RIKEN IMS AnnualReport 2021

43/104

The inflammatory response is an important host defense mechanism, initi-UbNF-B p65UbNF-B p6537Inflammatory responsesubiquitin ligase complexScaffordproteinSubstraterecruiting component Toll–like receptorNF-B p65nucleusNF-B p65PDLIM2-containingUbiquitinrecruiting component PDLIM2E2UbCullin1skp1Fbxo16degradationInflammatory responsesDendriticcellsCullin1skp1Fbxo16PDLIM2cytoplasmPML nuclearbodiesproteasomeated by dendritic cells, to eliminate invading microbial pathogens. How-ever, these responses must be terminated at the appropriate time, otherwise excessive responses may cause massive damage to the host and lead to autoim-mune diseases, indicating that negative regulatory systems for inflammation are critical to prevent immunopathology. Our research goal is to identify a series of key negative regulators of inflammation and clarify the complete picture of the molecular mechanisms for regulating inflammatory responses.We previously identified PDLIM2 (PDZ and LIM domain-containing pro-tein-2), a nuclear protein that belongs to a large family of LIM proteins, as a key factor negatively regulating inflammatory responses. PDLIM2 is a ubiquitin E3 ligase for the p65 subunit of NF-κB in dendritic cells that negatively regulates NF-κB-mediated inflammation. In general, ubiquitin E3 ligase binds to E2, a ubiquitin-conjugating enzyme, and forms a functional ubiquitin ligase complex with Cullin, a scaffold protein. This complex provides a platform onto which an F-box-containing protein can be added to bind to substrates. By screening using siRNA for F-box-containing proteins, we have identified Fbxo16 as a substrate-binding component in the PDLIM2-containing ubiquitin E3 ligase complex. Fbxo16 binds to PDLIM2 and synergistically promotes polyubiquitination and degradation of p65 together with PDLIM2. Notably, knockdown of Fbxo16 by siRNA in dendritic cells impaired PDLIM2-mediated degradation of p65 and thus enhanced LPS-induced production of proinflammatory cytokines such as IL-6 and IL-12. Moreover, Fbxo16 also binds to IRF3/7 transcription factors and promotes their proteasome-dependent degradation, thereby suppressing IRF3/7-mediated signaling. Consistently, knockdown of Fbxo16 in dendritic cells augmented Toll-like receptor-mediated expression of Type I interferon, IFNβ/α. These results delineate a novel role of Fbxo16, as a component of a ubiquitin E3 ligase complex, in negatively regulating NF-κB- and IRF3/7-medi-ated innate immune responses in dendritic cells, cooperatively with PDLIM2.Figure: PDLIM2 and Fbxo16 form a functional ubiquitin E3 ligase complex targeting NF-κB p65 for degradationPDLIM2 binds to E2, a ubiquitin conjugating enzyme, and forms a functional ubiquitin E3 ligase complex with Cullin1, a scaffold protein providing a platform consist-ing of the complex, Fbxo16, a component to bind to sub-strates, and skp1, an adaptor protein, and cooperatively promotes polyubiquitination and proteasomal degrada-tion of the p65 subunit of the NF-κB transcription factor, thereby suppressing NF-κB-mediated inflammatory responses.Recent Major PublicationsTakemori T, Sugimoto-Ishige A, Nishitsuji H, Futamura Y, Harada M, Kimura-Someya T, Matsumoto T, Honma T, Takana M, Yaguchi M, Isono K, Koseki H, Osada H, Miki D, Saito T, Tanaka T, Fukami T, Goto T, Shirouzu M, Shi-motohno K, Chayama K. Establishment of a monoclonal antibody against human NTCP that blocks HBV infection. J Virol JV10168621 (Epub ahead of print) (2022)Sugimoto-Ishige A, Harada M, Tanaka M, Terooatea T, Adachi Y, Takahashi Y, Tanaka T, Burrows PD, Hikidda M, Takemori T. Bim establishes the B cell repertories from early to late in the immune response. Int Immunol 33, 79-90 (2020)Jodo A, Shibazaki A, Onuma A, Kaishi T, Tanaka T. PDLIM7 synergizes with PDLIM2 and p62/Sqstm1 to inhibit in-flammatory signaling by promoting degradation of the p65 subunit of NF-κB. Front Immunol 11, 1559 (2020)Laboratory for Inflammatory RegulationTeam Leader: Takashi Tanaka