Supplementary Figure 8: Proposed working model for negative regulation of TLR-triggered NEMO/NF-κB activation and control of autoimmunity by Rhbdd3.

(a) (I) Activation of NEMO/NF-κB pathway upon TLR stimulation. Stimulation of TLR agonists activates IRAK1 and TRAF6. TRAF6 acts as an E3 ubiquitin ligase to activate the TAK1 complex, inducing IKK complex consisting of IKKα, IKKβ and NEMO. Activated IKK complex then phosphorylates IκB and leads to its dissociation from NF-κB. Freed NF-κB translocates into the nucleus and eventually initiates production of proinflammatory cytokines such as IL-6. (II) Inhibition of TLR-triggered NEMO/NF-κB activation by Rhbdd3. TLR signal promotes aggregation of Rhbdd3 in endosomes. Rhbdd3 directly binds to K27-linked polyubiquitin chains on K302 of NEMO via UBA domain. Rhbdd3 also recruits and interacts with A20 via K27-linked polyubiquitin chains on K268 and facilitates A20-mediated K63-linked deubiquitination on NEMO. Above signals lead to inhibition of IκBα degradation and NF-κB activation, and eventually inhibit the production of proinflammatory cytokines such as IL-6. (b) Activation of DC via TLRs leads to activation of NF-κB and production of IL-6, which then potently promotes Th17 generation while inhibits Treg generation, contributing to the development of autoimmune diseases. Rhbdd3 accumulates in endosome upon TLR stimulation and selectively inhibits TLR-triggered NF-κB activation and IL-6 production in DC by interacting with A20 and NEMO and facilitating K63-linked deubiquitination of NEMO. The inhibition of IL-6 production from DC by Rhbdd3 leads to the suppression of Th17 generation but promotion of Treg generation, and eventually contributing to the control of autoimmunity and prevention of autoimmune diseases. Abbreviations: IκBα, Inhibitor of NF-κB alpha; IKKα/β, IκB kinase α/β IRAK1, Interleukin-1 receptor-associated kinase 1; K27/K63, K27/K63-linked polyubiquitin chains; TAB1, TAK-binding protein 1; TAB2/3, TAK-binding protein 2/3 ; TAK1, Transforming growth factor β-activated kinase 1; TRAF6, TNF receptor-associated factor 6; NEMO, NF-κB essential modulator; NF-κB, Nuclear factor kappa B; UBA, Ubiquitin-binding associated domain.