Journal: Frontiers in Immunology

Article Title: Ubiquitin-Specific Protease 14 Negatively Regulates Toll-Like Receptor 4-Mediated Signaling and Autophagy Induction by Inhibiting Ubiquitination of TAK1-Binding Protein 2 and Beclin 1

doi: 10.3389/fimmu.2017.01827

Figure Lengend Snippet: Molecular interactions among tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6), Beclin 1, and ubiquitin-specific protease 14 (USP14). (A) Truncated mutants of Beclin 1. Myc-tagged Beclin 1 truncated mutants were generated as described in Section “ .” BH3, Bcl-2 Homology 3; CCD, coiled coil domain; ECD, evolutionarily conserved domain. (B) Expression vectors of Myc-tagged Beclin 1 wild type (wt) and truncated mutants were co-transfected with Flag-tagged USP14 plasmid into HEK293T cells followed by immunoprecipitation (IP) and western blotting analyses. (C) Myc-tagged Beclin 1 wt, Myc-tagged Beclin 1 truncated mutants, and mock as control plasmid were co-transfected with Flag-tagged TRAF6 into HEK293T cells followed by IP and western blotting analyses. (D) Flag-tagged TRAF6 wt, Flag-tagged TRAF6 truncated mutants, and mock as control plasmid were co-transfected with Myc-tagged Beclin 1 into HEK293T cells followed by IP and western blotting analyses. (E) Flag-tagged TRAF6 wt, Flag-tagged TRAF6 truncated mutants, and mock as control plasmid were co-transfected with Myc-tagged USP14 into HEK293T cells followed by IP and western blotting analyses. (F) A schematic model showing molecular interactions among TRAF6, Beclin 1, and USP14. TRAF-C, C-terminal TRAF domain.

Article Snippet: Myc-tagged Beclin 1 vector was generated by PCR using Flag-tagged Beclin 1 as a template and inserted into pCMV-Myc (Addgene). pCDNA3-USP14 vector was kindly provided by Dr. M. J. Lee (Seoul National University College of Medicine, Seoul, Korea).

Techniques: Generated, Expressing, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot

Journal: Frontiers in Immunology

Article Title: Ubiquitin-Specific Protease 14 Negatively Regulates Toll-Like Receptor 4-Mediated Signaling and Autophagy Induction by Inhibiting Ubiquitination of TAK1-Binding Protein 2 and Beclin 1

doi: 10.3389/fimmu.2017.01827

Figure Lengend Snippet: Ubiquitin-specific protease 14 (USP14) inhibits ubiquitination of Beclin 1. (A) Myc-tagged Beclin 1, Flag-tagged tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6), and mock as control plasmid were co-transfected with Flag-tagged USP14 plasmid at different concentrations. At 38 h post-transfection, transfected cells were extracted, immunoprecipitated with anti-Myc antibody, and subjected to immuno-blotting (IB) assay using anti-USP14, anti-TRAF6, anti-Flag, or anti-Myc antibody. *The intensity of Flag-USP14 was measured by ImageJ. Fold change relative to Flag-USP14 of lane 3 was calculated. **The intensity of Flag-TRAF6 was measured by ImageJ. Fold change relative to Flag-TRAF6 of lane 2 was calculated. (B) Myc-tagged Beclin 1, HA-tagged Ub, Flag-tagged TRAF6, and mock as control plasmid were co-transfected with Flag-tagged USP14 vector at different concentrations. At 38 h post-transfection, transfected cells were extracted, immunoprecipitated with anti-Myc antibody, and subjected to IB assay using anti-HA, anti-Myc, anti-USP14, or anti-TRAF6 antibody. (C) A schematic model showing the inhibition of Beclin 1 ubiquitination by USP14.

Article Snippet: Myc-tagged Beclin 1 vector was generated by PCR using Flag-tagged Beclin 1 as a template and inserted into pCMV-Myc (Addgene). pCDNA3-USP14 vector was kindly provided by Dr. M. J. Lee (Seoul National University College of Medicine, Seoul, Korea).

Techniques: Plasmid Preparation, Transfection, Immunoprecipitation, Inhibition

Journal: Frontiers in Immunology

Article Title: Ubiquitin-Specific Protease 14 Negatively Regulates Toll-Like Receptor 4-Mediated Signaling and Autophagy Induction by Inhibiting Ubiquitination of TAK1-Binding Protein 2 and Beclin 1

doi: 10.3389/fimmu.2017.01827

Figure Lengend Snippet: Ubiquitin-specific protease 14 (USP14) negatively regulates autophagy induction and toll-like receptor 4 (TLR4)-mediated signaling. (A) Upon TLR4 stimulation, tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) interacts with Beclin 1 through its coiled coil (CC) domain, induces ubiquitination of Beclin 1, and facilitate autophagy induction (upper). In contrast, USP14 competitively interacts with Beclin 1 to the CC domain of TRAF6. This leads to the suppression of Beclin 1 ubiquitination by TRAF6, resulting in the inhibition of autophagy induction (down). (B) Following TLR4 stimulation, ubiquitinated TRAF6 is associated with the TAB 2–TAK1–TAB 1 complex through an interaction between its polyubiquitinated chain and TAB 2. TAB 2 is then ubiquitinated and the complex facilitates the activation of TAK1. Simultaneously, the IKK complex is associated with the former complex through the polyubiquitinated chain and TAB 2, leading to the activations of nuclear factor-kappa B (NF-κB) and p38 (left). In contrast, the interaction between TAB 2 and USP14 may lead to the deubiquitination of TAB 2, thus inhibiting the association of IKKs, which inhibits the activation of NF-κB and p38 (right).

Article Snippet: Myc-tagged Beclin 1 vector was generated by PCR using Flag-tagged Beclin 1 as a template and inserted into pCMV-Myc (Addgene). pCDNA3-USP14 vector was kindly provided by Dr. M. J. Lee (Seoul National University College of Medicine, Seoul, Korea).

Techniques: Inhibition, Activation Assay