Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Nonproteolytic ubiquitination regulates chromatin occupancy by the NCoR/SMRT/HDAC3 corepressor complex in MCF-7 breast cancer cells.

doi: 10.1073/pnas.2502805122

Figure Lengend Snippet: Fig. 5. (A) Western blotting with anti-TRAF6 Ab to visualize the nuclear translocation of TRAF6 in MCF-7 cells treated with IL-1β for 5 min. WBs for HDAC2 and β-tubulin were run on the cytoplasmic and nuclear extracts as a control. (B) TRAF6 interaction with NCoR in IL-1β-treated MCF7 cells by immunoprecipitation with anti-NCoR Ab. (C) Graphic representation of the NCoR protein showing the putative TRAF6 interaction motif, and sequence alignment of similar motifs found in human RIP2, IRAK2, and NCoR across species. (D) Interaction between TRAF6 and the identified interaction motif in HEK293T cells transfected with Myc-TRAF6 and wild type (Left) or PxExxDAxAxxA mutant (Right) GAL4-NCoR516-811 plasmids. Immunoprecipitation was performed either with anti-Myc, anti-GAL4, or control IgG, and blotted with anti-GAL4, anti-TRAF6, or anti-Myc Ab. (E) ChIP experiment showing rapid and transient recruitment of TRAF6 and Ubc13 to the BMP7 promoter in response to IL-1β in MCF7 pretreated with E2 for 1 h. (F) Validation of siRNA efficiency in MCF7 cells. Cells are transfected with siRNA (siCtl, siTRAF6, or siUbc13) for 48 h and relative expression measured by RTqPCR, with normalization to b-actin. (G) BMP7 derepression by IL-1β, as measured by RT-PCR, requires both TRAF6 and Ubc13. MCF7 cells transfected with either control, TRAF6, or Ubc13 siRNA, were treated with E2 and/or IL-1β for 6 h. (H) Overexpression of dominant-negative TRAF6 or mutated TAB2 abrogates derepression by IL-1β in U2OS-ERα cells transiently transfected with BMP7promoter reporter and treated with E2 and vehicle (PBS) or IL-1β for 36 h. Luciferase activity was assayed and normalized to b-gal activity. (I) MEKK1’s ubiquitin-interacting motif (UIM) interacts with K63-linked, but not K48-linked, polyubiquitin chains. GST and GST-MEKK1-UIM proteins were expressed in bacteria, purified, and used for GST pulldown with recombinant K63- (Left) or K48-linked (Right) polyubiquitin chains, followed by western blotting with anti-ubiquitin or anti-GST Ab. (J) Ubc13 is required for accumulation of K63-linked polyubiquitin chains and for the recruitment of MEKK1 to the BMP7 promoter in MCF7 cells transfected with control or Ubc13 siRNA and pretreated with E2 for 1 h prior to treatment with IL-1β. ChIP was performed with anti-K63-ubiquitin or anti-MEKK1 Ab or protein A beads alone as control. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01 vs. vehicle control, Student’s t test.

Article Snippet: Commercial antibodies used for western blotting, immunoprecipitation, and ChIP experiments include HDAC3 rabbit polyclonal antibody #ab7030 (Abcam); anti- HDAC3(H- 99) rabbit polyclonal antibody #sc- 11417 (Santa Cruz Biotechnology); anti- ubiquitin mouse monoclonal antibody (P4D1 clone, Cell Signaling Technology); anti- β- tubulin mouse monoclonal antibody #T0198 (Sigma- Aldrich); anti- HDAC2 rabbit polyclonal antibody #ab16032 (Abcam); anti- TAB2 mouse monoclonal antibody sc- 398188, goat polyclonal antibody sc- 11850, and rabbit polyclonal sc- 20756 (Santa Cruz Biotechnology); anti- K63 ubiquitin rabbit monoclonal antibody #05- 1308 (Millipore); anti- TRAF6 rabbit polyclonal antibody sc- 7221 (Santa Cruz Biotechnology); anti- Ubc13 rabbit polyclonal antibody #10243- 1- AP (ProteinTech); anti- Ubc13 (131- 148) rabbit polyclonal antibody #PA1- 41188 (Thermo Scientific); anti- FLAG mouse monoclonal antibody, clone M2, #F3165, and anti- Flag- HRP #A8592 (Sigma- Aldrich); antiMYC(9E10) mouse monoclonal antibody #sc- 40 (Santa Cruz Biotechnology); and anti- GST(56C1) mouse monoclonal antibody #sc- 80998 (Santa Cruz Biotechnology).

Techniques: Western Blot, Translocation Assay, Control, Immunoprecipitation, Sequencing, Transfection, Mutagenesis, Biomarker Discovery, Expressing, Reverse Transcription Polymerase Chain Reaction, Over Expression, Dominant Negative Mutation, Luciferase, Activity Assay, Ubiquitin Proteomics, Bacteria, Purification, Recombinant

Journal: STAR protocols

Article Title: Protocol for evaluating the E3 ligase activity of BRCA1-BARD1 and its variants by nucleosomal histone ubiquitylation.

doi: 10.1016/j.xpro.2024.103294

Figure Lengend Snippet: Figure 5. Purification of UBE2D3 and other E2s (A) Whole cell lysate (WCL) (6 mL), cell pellet (P) (6 mL), cell lysate supernatant (S) (3 mL), Ni-NTA resin flowthrough (FT) (3 mL), and Ni-NTA elution fractions (lanes 6–11) (3 mL) of UBE2D3 were analyzed by SDS-PAGE and Coomassie staining. (B) The UBE2D3 elution fractions from SP cation affinity chromatography (3 mL) were analyzed by SDS-PAGE and Coomassie staining. (C) The UBE2D3 elution fractions from size exclusion chromatography (SEC) (3 mL) were analyzed by SDS-PAGE and Coomassie staining. (D) SDS-PAGE of purified E2s (UBE2D3, UBE2W, UBE2N, UBE2E3, UBE2V2, UBE2E2, UBE2E1, UBE2B), Lane 1 shows MW markers.

Article Snippet: The plasmids of pFastbac-Flag-BRCA1 (modified based on the plasmid from Jeffrey Parvin; deposited in Addgene (#223228)) and pFastBac-TwinStrepTag-BARD1 (Addgene#137166) are used for Flag-BRCA1 and Twin-StrepTag-BARD1 expression in insect cells.8,9 c. The expression plasmids for human E1 (pET3a-hUBA1 (Addgene#63571)), E2s (pET28a- UBE2D3 (Addgene#12643), pET15-UBE2W, pET24a-UBE2E3, pET24a-UBE2E2, pET24aUBE2E1, pET24a-UBE2N, pET24a-UBE2V2), and Ubiquitin (pET15-Ub (Addgene#12647)) were described before.5,10–12 Plasmids of pDEST17-UBE2B (Addgene#15781) was from Addgene.13 Note: pET15-UbE2W (Addgene#15809); pDEST17-UbE2E3 (Addgene#15789); pDEST17UbE2E2 (Addgene#15788); pDEST17-UbE2E1 (Addgene#15787); pET21(+)-His6-Ubc13/ UBE2N (Addgene#212715); MBP-His6-TEV-MMS2/UBE2V2 (Addgene#25465) are available from Addgene.13

Techniques: SDS Page, Staining, Chromatography, Size-exclusion Chromatography

Journal: STAR protocols

Article Title: Protocol for evaluating the E3 ligase activity of BRCA1-BARD1 and its variants by nucleosomal histone ubiquitylation.

doi: 10.1016/j.xpro.2024.103294

Figure Lengend Snippet: Figure 6. In vitro ubiquitylation of histone H2A by BRCA1-BARD1 and its variants (A) Schematic of ubiquitination assay mediated by E3 BRCA1-BARD1 with NCP as a substrate. (B) Representative nucleosome ubiquitylation assays monitoring H2A-Ub efficiency of BRCA1-BARD1 in conjunction with different E2s. UBE2B that does not bind BRCA1-BARD1 was included as a negative control. BC1-BD1, BRCA1-BARD1. (C) The BRCA1-BARD1 RING structure showing the locations of BRCA1 residues I26, L63, and K65 with the E2 binding surface indicated by dotted lines (PDB:7JZV). (D) Representative nucleosome ubiquitylation assays monitoring H2A-Ub efficiency for wild-type or mutant BRCA1-BARD1 in conjunction with NCP as a substrate and E2s: UBE2D3 (top) and UBE2D3 + UBE2N-UBE2V2 (bottom).

Article Snippet: The plasmids of pFastbac-Flag-BRCA1 (modified based on the plasmid from Jeffrey Parvin; deposited in Addgene (#223228)) and pFastBac-TwinStrepTag-BARD1 (Addgene#137166) are used for Flag-BRCA1 and Twin-StrepTag-BARD1 expression in insect cells.8,9 c. The expression plasmids for human E1 (pET3a-hUBA1 (Addgene#63571)), E2s (pET28a- UBE2D3 (Addgene#12643), pET15-UBE2W, pET24a-UBE2E3, pET24a-UBE2E2, pET24aUBE2E1, pET24a-UBE2N, pET24a-UBE2V2), and Ubiquitin (pET15-Ub (Addgene#12647)) were described before.5,10–12 Plasmids of pDEST17-UBE2B (Addgene#15781) was from Addgene.13 Note: pET15-UbE2W (Addgene#15809); pDEST17-UbE2E3 (Addgene#15789); pDEST17UbE2E2 (Addgene#15788); pDEST17-UbE2E1 (Addgene#15787); pET21(+)-His6-Ubc13/ UBE2N (Addgene#212715); MBP-His6-TEV-MMS2/UBE2V2 (Addgene#25465) are available from Addgene.13

Techniques: In Vitro, Ubiquitin Proteomics, Negative Control, Binding Assay, Mutagenesis

Journal: PLoS Pathogens

Article Title: HTLV-1 Tax Stimulates Ubiquitin E3 Ligase, Ring Finger Protein 8, to Assemble Lysine 63-Linked Polyubiquitin Chains for TAK1 and IKK Activation

doi: 10.1371/journal.ppat.1005102

Figure Lengend Snippet: (A) Ubc13-containing E2 conjugating enzyme complexes enhanced IKK activation by Tax in cell-free HeLa-G S100 extracts. The HeLa-G cytosolic S100 extract [ , ] was incubated with recombinant TaxH 6 alone (lanes 2), Ubc13, Ubc13:Uev2, or Ubc13:Uev1AH 6 with (lanes 4–5) or without TaxH 6 (lanes 6–8) as indicated at 30°C for 1 hour. IKK activation was detected by immunoblot with anti-p-IκBα. Uev1A was detected using anti-poly-Histidine antibody. (B & C) IKK activation by Tax is reduced by the depletion of Ubc13, Uev1A, or Uev2, but restored by exogenously added Ubc13, Uev2 or Uev1A. ( B ) HeLa-G, Ubc13 KD , Uev1A KD and Uev2 KD S100 extracts were generated and incubated with (lanes 2, 4, 6, and 8) or without (lanes 1, 3, 5, and 7) recombinant TaxH 6 , or ( C ) in the absence of exogenous factors (lanes 1, 4, and 7), in the presence of Tax without (lanes 2, 5, and 8) or with Ubc13, Uev2, or Ubc13:Uev1A H 6 (lanes 3, 6, and 9) as indicated at 30°C for 1 hour. Uev1A protein was not detectable in immunoblot, and Uev1A KD is determined via mRNA levels shown in . (D) Tax-induced IKK activation is attenuated in HeLa-G cells knocked down for Ubc13, Uev2, or Uev1A expression. The Ubc13 KD , Uev1A KD and Uev2 KD cell clones were transiently co-transfected with E-Selectin-Luc with or without Tax (Materials and Methods). Firefly luciferase activity was measured at 48 hours post-transfection. Fold of activation was calculated as the ratio of luciferase activities in cells with Tax over those in cells without Tax.

Article Snippet: Ubc13, IKKα, IKKβ, TAK1, p100/p52, p-IκBα, p-IKKα/β, p-TAK1, p-JNK/SAPK, p-p38, p-p100 antibodies were from Cell Signaling Technology.

Techniques: Activation Assay, Incubation, Recombinant, Western Blot, Generated, Expressing, Clone Assay, Transfection, Luciferase, Activity Assay

Journal: PLoS Pathogens

Article Title: HTLV-1 Tax Stimulates Ubiquitin E3 Ligase, Ring Finger Protein 8, to Assemble Lysine 63-Linked Polyubiquitin Chains for TAK1 and IKK Activation

doi: 10.1371/journal.ppat.1005102

Figure Lengend Snippet: (A) In vitro assembly of unanchored K63-pUb by RNF8 and RNF8 345–485 in the presence or absence of Tax. Each ubiquitination reaction contained E1, ubiquitin, ATP, TaxH 6 , RNF8 or RNF8 345–485 , and Ubc13:Uev1A or Ubc13:Uev2 as indicated and incubated at 37 ° C for 4 hours. Reactions products were resolved in 15% (upper panel), 4–20% (middle panel), and 4–20% (bottom panel) polyacrylamide gels, respectively. They are probed with indicated antibodies (ubiquitin, upper and middle panels and K63-pUb, bottom panel). Reactions in lanes 2–6 and 7–11 contained Ubc13:Uev1A and Ubc13:Uev2 E2 enzyme complexes respectively. RNF8 and RNF8 345–485 were used in reactions in lanes 3, 4, 8, 9 and 5, 6, 10, 11 respectively. (B) Polyubiquitin chain assembly by RNF8 and Ubc13:Uev1A in the presence of Tax requires ubiquitin molecules that contain lysine at amino acid residue 63. In vitro reactions were carried out as described in (A) with RNF8, Ubc13:Uev1A, ATP and E1, in the presence (lanes 2–7) or absence (lane 1) of TaxH 6 . Different variants of ubiquitin were used: wild-type ubiquitin (WT); K63R and K43R, mutants with lysine to arginine substitution at amino acid residue 63 and 48 respectively; K63 and K48, mutants with all lysine residues substituted by arginine except residue 63 and 48; and K0, a mutant with all lysine residues substituted by arginine. The protein concentration of the K63R ubiquitin mutant was based on the data sheet provided by the vender and was approximately ~1/4 of the other ubiquitin mutants, making it harder to detect by immunoblotting. (C) HeLa-G cells (10 5 ) were co-transfected with Ub-HA (50 ng/ml), Tax (50 ng/ml), and/or increasing amounts of RNF8 (0, 50, 100, 200 ng/ml) as indicated for 48 hours. Cells were lysed with SDS sample buffer and immunoblotted for the indicated proteins.

Article Snippet: Ubc13, IKKα, IKKβ, TAK1, p100/p52, p-IκBα, p-IKKα/β, p-TAK1, p-JNK/SAPK, p-p38, p-p100 antibodies were from Cell Signaling Technology.

Techniques: In Vitro, Incubation, Mutagenesis, Protein Concentration, Western Blot, Transfection

Journal: PLoS Pathogens

Article Title: HTLV-1 Tax Stimulates Ubiquitin E3 Ligase, Ring Finger Protein 8, to Assemble Lysine 63-Linked Polyubiquitin Chains for TAK1 and IKK Activation

doi: 10.1371/journal.ppat.1005102

Figure Lengend Snippet: Tax interacts with and stimulates RNF8 and Ubc13:Uev1A/2 to assemble free K63-pUb chains, which serve as platforms for TAK1 and IKK to convene and activate the canonical NF-κB and other signaling pathways.

Article Snippet: Ubc13, IKKα, IKKβ, TAK1, p100/p52, p-IκBα, p-IKKα/β, p-TAK1, p-JNK/SAPK, p-p38, p-p100 antibodies were from Cell Signaling Technology.

Techniques: