWE1 PAb (fifth panel), anti-c-Myc MAb (sixth panel), and anti-GAPDH MAb (seventh panel). GAPDH served as a loading control. (C) HepG2 cells were transfected with 40 nM HUWE1 siRNA or control siRNA. At 24 h post-siRNA transfection, cells were co-transfected with pCAG-FLAG-Nrf2, pRK5-HA-Ub-K6, pEF1A-HBx-Myc-His 6 , and the HUWE1 siRNA-resistant plasmid pCAG-HUWE1_R. At 48 h posttransfection, cells were harvested. Cell lysates were subjected to immunoprecipitation with anti-FLAG beads, followed by immunoblotting with anti-HA PAb (top panel) or anti-FLAG MAb (second panel). Input samples were immunoblotted with anti-HA PAb (third panel), anti-Nrf2 MAb (fourth panel), anti-HUWE1 PAb (fifth panel), anti-c-Myc MAb (sixth panel), or anti-GAPDH MAb (seventh panel). GAPDH served as a loading control. HUWE1_R, siRNA-resistant HUWE1. (D) HepG2 cells were co-transfected with pCAG-FLAG-Nrf2, pRK5-HA-Ub-K6, pEF1A-HBx-Myc-His 6 and either pCAG-HUWE1(WT) or the catalytically inactive mutant pCAG-HUWE1(C4341A). At 48 h posttransfection, cells were harvested. Cell lysates were subjected to immunoprecipitation with anti-FLAG beads, followed by immunoblotting with anti-HA PAb (top panel) or anti-Nrf2 MAb (second panel). Input samples were immunoblotted with anti-HA PAb (third panel), anti-HUWE1 PAb (fourth panel), anti-Nrf2 MAb (fifth panel), anti-c-Myc MAb (sixth panel), or anti-GAPDH MAb (seventh panel). GAPDH served as a loading control. Data in panels A to D are representative of three independent experiments.

Article Snippet: The HUWE1 inhibitor BI8626 (TargetMol Chemicals, Wellesley Hills, MA) were dissolved in dimethyl sulfoxide (DMSO).

Techniques: Transfection, Gene Expression, Immunoprecipitation, Western Blot, Control, Plasmid Preparation, Mutagenesis

Journal: bioRxiv

Article Title: E3 ubiquitin ligase HUWE1 mediates K6-linked polyubiquitylation and stabilization of Nrf2 in an HBx-dependent manner, thereby inhibiting hepatitis B virus replication

doi: 10.64898/2026.04.20.719611

Figure Lengend Snippet: (A) HepG2 cells were co-transfected with pCAG-HA-HUWE1 and pEF1A-HBx-Myc-His 6 . At 48 h posttransfection, cells were harvested. Cell lysates were immunoprecipitated with anti-HA PAb, followed by immunoblotting with anti-c-Myc MAb (top panel) or anti-HA PAb (second panel). Input samples were immunoblotted with anti-HA PAb (third panel), anti-c-Myc MAb (fourth panel), and anti-GAPDH MAb (fifth panel). GAPDH served as a loading control. (B) HepG2 cells were co-transfected with pCAG-HA-HUWE1 and either pUC19-HBV-C-AT_JPN or pUC19-HBV-C-AT_JPN (ΔHBx). At 48 h posttransfection, cells were harvested. Cell lysates were immunoprecipitated with anti-HA PAb, followed by immunoblotting with anti-HBx PAb (top panel), anti-HBc MAb (second panel), or anti-HA PAb (third panel). Input samples were immunoblotted with anti-HA PAb (fourth panel), anti-HBx PAb (fifth panel), anti-HBc Mab (sixth panel), and anti-GAPDH MAb (seventh panel). GAPDH served as a loading control. (C) HepG2 cells were transfected with pEF1A-HBx-Myc-His 6 . At 48 h posttransfection, cells were harvested. Cell lysates were immunoprecipitated with anti-HUWE1 PAb or rabbit IgG (control), followed by immunoblotting with anti-c-Myc MAb (top panel) or anti-HUWE1 PAb (second panel). Input samples were immunoblotted with anti-c-Myc MAb (third panel), anti-HUWE1 PAb (fourth panel), and anti-GAPDH MAb (fifth panel). GAPDH served as a loading control. Data are representative of three independent experiments.

Article Snippet: The HUWE1 inhibitor BI8626 (TargetMol Chemicals, Wellesley Hills, MA) were dissolved in dimethyl sulfoxide (DMSO).

Techniques: Transfection, Immunoprecipitation, Western Blot, Control

Journal: bioRxiv

Article Title: E3 ubiquitin ligase HUWE1 mediates K6-linked polyubiquitylation and stabilization of Nrf2 in an HBx-dependent manner, thereby inhibiting hepatitis B virus replication

doi: 10.64898/2026.04.20.719611

Figure Lengend Snippet: (A) HepG2 cells were transfected with pCAG-HA-HUWE1 alone, pEF1A-HBx-Myc-His6 alone, or both plasmids. At 48 h posttransfection, cells were fixed and subjected to indirect immunofluorescence staining with anti-HUWE1 rabbit PAb followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG (green), and anti-c-Myc mouse MAb followed by Alexa Fluor 594-conjugated goat anti-mouse IgG (red). Nuclei were counterstained with Hoechst 33342 (blue). Scale bar, 10 μm. (B) HepG2 cells were co-transfected with pCAG-HA-HUWE1 and pEF1A-HBx-Myc-His 6 . At 48 h posttransfection, cells were fixed and subjected to an in situ proximity ligation assay (PLA) using both anti-HA rabbit PAb and anti-c-Myc mouse MAb. Nuclei were counterstained with Hoechst 33342 (blue). Scale bar, 10 μm. Data in panels A and B are representative of three independent experiments.

Article Snippet: The HUWE1 inhibitor BI8626 (TargetMol Chemicals, Wellesley Hills, MA) were dissolved in dimethyl sulfoxide (DMSO).

Techniques: Transfection, Immunofluorescence, Staining, In Situ, Proximity Ligation Assay

Journal: bioRxiv

Article Title: E3 ubiquitin ligase HUWE1 mediates K6-linked polyubiquitylation and stabilization of Nrf2 in an HBx-dependent manner, thereby inhibiting hepatitis B virus replication

doi: 10.64898/2026.04.20.719611

Figure Lengend Snippet: (A) HepG2 cells were co-transfected with pCAG-HA-HUWE1, pCAG-FLAG-Nrf2, and pEF1A-HBx-Myc-His 6 . At 48 h posttransfection, cells were harvested. Cell lysates were immunoprecipitated with anti-HA PAb, followed by immunoblotting with anti-Nrf2 MAb (top panel), anti-c-Myc MAb (second panel), and anti-HUWE1 PAb (third panel). Input samples were immunoblotted with the indicated antibodies, and GAPDH served as a loading control. (B) HepG2 cells were co-transfected with pCAG-FLAG-Nrf2 and pCAG-HA-HUWE1 and either pEGFP-C3 (upper panel) or pEGFP-C3-HBx (bottom panel). At 48 h posttransfection, the cells were fixed and incubated with anti-FLAG mouse MAb and anti-HA rabbit PAb, followed by an in situ PLA. Scale bar, 10 μm. (C) HepG2 cells were co-transfected with pCAG-FLAG-Nrf2, pRK5-HA-Ub-K6, pCAG-HUWE1, and pEF1A-HBx-Myc-His 6. At 48 h posttransfection, cells were harvested. Cell lysates were immunoprecipitated with anti-FLAG beads, followed by immunoblotting with anti-HA PAb (top panel) or anti-Nrf2 MAb (second panel). Input samples were immunoblotted with anti-HA PAb (third panel), anti-Nrf2 MAb (fourth panel), anti-HUWE1 PAb (fifth panel), anti-c-Myc MAb (sixth panel), or anti-GAPDH MAb (seventh panel). GAPDH served as a loading control. Data in panels A to C are representative of three independent experiments.

Article Snippet: The HUWE1 inhibitor BI8626 (TargetMol Chemicals, Wellesley Hills, MA) were dissolved in dimethyl sulfoxide (DMSO).

Techniques: Transfection, Immunoprecipitation, Western Blot, Control, Incubation, In Situ

Journal: bioRxiv

Article Title: E3 ubiquitin ligase HUWE1 mediates K6-linked polyubiquitylation and stabilization of Nrf2 in an HBx-dependent manner, thereby inhibiting hepatitis B virus replication

doi: 10.64898/2026.04.20.719611

Figure Lengend Snippet: (A to D) HepG2 cells were transfected with 40 nM HUWE1 siRNA or control siRNA. At 24 h post-siRNA transfection, cells were co-transfected with pCAG-FLAG-Nrf2 and either pEF1A-Myc-His 6 (panels A and B) or pEF1A-HBx-Myc-His 6 (panels C and D). At 48 h posttransfection, cells were treated with 100 µg/mL cycloheximide (CHX) for 0, 1, 2, 4, or 6 h prior to harvesting. Cell lysates were then subjected to immunoblotting with the indicated antibodies. GAPDH served as a loading control. (B, D) Specific signals were quantified by densitometry, and the percentage of remaining FLAG-Nrf2 at each time point was compared with that at time zero. Closed triangles (▴) indicate control siRNA, and closed circles (●) indicate HUWE1 siRNA. Data are mean ± standard errors of the mean (SEM) of three independent experiments. * P < 0.05 compared with the control siRNA by Student’s t test. (E) HepG2 cells were transfected with 40 nM HUWE1 siRNA or control siRNA. At 24 h post-siRNA transfection, cells were further transfected with pCAG-FLAG-Nrf2 and either pEF1A-Myc-His 6 or pEF1A-HBx-Myc-His 6 . At 48 h posttransfection, cells were harvested and subjected to subcellular fractionation. Cytoplasmic and nuclear factions were analyzed by immunoblotting with the indicated antibodies. The relative levels of nuclear Nrf2 were quantified by densitometry and are indicated below each lane. IĸBα and histone H3 served as cytoplasmic and nuclear markers, respectively. The western blots are representative of three independent experiments.

Article Snippet: The HUWE1 inhibitor BI8626 (TargetMol Chemicals, Wellesley Hills, MA) were dissolved in dimethyl sulfoxide (DMSO).

Techniques: Transfection, Control, Western Blot, Fractionation

Journal: bioRxiv

Article Title: E3 ubiquitin ligase HUWE1 mediates K6-linked polyubiquitylation and stabilization of Nrf2 in an HBx-dependent manner, thereby inhibiting hepatitis B virus replication

doi: 10.64898/2026.04.20.719611

Figure Lengend Snippet: (A to C) HepG2 cells were transfected with 40 nM HUWE1 siRNA or control siRNA. At 24 h post-siRNA transfection, cells were co-transfected with pUC19-HBV-D-IND60 and the HUWE1 siRNA-resistant plasmid pCAG-HUWE1_R. At 48 h post-plasmid transfection, cells were harvested. Total RNA was extracted, and HBV RNA (A) and pgRNA (B) were quantified by RT-qPCR and normalized to GAPDH mRNA. Data represent the means ± SEM from three biological replicates. Values for control cells were arbitrarily set to 1.0. * P < 0.05 by Student’s t test. HUWE1_R, siRNA-resistant HUWE1. (C) Cell lysates were subjected to immunoblotting using anti-HUWE1 PAb (top panel), anti-HBc MAb (middle panel), and anti-GAPDH MAb (bottom panel), with GAPDH as the loading control. The relative levels of HBc were quantified by densitometry and are indicated below each lane. (D-F) Hep38.7-Tet cells were transfected with 40 nM HUWE1 siRNA or control siRNA and subsequently cultured for 7 days without doxycycline to induce HBV replication. Total RNA was extracted, and HBV RNA (D) and pgRNA (E) were quantified by RT-qPCR. Data represent the means ± SEM from three biological replicates. Values for control cells were arbitrarily set to 1.0. * P < 0.05 by Student’s t test. (F) Cell lysates were subjected to immunoblotting using anti-HUWE1 PAb (top panel), anti-HBc MAb (middle panel), and anti-GAPDH MAb (bottom panel), with GAPDH as the loading control. The relative levels of HBc were quantified by densitometry and are indicated below each lane. (G) HepG2-NTCP-Myc-His 6 cells were transfected with 40 nM HUWE1 siRNA or control siRNA. At 48 h posttransfection, cells were infected with recombinant HBV expressing the NanoLuc (NL) reporter gene (HBV/NL) at 100 genome equivalents (GEq)/cell. At 4 days postinfection (dpi), cells were lysed to measure NL activity as an indicator of HBV replication. Results represent the mean ± SEM from three biological replicates. Values for control cells were arbitrarily set to 1.0. * P < 0.05 by Student’s t test. (H-J) HepG2-NTCP-Myc-His 6 cells were transfected with 40 nM HUWE1 siRNA or control siRNA. At 48 h posttransfection, cells were infected with HBV at 1,000 GEq/cell, and harvested at 5 dpi. Total RNA was extracted, and HBV RNA (H) and pgRNA (I) were quantified by qPCR. Data represent the means ± SEM from three biological replicates. Values for control cells were arbitrarily set to 1.0. * P < 0.05 by Student’s t test. (J) Cell lysates were subjected to immunoblotting using anti-HUWE1 PAb (top panel), anti-HBc MAb (middle panel), and anti-GAPDH MAb (bottom panel), with GAPDH as the loading control. The relative levels of HBc were quantified by densitometry and are indicated below each lane. (K-L) PXB cells were transfected with 40 nM HUWE1 siRNA or control siRNA. At 48 h posttransfection, cells were infected with HBV at 1,000 GEq/cell and harvested at 5 dpi. Total RNA was extracted, and HUWE1 mRNA (K) was quantified by RT-qPCR. Data represent the means ± SEM from three biological replicates. Values for control cells were arbitrarily set to 1.0. * P < 0.05 by Student’s t test. (L) Cell lysates were subjected to immunoblotting using anti-HUWE1 PAb (top panel), anti-HBc MAb (middle panel), and anti-GAPDH MAb (bottom panel), with GAPDH as the loading control. The relative levels of HBc were quantified by densitometry and are indicated below each lane.

Article Snippet: The HUWE1 inhibitor BI8626 (TargetMol Chemicals, Wellesley Hills, MA) were dissolved in dimethyl sulfoxide (DMSO).

Techniques: Transfection, Control, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Cell Culture, Infection, Recombinant, Expressing, Activity Assay

Journal: bioRxiv

Article Title: E3 ubiquitin ligase HUWE1 mediates K6-linked polyubiquitylation and stabilization of Nrf2 in an HBx-dependent manner, thereby inhibiting hepatitis B virus replication

doi: 10.64898/2026.04.20.719611

Figure Lengend Snippet: (A) HepG2 cells were co-transfected with pCAG-FLAG-Nrf2, pRK5-HA-Ub-K6, and pEF1A-HBx-Myc-His 6 . At 24 h posttransfection, cells were treated with 10 µM BI8626 or equivalent volume of dimethyl sulfoxide (DMSO; vehicle control) for 48 h prior to harvesting. Cell lysates were immunoprecipitated with anti-FLAG beads and subjected to immunoblotting with anti-HA PAb (top panel) or anti-Nrf2 MAb (second panel). Input samples were immunoblotted with the indicated antibodies. GAPDH served as a loading control. (B) Cell viability of HepG2 cells treated with 10 µM BI8626 for 48 h, assessed by CellTiter-Glo v2.0 ATP assay. Results represent the mean values ± SEM from three biological replicates. (C to E) HepG2-NTCP-Myc-His 6 cells were infected with HBV at 1,000 GEq/cell. At 24 h postinfection, cells were treated with 50 µM BI8626 and maintained until harvesting. At 10 dpi, total RNA was extracted, and HBV RNA (C) and pgRNA (D) were quantified by RT-qPCR. Data represent the means ± SEM from three biological replicates. Values for control cells were arbitrarily set to 1.0. * P < 0.05 by Student’s t test. (E) Cell lysates were analyzed by immunoblotting with anti-HUWE1 PAb (top panel), anti-HBc MAb (middle panel), and anti-GAPDH MAb (bottom panel), with GAPDH serving as a loading control. The relative levels of HBc were quantified by densitometry and are indicated below each lane. (F) Cell viability of HepG2-NTCP-Myc-His 6 cells treated with 50 µM BI8626 for 48 h, assessed by CellTiter-Glo 2.0. Data represent the means ± SEM from three biological replicates.

Article Snippet: The HUWE1 inhibitor BI8626 (TargetMol Chemicals, Wellesley Hills, MA) were dissolved in dimethyl sulfoxide (DMSO).

Techniques: Transfection, Control, Immunoprecipitation, Western Blot, ATP Assay, Infection, Quantitative RT-PCR

Journal: bioRxiv

Article Title: E3 ubiquitin ligase HUWE1 mediates K6-linked polyubiquitylation and stabilization of Nrf2 in an HBx-dependent manner, thereby inhibiting hepatitis B virus replication

doi: 10.64898/2026.04.20.719611

Figure Lengend Snippet: Under normal conditions (upper panel), HUWE1 does not associate with Nrf2. Keap1 bridges Nrf2 to the Cullin3 E3 ligase complex, leading to K48-linked polyubiquitylation and proteasomal degradation of Nrf2. Upon HBV infection (lower panel), HBx recruits HUWE1 to form a ternary HUWE1/HBx/Nrf2 complex in the cytoplasm. HUWE1 then adds K6-linked polyubiquitylation chains to Nrf2, switching the ubiquitin code from K48 to K6 linkage. K6-linked polyubiquitylation stabilizes Nrf2, promotes its nuclear translocation, and leads transcriptional activation of antioxidant response element (ARE)-driven genes. In the nucleus, Nrf2 suppresses HBV core, PreS1, and PreS2/S promoter activities, thereby suppressing HBV replication. Meanwhile, Nrf2 also suppresses HBV replication through its target gene Prdx1, which facilitates HBV RNA degradation, as we reported previously .

Article Snippet: The HUWE1 inhibitor BI8626 (TargetMol Chemicals, Wellesley Hills, MA) were dissolved in dimethyl sulfoxide (DMSO).

Techniques: Infection, Ubiquitin Proteomics, Translocation Assay, Activation Assay

Journal: The Journal of Biological Chemistry

Article Title: CLDN5 as a novel modulator of podocyte adhesion to extracellular matrix via β1-integrin binding

doi: 10.1016/j.jbc.2026.111163

Figure Lengend Snippet: CLDN5 protects β1-integrin from HUWE1-mediated ubiquitination and degradation . A , Western blot with densitometric analysis of β1-integrin in isolated podocytes treated with 20 μg/ml cycloheximide (CHX) for 0, 4, and 8 h, CHX was used to inhibit new protein synthesis and assess β1-integrin protein stability. The line graph shows relative β1-integrin protein levels, quantified as the β1-integrin/β-actin ratio, with the 0-h point arbitrarily set to 100%. B , Western blot with densitometric analysis of β1-integrin in isolated podocytes treated with vehicle (control), 10 μM MG132, or 20 μM Chloroquine (CQ) for 8 h (n = 3). C , Ubiquitination levels of β1-integrin in isolated podocytes from WT and Cldn5 -KO mice (n = 3). D , mass spectrometry analysis of β1-integrin immunocomplexes precipitated from isolated glomerulus. E , single-cell RNA sequencing data for Huwe1 from healthy mouse kidneys, obtained from the Humphreys Lab's Online Analyzer for Kidney Single Cell Datasets. Pod, podocyte; MC, mesangial cell; EC, endothelial cell; PT, proximal tubule; LH (DL), descending loop of Henle; LH (AL), ascending loop of Henle; DCT, distal convoluted tubule; CNT, connecting tubule; CD-PC, collecting duct principal cell; IC-A, intercalated cells-α; IC-B, intercalated cells-β; Mø, macrophage. F , immunofluorescence analysis of HUWE1 in primary podocyte. Scale bars, 20 μm. ( G and H ) Co-immunoprecipitation (Co-IP) of CLDN5 and β1-integrin in isolated glomerulus from Cldn5 -KO ( G ) and WT ( H ) mice to assess the interaction of endogenous CLDN5 with β1-integrin. I , Co-IP of CLDN5 and HUWE1 in doubly transfected HEK293 cells shows no interaction between CLDN5 and HUWE1. ( J and K ) Western blot with densitometric analysis of β1-integrin in the isolated WT podocytes treated with lentivirus carrying either control- or Huwe1 -shRNA ( J ), or lentivirus carrying either control or Huwe1 coding sequence ( K ) (n = 3). L , Western blot with densitometric analysis of β1-integrin in the isolated Cldn5 -KO podocytes treated with lentivirus carrying either control- or Huwe1 -shRNA (n = 3). M , cellular abundance of β1-integrin in isolated podocytes co-transfected with Huwe1 and either WT or lysine-mutant β1-integrin gene (n = 3). For all immunoprecipitation or Co-IP analyses, antibodies used for immunoprecipitation are shown above the lanes; antibody for blot visualization is shown on the right . Control IPs with singly transfected cells (β1-integrin or CLDN alone) confirm specificity and exclude nonspecific binding. Whole-cell lysates (input) from singly and doubly transfected cells demonstrate expression levels of CLDNs and β1-integrin. Data are presented as mean ± SD. Statistical significance was determined by two-way ANOVA followed by Tukey’s post-test ( A , B ), two-tailed Student’s unpaired t test analysis ( C , J – L ), one-way ANOVA followed by Tukey’s post-test ( M ).

Article Snippet: For gene knockdown study, shRNA hairpin oligonucleotides targeting mouse Huwe1 ( NM_021523 ) and Cldn5 ( NM_013805 ) were synthesized by Sangon Biotech.

Techniques: Ubiquitin Proteomics, Western Blot, Isolation, Control, Mass Spectrometry, Single Cell, RNA Sequencing, Immunofluorescence, Immunoprecipitation, Co-Immunoprecipitation Assay, Transfection, shRNA, Sequencing, Mutagenesis, Binding Assay, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: Guardian ubiquitin E3 ligases target cancer-associated APOBEC3 deaminases for degradation to promote human genome integrity

doi: 10.1038/s41467-026-68420-5

Figure Lengend Snippet: A A monoclonal RKO cell line expressing DOX-inducible Cas9-P2A-BFP and a constitutive mCherry-A3H-II-P2A-EGFP-A3H-I dual reporter was generated (RKO-DOX-Cas9-dualA3H). EGFP-A3H-I and mCherry-A3H-II are synthesized in equimolar amounts, but EGFP-A3H-I shows low steady-state levels due to proteasomal degradation. B Schematic of the CRISPR/Cas9 FACS-based screening strategy. Cells were transduced with an sgRNA library targeting ubiquitin-proteasome and autophagy-related genes, selected with G418, induced with DOX for 3 or 6 days, and sorted for the top and bottom 1–2% of EGFP or mCherry fluorescence. sgRNA abundance was determined by next-generation sequencing and compared to unsorted controls. C Genes enriched in EGFP-A3H-I high populations at day 6 post induction, with adjusted p-values from MaGECK FDR analysis of three independent replicate sorts. D Heatmap of top genes on log 2 fold-change and p-value grouped by functional categories. Genes enriched in EGFP-A3H-I high cell populations 6 days post Cas9 induction with a log 2 fold-change >0.6, which were not enriched in mCherry high or GFP low on either day 3 (LFC > 0.45) or day 6 (LFC > 0.6). Adjusted p-values are based on MaGECK FDR analysis of three independent replicate sorts. Dashed lines indicate a log 2 fold-change <0.6. E RKO-DOX-Cas9-dualA3H cells were transduced with individual sgRNAs, induced with DOX for 6 days, and analyzed for EGFP-A3H-I and mCherry-A3H-II mean fluorescence intensity by flow cytometry, with F quantification (means and SD, two-way ANOVA with Šídák correction, ns: p ≥ 0.05, n = 3). G RKO cells expressing DOX-inducible Cas9 were transduced with sgRNAs targeting UBR4, UBR5, or HUWE1, individually or in combination. Following 6 days of DOX treatment, endogenous A3B protein levels were assessed by WB and H quantified (n = 2 biological replicates). Source data are provided as a file.

Article Snippet: Membranes were blocked in 5% BSA in PBS-T for 1 h at RT, and subsequently incubated with primary antibodies diluted in 5% BSA overnight at 4 °C (ARP10 Antibody (Novus, 1:1000), Anti-APOBEC3B Antibody (Abcam, 1:1000), Anti-APOBEC3G (D9C6Z) Rabbit mAb (Cell Signaling Technologies, 1:1000), Anti-MYC antibody (Sigma-Aldrich, 1:5000), HA-Tag (C29F4) Rabbit mAb (Cell Signaling Technology, 1:1000), HA-Tag (6E2) Mouse mAb (Cell Signaling Technology, 1:1000), OLLAS Epitope Tag Antibody (L2) (Novus, 1:4000), Anti-Penta·His Antibody (Quiagen, 1:10.000), LC3B Antibody (Cell Signaling Technology, 1:1000), Ubiquitin (P4D1) (Santa Cruz Biotechnology, 1:1000), Anti-UBR4/p600 antibody (Abcam, 1:1000), Rabbit anti-EDD1 Antibody (Bethyl, 1:1000), Rabbit anti-Lasu1/Ureb1 Antibody (HUWE1) (Bethyl, 1:1000), Monoclonal Anti-α-Tubulin antibody produced in mouse (Sigma-Aldrich, 1:1000), Lamin A/C Antibody (E-1) (Santa Cruz Biotechnology, 1:1000), Monoclonal Anti-Vinculin antibody (Sigma-Aldrich, 1:1000), Anti-beta Actin antibody (HRP) (Abcam, 1:20000)).

Techniques: Expressing, Generated, Synthesized, CRISPR, Transduction, Ubiquitin Proteomics, Fluorescence, Next-Generation Sequencing, Functional Assay, Flow Cytometry

Journal: Nature Communications

Article Title: Guardian ubiquitin E3 ligases target cancer-associated APOBEC3 deaminases for degradation to promote human genome integrity

doi: 10.1038/s41467-026-68420-5

Figure Lengend Snippet: A Overview of TurboID principle. B – F Polyclonal RKO-DOX-TID-A3H-I/II/GFP were treated with DOX for 2 days to achieve similar protein levels. Cells were treated with EPOX for 5 h. and supplemented with biotin during the last 15 min. Biotinylated proteins were purified under denaturing conditions and quantified by nLC-MS/MS (mean and SD, n = 3 biological replicates, moderated t-statistics via the limma-trend method with Benjamini–Hochberg multiple testing correction). B Differentially enriched proteins in A3H-I/GFP (light blue, LFC > 1, p-value < 0.01) and A3H-II/GFP (dark blue, LFC > 1, p-value < 0.01) were compared. C GO terms for biological processes (GO:BP) of differentially enriched proteins in A3H-I/GFP (light blue, LFC > 1, p-value < 0.01, input: 170 factors derived from B ) and A3H-II/GFP (dark blue, LFC > 1, p-value < 0.01, input: 52 factors derived from B ). D Differential expression of TID-A3H-I, or ( E ) TID-A3H-II interactors relative to TID-GFP (n = 3). Light blue dots mark factors of enriched proteins in TID-A3H-I samples relative to TID-GFP. Highlighted are the top 20 A3H-I-specific proteins displayed according to the following criteria: A3H-I/GFP LFC > 1, p-value < 0.01, which are not enriched in A3H-II/GFP LFC > 1, p-value < 0.01. F Heatmap of enriched proteins in TID-A3H-I samples, or TID-A3H-II samples relative to TID-GFP. Top 20 A3H-I-specific proteins displayed (A3H-I/GFP LFC > 1, p-value < 0.01), which are not enriched in A3H-II/GFP (LFC > 1, p-value < 0.01). HEK-293T cells transfected with different amounts of plasmids encoding G 3xHA-A3H-I/II, or H 3xHA-GFP or A3B-3xHA to achieve similar steady-state protein levels. 3xHA-tagged proteins were immunoprecipitated, and their interaction with endogenous UBR5 and HUWE1 determined by WB. Source data are provided as a file.

Article Snippet: Membranes were blocked in 5% BSA in PBS-T for 1 h at RT, and subsequently incubated with primary antibodies diluted in 5% BSA overnight at 4 °C (ARP10 Antibody (Novus, 1:1000), Anti-APOBEC3B Antibody (Abcam, 1:1000), Anti-APOBEC3G (D9C6Z) Rabbit mAb (Cell Signaling Technologies, 1:1000), Anti-MYC antibody (Sigma-Aldrich, 1:5000), HA-Tag (C29F4) Rabbit mAb (Cell Signaling Technology, 1:1000), HA-Tag (6E2) Mouse mAb (Cell Signaling Technology, 1:1000), OLLAS Epitope Tag Antibody (L2) (Novus, 1:4000), Anti-Penta·His Antibody (Quiagen, 1:10.000), LC3B Antibody (Cell Signaling Technology, 1:1000), Ubiquitin (P4D1) (Santa Cruz Biotechnology, 1:1000), Anti-UBR4/p600 antibody (Abcam, 1:1000), Rabbit anti-EDD1 Antibody (Bethyl, 1:1000), Rabbit anti-Lasu1/Ureb1 Antibody (HUWE1) (Bethyl, 1:1000), Monoclonal Anti-α-Tubulin antibody produced in mouse (Sigma-Aldrich, 1:1000), Lamin A/C Antibody (E-1) (Santa Cruz Biotechnology, 1:1000), Monoclonal Anti-Vinculin antibody (Sigma-Aldrich, 1:1000), Anti-beta Actin antibody (HRP) (Abcam, 1:20000)).

Techniques: Purification, Tandem Mass Spectroscopy, Derivative Assay, Quantitative Proteomics, Transfection, Immunoprecipitation

Journal: Nature Communications

Article Title: Guardian ubiquitin E3 ligases target cancer-associated APOBEC3 deaminases for degradation to promote human genome integrity

doi: 10.1038/s41467-026-68420-5

Figure Lengend Snippet: A RKO-mCherry-P2A-EGFP-A3H cells expressing the indicated EGFP-tagged A3H variants were treated for 5 h with EPOX or CHX. mCherry and EGFP-A3H MFI was measured by flowcytometry (means and SD, two-way ANOVA with Tukey-correction, ns: p ≥ 0.05, n = 3). B HEK-293T cells were transfected with varying amounts of plasmids encoding 3xHA-tagged A3H to achieve comparable protein levels. After 5 h of EPOX treatment, sub-cellular fractions were analyzed by WB and quantified (means and SD, two-way ANOVA with Tukey correction, ns: p ≥ 0.05, n = 3). HEK-293T cells were transfected as in ( B ), treated with EPOX, and 3xHA-tagged proteins were immunoprecipitated to assess ( C ) ubiquitination or ( D ) interaction with UBR5 and HUWE1 by WB. E , F HEK-293T cells transiently expressing mCherry-P2A-3xHA-tagged A3G WT or RNA-binding mutants were treated with EPOX for 5 h, followed by WB analysis and quantification (means and SD, multiple unpaired two-sided t-tests with Šídák correction, ns: p ≥ 0.05, n = 3). G , H Cells were transfected as in ( E ) with adjusted plasmid amounts to equalize protein levels, followed by EPOX treatment and immunoprecipitation to assess ( G ) ubiquitination or ( H ) interaction with UBR5 and HUWE1. I HEK-293T cells expressing A3B-3xHA were treated with EPOX, with or without RNase A treatment prior to immunoprecipitation. J , K HEK-293T cells were transfected with different amounts of plasmids expressing the indicated 3xHA-tagged A3H constructs to achieve similar steady-state protein levels. Following 5 h. of EPOX treatment, cellular fractions were extracted, analyzed by WB, and quantified (means and SD, 2-way ANOVA; not corrected for multiple comparisons, ns: p ≥ 0.05, n = 3 biological replicates for ( J ), n = 2 biological replicates for ( K )). Source data are provided as a file.

Article Snippet: Membranes were blocked in 5% BSA in PBS-T for 1 h at RT, and subsequently incubated with primary antibodies diluted in 5% BSA overnight at 4 °C (ARP10 Antibody (Novus, 1:1000), Anti-APOBEC3B Antibody (Abcam, 1:1000), Anti-APOBEC3G (D9C6Z) Rabbit mAb (Cell Signaling Technologies, 1:1000), Anti-MYC antibody (Sigma-Aldrich, 1:5000), HA-Tag (C29F4) Rabbit mAb (Cell Signaling Technology, 1:1000), HA-Tag (6E2) Mouse mAb (Cell Signaling Technology, 1:1000), OLLAS Epitope Tag Antibody (L2) (Novus, 1:4000), Anti-Penta·His Antibody (Quiagen, 1:10.000), LC3B Antibody (Cell Signaling Technology, 1:1000), Ubiquitin (P4D1) (Santa Cruz Biotechnology, 1:1000), Anti-UBR4/p600 antibody (Abcam, 1:1000), Rabbit anti-EDD1 Antibody (Bethyl, 1:1000), Rabbit anti-Lasu1/Ureb1 Antibody (HUWE1) (Bethyl, 1:1000), Monoclonal Anti-α-Tubulin antibody produced in mouse (Sigma-Aldrich, 1:1000), Lamin A/C Antibody (E-1) (Santa Cruz Biotechnology, 1:1000), Monoclonal Anti-Vinculin antibody (Sigma-Aldrich, 1:1000), Anti-beta Actin antibody (HRP) (Abcam, 1:20000)).

Techniques: Expressing, Transfection, Immunoprecipitation, Ubiquitin Proteomics, RNA Binding Assay, Plasmid Preparation, Construct

Journal: Nature Communications

Article Title: Guardian ubiquitin E3 ligases target cancer-associated APOBEC3 deaminases for degradation to promote human genome integrity

doi: 10.1038/s41467-026-68420-5

Figure Lengend Snippet: A 10x-His-MBP-A3H-I, 10x-His-MBP-A3H-II and 10xHis-MBP-A3H-II-RBM were expressed in E. coli , purified by HisTrap and subsequent gel filtration, after which purified proteins were analyzed by PAGE and Coomassie staining. B Purified proteins were analyzed by denaturing Urea-TBE PAGE followed by SYBR Gold staining. In vitro ubiquitination assays were performed with recombinant C UBR5, D HUWE1, or E UBR4 and WT A3H-I/II or A3H-RBM as substrates, and in the presence of DyLight488-labeled recombinant ubiquitin. Subsequently, A3H was immunoprecipitated using anti-MBP-coupled beads and the ubiquitination pattern visualized by fluorescent imaging for DyLight488. In vitro ubiquitination assay with recombinant F UBR5, G HUWE1, H UBR4 and A3H-I/II WT or A3H-RBM in the absence or presence of RNase A. Ubiquitinated A3H was visualized as in ( C – E ). I , J Sucrose gradient binding assays of UBR5 and recombinant A3H proteins. J Recombinant A3H was pre-incubated with RNase A. K Analytical size exclusion chromatography of C-terminally tagged hHUWE1 (inactive) and recombinant A3H. Source data are provided as a file.

Article Snippet: Membranes were blocked in 5% BSA in PBS-T for 1 h at RT, and subsequently incubated with primary antibodies diluted in 5% BSA overnight at 4 °C (ARP10 Antibody (Novus, 1:1000), Anti-APOBEC3B Antibody (Abcam, 1:1000), Anti-APOBEC3G (D9C6Z) Rabbit mAb (Cell Signaling Technologies, 1:1000), Anti-MYC antibody (Sigma-Aldrich, 1:5000), HA-Tag (C29F4) Rabbit mAb (Cell Signaling Technology, 1:1000), HA-Tag (6E2) Mouse mAb (Cell Signaling Technology, 1:1000), OLLAS Epitope Tag Antibody (L2) (Novus, 1:4000), Anti-Penta·His Antibody (Quiagen, 1:10.000), LC3B Antibody (Cell Signaling Technology, 1:1000), Ubiquitin (P4D1) (Santa Cruz Biotechnology, 1:1000), Anti-UBR4/p600 antibody (Abcam, 1:1000), Rabbit anti-EDD1 Antibody (Bethyl, 1:1000), Rabbit anti-Lasu1/Ureb1 Antibody (HUWE1) (Bethyl, 1:1000), Monoclonal Anti-α-Tubulin antibody produced in mouse (Sigma-Aldrich, 1:1000), Lamin A/C Antibody (E-1) (Santa Cruz Biotechnology, 1:1000), Monoclonal Anti-Vinculin antibody (Sigma-Aldrich, 1:1000), Anti-beta Actin antibody (HRP) (Abcam, 1:20000)).

Techniques: Purification, Filtration, Staining, In Vitro, Ubiquitin Proteomics, Recombinant, Labeling, Immunoprecipitation, Imaging, Binding Assay, Incubation, Size-exclusion Chromatography

Journal: Nature Communications

Article Title: Guardian ubiquitin E3 ligases target cancer-associated APOBEC3 deaminases for degradation to promote human genome integrity

doi: 10.1038/s41467-026-68420-5

Figure Lengend Snippet: A Schematic of mutREAD sequencing to detect APOBEC signature mutations. UNG2-deficient RKO cells expressing DOX-inducible Cas9 and with or without mCherry-P2A-3xHA-A3H-I overexpression were transduced with sgRNAs targeting UBR4 , UBR5 , or HUWE1 . Following sorting for sgRNA-positive cells, gene editing was induced with DOX for up to 10 days, after which genomic DNA was isolated for mutREAD sequencing. B Fraction of APOBEC signature related mutations over all identified mutations in mutREAD sequencing control samples (n = 3 biological replicates, two-sided Fisher’s exact test, no adjustments for multiple comparisons, test statistic = Odds Ratio = 0.5588, p-value = 4.41 × 10 −72 , CI low = 0.5239, CI high = 0.5960). C Best-subset signature refitting of samples expressing exogenous A3H-I, averaged per genotype, using APOBEC-associated and colon carcinoma signatures. Each bar represents the mean of three technical replicates scaled to one. D Fraction of APOBEC-related mutations in samples with exogenous A3H-I expression (n = 3 biological replicates, two-sided Fisher’s exact test with Bonferroni correction; odds ratio, 95% confidence intervals, and exact p-values shown). E Pentanucleotide context preference of APOBEC mutations in control samples expressing A3H-I. F Schematic of mutational signature analysis using PCAWG data. G TCGA and ICGC cancer samples were grouped by wild-type or mutated status of UBR4 , UBR5 , and HUWE1 . Mutational signatures were normalized to the total number of mutations in each sample. The level of SBS13 APOBEC signature was compared between the groups. “E3s comb ” are all samples in which at least one of the E3s is mutated. Wilcoxon rank-sum test, two-sided, ns: p > 0.05, n = 2703 samples). Box plots show median, interquartile range, and whiskers to 1.5× IQR; y-axis is log10-transformed. H Model: lack of RNA-binding determines A3 nuclear localization and simultaneously targeting by th eE3 ligases, ensuring low nuclear A3 levels.

Article Snippet: Membranes were blocked in 5% BSA in PBS-T for 1 h at RT, and subsequently incubated with primary antibodies diluted in 5% BSA overnight at 4 °C (ARP10 Antibody (Novus, 1:1000), Anti-APOBEC3B Antibody (Abcam, 1:1000), Anti-APOBEC3G (D9C6Z) Rabbit mAb (Cell Signaling Technologies, 1:1000), Anti-MYC antibody (Sigma-Aldrich, 1:5000), HA-Tag (C29F4) Rabbit mAb (Cell Signaling Technology, 1:1000), HA-Tag (6E2) Mouse mAb (Cell Signaling Technology, 1:1000), OLLAS Epitope Tag Antibody (L2) (Novus, 1:4000), Anti-Penta·His Antibody (Quiagen, 1:10.000), LC3B Antibody (Cell Signaling Technology, 1:1000), Ubiquitin (P4D1) (Santa Cruz Biotechnology, 1:1000), Anti-UBR4/p600 antibody (Abcam, 1:1000), Rabbit anti-EDD1 Antibody (Bethyl, 1:1000), Rabbit anti-Lasu1/Ureb1 Antibody (HUWE1) (Bethyl, 1:1000), Monoclonal Anti-α-Tubulin antibody produced in mouse (Sigma-Aldrich, 1:1000), Lamin A/C Antibody (E-1) (Santa Cruz Biotechnology, 1:1000), Monoclonal Anti-Vinculin antibody (Sigma-Aldrich, 1:1000), Anti-beta Actin antibody (HRP) (Abcam, 1:20000)).

Techniques: Sequencing, Expressing, Over Expression, Transduction, Isolation, Control, Transformation Assay, RNA Binding Assay