Person:

Chun, Eunyoung

A TRAF6-ECSIT complex is crucial for the generation of mitochondrial reactive oxygen species (mROS) and nuclear factor-kappa B (NF-κB) activation induced by Toll-like receptor 4 (TLR4). Peroxiredoxin-6 (Prdx6) as a member of the peroxiredoxin family of antioxidant enzymes is involved in antioxidant protection and cell signaling. Here, we report on a regulatory role of Prdx6 in mROS production and NF-κB activation by TLR4. Prdx6 was translocated into the mitochondria by TLR4 stimulation and Prdx6-knockdown (Prdx6KD) THP-1 cells had increased level of mitochondrial reactive oxygen species levels and were resistant to Salmonella typhimurium infection. Biochemical studies revealed Prdx6 interaction with the C-terminal TRAF-C domain of TRAF6, which drove translocation into the mitochondria. Interestingly, Prdx6 competitively interacted with ECSIT to TRAF6 through its C-terminal TRAF-C domain, leading to the interruption of TRAF6-ECSIT interaction. The inhibitory effect was critically implicated in the activation of NF-κB induced by TLR4. Overexpression of Prdx6 led to the inhibition of NF-κB induced by TLR4, whereas Prdx6KD THP-1 cells displayed enhanced production of pro-inflammatory cytokines including interleukin-6 and -1β, and the up-regulation of NF-κB-dependent genes induced by TLR4 stimulation. Taken together, the data demonstrate that Prdx6 interrupts the formation of TRAF6-ECSIT complex induced by TLR4 stimulation, leading to suppression of bactericidal activity because of inhibited mROS production in mitochondria and the inhibition of NF-κB activation in the cytoplasm.

Cereblon (CRBN) is a substrate receptor protein for the CRL4A E3 ubiquitin ligase complex. In this study, we report on a new regulatory role of CRBN in TLR4 signaling. CRBN overexpression leads to suppression of NF-κB activation and production of pro-inflammatory cytokines including IL-6 and IL-1β in response to TLR4 stimulation. Biochemical studies revealed interactions between CRBN and TAK1, and TRAF6 proteins. The interaction between CRBN and TAK1 did not affect the association of the TAB1 and TAB2 proteins, which have pivotal roles in the activation of TAK1, whereas the CRBN-TRAF6 interaction critically affected ubiquitination of TRAF6 and TAB2. Binding mapping results revealed that CRBN interacts with the Zinc finger domain of TRAF6, which contains the ubiquitination site of TRAF6, leading to attenuation of ubiquitination of TRAF6 and TAB2. Functional studies revealed that CRBN-knockdown THP-1 cells show enhanced NF-κB activation and p65- or p50-DNA binding activities, leading to up-regulation of NF-κB-dependent gene expression and increased pro-inflammatory cytokine levels in response to TLR4 stimulation. Furthermore, Crbn−/− mice exhibit decreased survival in response to LPS challenge, accompanied with marked enhancement of pro-inflammatory cytokines, such as TNF-α and IL-6. Taken together, our data demonstrate that CRBN negatively regulates TLR4 signaling via attenuation of TRAF6 and TAB2 ubiquitination.

Ubiquitin-specific protease 14 (USP14), one of three proteasome-associated deubiquitinating enzymes, has multifunctional roles in cellular context. Here, we report a novel molecular mechanism and function of USP14 in regulating autophagy induction and nuclear factor-kappa B (NF-κB) activation induced by toll-like receptor (TLR) 4 (TLR4). USP14 interacted with tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) and interrupted the association of Beclin 1 with TRAF6, leading to inhibition of TRAF6-mediated ubiquitination of Beclin 1. Reduced expression of USP14 in USP14-knockdown (USP14KD) THP-1 cells enhanced autophagy induction upon TLR4 stimulation as shown by the increased conversion of cytosolic LC3-I to membrane-bound LC3-II. Moreover, USP14KD human breast carcinoma MDA-MB-231 cells and USP14KD human hepatic adenocarcinoma SK-HEP-1 cells showed increased cell migration and invasion, indicating that USP14 is negatively implicated in the cancer progression by the inhibition of autophagy induction. Furthermore, we found that USP14 interacted with TAK1-binding protein (TAB) 2 protein and induced deubiquitination of TAB 2, a key factor in the activation of NF-κB. Functionally, overexpression of USP14 suppressed TLR4-induced activation of NF-κB. In contrast, USP14KD THP-1 cells showed enhanced activation of NF-κB, NF-κB-dependent gene expression, and production of pro-inflammatory cytokines such as IL-6, IL-1β, and tumor necrosis factor-α. Taken together, our data demonstrate that USP14 can negatively regulate autophagy induction by inhibiting Beclin 1 ubiquitination, interrupting association between TRAF6 and Beclin 1, and affecting TLR4-induced activation of NF-κB through deubiquitination of TAB 2 protein.