PMC

Disruption of interactions between E2 and E3 enzymes in the TNFR and TLR4/IL-1R pathways by A20 and TAX1BP1. (A) Kinetics of TRAF6, Ubc13, UbcH5c, Itch, RNF11, A20, and TAX1BP1 interactions in control and A20-deficient MEFs. A20^+/+ and A20^−/− MEFs were stimulated with IL-1 for the indicated times. Proteins from lysates were immunoprecipitated with TRAF6 antibody and detected by immunoblotting with antibodies to A20, Ubc13, UbcH5c, Itch, RNF11, TAX1BP1, or TRAF6. Lysates were subjected to immunoblotting with antibodies to IκBα, A20, TAX1BP1, Ubc13, UbcH5c, RNF11, Itch, and β-actin. (B) Specificity of TRAF6, Ubc13, A20, UbcH5c, and TAX1BP1 interactions. A20^+/+ MEFs were stimulated with IL-1 for the indicated times. Proteins from lysates were immunoprecipitated with TRAF6 or control rabbit antibody [Cont. IgG (immunoglobulin G)] and detected by immunoblotting with antibodies to A20, Ubc13, UbcH5c, TAX1BP1, or TRAF6. Lysates were subjected to immunoblotting with antibodies to IκBα and β-actin. (C) Kinetics of TRAF2, Ubc13, Itch, RNF11, A20, and TAX1BP1 interactions in control and A20-deficient MEFs. A20^+/+ and A20^−/− MEFs were stimulated with TNF-α, and proteins from lysates were immunoprecipitated with TRAF2 antibody followed by immunoblotting with antibodies to A20, Ubc13, Itch, RNF11, TAX1BP1, and TRAF2. Lysates were subjected to immunoblotting with antibodies to IκBα, TAX1BP1, A20, Ubc13, RNF11, Itch, and β-actin. (D and E) Interaction of TAX1BP1 with Ubc13. A20^+/+ and A20^−/− MEFs were stimulated with TNF-α (D) or LPS (E) for 30 and 60 min, and proteins from lysates were immunoprecipitated with antibody to TAX1BP1, followed by immunoblotting with antibodies to Ubc13, UbcH5c, or TAX1BP1. Lysates were subjected to immunoblotting with antibodies to IκBα, A20, TAX1BP1, Ubc13, and β-actin. The results shown are representative of three independent experiments. IP, immunoprecipitation; IB, immunoblot.

Requirement of A20 zinc finger 4 for TAX1BP1 binding and degradation of Ubc13 and UbcH5c. (A) Schematic of A20 deletion mutants. (B) Requirement of A20 ZnF4 for the degradation of Ubc13. A20^−/− MEFs were transiently transfected with the indicated mutants. After 36 hours, cells were treated with TNF-α for the indicated times. Lysates were subjected to immunoblotting with antibodies to Ubc13, IκBα, β-actin, or Flag. (C) A20 DUB and ZnF4 mutants do not interact with Ubc13. A20^−/− MEFs were transiently transfected with Flag-A20, Flag-A20 (C103A), and Flag-A20 ZnF4 mutant (C624A and C627A). After 36 hours, cells were stimulated with LPS for the indicated times, and proteins from lysates were immunoprecipitated with Ubc13 antibody and detected by immunoblotting with antibodies to A20 or Ubc13. Lysates were subjected to immunoblotting with antibodies to Flag, IκBα, and β-actin. (D) A20 ZnF4 mutant does not interact with Ubc13. A20^−/− MEFs were transiently transfected with Flag-A20 and Flag-A20 ZnF4 mutant (C624A and C627A). After 36 hours, cells were stimulated with TNF-α for 60 min, and proteins from lysates were immunoprecipitated with Ubc13 antibody and detected by immunoblotting with antibodies to Flag or Ubc13. Lysates were subjected to immunoblotting with antibodies to Flag, IκBα,and β-actin. (E) A20 DUB and ZnF4 mutants do not interact with UbcH5c. A20^−/− MEFs were transiently transfected with plasmids as described in (C). After 36 hours, cells were stimulated with IL-1 for the indicated times, and proteins from lysates were immunoprecipitated with UbcH5c antibody and detected by immunoblotting with antibodies to A20 or UbcH5c. Lysates were subjected to immunoblotting with antibodies to Flag, IκBα,and β-actin. (F) Differential requirement for the A20 OTU domain and C-terminal zinc fingers in binding to TAX1BP1, Ubc13, and UbcH5c. A20^−/− MEFs were transiently transfected with empty vector, Flag-A20, Flag-A20 (547-775) or Flag-A20Δ547-699. After 36 hours, cells were treated with IL-1 for either 30 or 60 min. Proteins from lysates were immunoprecipitated with antibody to Flag, followed by immunoblotting with antibodies to TAX1BP1, UbcH5c, or Ubc13. Lysates were subjected to immunoblotting with antibodies to IκBα, Flag, and β-actin. (G) A20 ZnF4 mutant does not interact with TAX1BP1. A20^−/− MEFs were transiently transfected with plasmids as described in (C). After 36 hours, cells were stimulated with LPS for the indicated times, and proteins from lysates were immunoprecipitated with TAX1BP1 antibody and detected by immunoblotting with antibodies to A20 and TAX1BP1. Lysates were subjected to immunoblotting with antibodies to Flag, IκBα,and β-actin. The results shown are representative of three independent experiments.

(A) Annexin V staining demonstrates increased apoptosis with A20 targeting in glioma stem cells isolated from a D317 glioma xenograft or a T3359 patient specimen passaged short term in immunocompromised mice. An asterisk (*) indicates p<0.01 with t-test comparison of nontargeting (NT) shRNA control and A20 shRNA. (B) Annexin V staining demonstrates increased apoptosis with A20 targeting in tumor subfractions isolated from a T0066-002 patient specimen. An asterisk (*), p<0.05 with ANOVA comparison of nontargeting shRNA control to the indicated A20 shRNA. (C) Caspase activity relative to cell number as determined in the cell titer assay increased in A20 shRNA infected glioma stem cells isolated directly from a T0066-022 patient specimen, but not in matched non-stem glioma cells. An asterisk (*) indicates p<0.05 with t-test comparison of non-targeting shRNA control to A20 shRNA. (D) Caspase activity relative to cell number as determined in the cell titer assay increased in A20 shRNA infected glioma stem cells isolated from a T3359 patient specimen passaged short-term in immunocompromised mice. An asterisk (*) indicates p<0.05 with ANOVA comparison of non-targeting shRNA control to the indicated A20 shRNA. (E) TUNEL staining demonstrates increased apoptosis in A20 infected glioma stem cells isolated from a T4105 patient specimen passaged short term in immunocompromised mice. (F) Targeting A20 in GSCs isolated from CCF1863 or T3359 patient specimens passaged short term in immunocompromised mice decreased phosphorylation of p65/RelA on Serine 536 as determined via Western blot analysis.

A) A20 inhibits TLR4-mediated NF-κB activation by targeting TRAF6 for inactivation. A20 may directly disassemble K63-linked polyubiquitin chains from TRAF6 via its OTU domain. In addition, A20 disrupts the interactions between TRAF6 and its E2 enzymes Ubc13 and UbcH5, and later triggers the K48-linked polyubiquitination and degradation of the E2 enzymes. A20 is dependent on the ubiquitin-binding adaptor molecule TAX1BP1 to disrupt interactions between TRAF6 and its E2 enzymes Ubc13 and UbcH5. B) A20 inhibits RIG-I-dependent antiviral signaling by disrupting TRAF3 binding to TBK1 and IKKε, resulting in the inhibition of K63-linked polyubiquitination of TBK1 and IKKε. A20 also requires TAX1BP1 to inhibit antiviral signaling.