Fig. 6

Ubiquitin-specific peptidase 17 disrupts formation of the TNFR-associated factor 2/TNFR-associated factor 3 complex and stabilizes their client proteins. a TNFR-associated factor (TRAF) 2-binding and TRAF3-binding motifs were identified in ubiquitin-specific peptidase 17 (USP17) as shown in the schematic diagram. USP Ubiquitin-specific protease, HAMBs hyaluronan-binding motifs. Numbers show the amino-acid residue positions. b USP17 was coexpressed with TRAF2 (left panel) or TRAF3 (right panel) in HEK293 cells, and their interaction was analyzed by immunoprecipitation and immunoblotting with the antibodies indicated. c Different amino-acid residues in the two TRAF2-binding motifs of USP17 were mutated. Wild-type (wt) USP17 and USP17 mutants were coexpressed with TRAF2 in HEK293 cells, and the requirement of the amino-acid residues in the binding motif of USP17 for TRAF2 binding was analyzed by immunoprecipitation and immunoblotting with the antibodies indicated. d Different amino-acid residues in the TRAF3-binding motifs of USP17 were mutated. Wt USP17 and these mutants were coexpressed with TRAF3 in HEK293 cells, and the requirement of the amino-acid residues in the binding motif of USP17 for TRAF3 binding was analyzed by immunoprecipitation and immunoblotting with the antibodies indicated. e HEK293 cells were co-transfected with a nuclear factor-κB (NF-κB)-controlled luciferase-reporter plasmid and expression vectors encoding wt USP17 and different USP17 mutants. These cells were treated with or without interleukin (IL)-1β (10 ng/ml) and the relative luciferase activities were analyzed to determine the requirement of TRAF2 and TRAF3 binding for regulating NF-κB activation by USP17. f HEK293 cells were co-transfected with expression vectors for wt USP17, C89S USP17, UB-K63, TRAF2 (left panel), or TRAF3 (right panel) and treated with MG132. The effects of wt USP17 and C89S USP17 on the K63-linked ubiquitination of TRAF2 and TRAF3 were analyzed by immunoprecipitation and immunoblotting with the antibodies indicated. g HEK293 cells were co-transfected with an NF-κB-controlled luciferase-reporter plasmid and expression vectors encoding wt USP17 and C89S USP17. These cells were treated with or without IL-1β (10 ng/ml) and the relative luciferase activities were analyzed to determine the requirement of deubiquitinase activity for regulating NF-κB activation by USP17. h HEK293 cells were co-transfected with expression vectors for USP17, TRAF2, and TRAF3, and the effects of USP17 on blocking TRAF2/TRAF3 complex formation (top panel) and stabilizing the client proteins of this complex (bottom panel) were analyzed by immunoprecipitation and immunoblotting with the antibodies indicated. Each set of blots is representative of three independent experiments. In the bar figures, the data represent mean ± standard deviation of three independent experiments, *P < 0.05; **P < 0.01 compared with the wt USP17 group (c, d), or the group without IL-1β treatment (e, g)