Cereblon negatively regulates TLR4 signaling through the attenuation of ubiquitination of TRAF6

Abstract

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.

Inhibition of TRAF6 ubiquitin-ligase activity by PRDX1 leads to inhibition of NFKB activation and autophagy activation

“…Luciferase reporter assay was performed as described previously [23][24][25]46]. Briefly, cells were transiently transfected with or without appropriate vectors indicated in each experiment using Lipofectamine 2000 (Thermo Fisher Scientific, 11,668,019) or Neon® Transfection System (Thermo Fisher Scientific, MPK5000).…”

“…Cells were then treated with or without LPS (200 ng/ml) for 6 h. Nuclear proteins were then prepared using CelLytic NuCLEAR extraction kit (Sigma, NXTRACT) per the manufacturer's protocol. Activity of transcription factor RELA was determined using Trans AM NFKB transcription factor assay kit (Active Motif North America, 40,596) per the manufacturer's instructions [24,46].…”

Ligand-mediated protein degradation reveals functional conservation among sequence variants of the CUL4-type E3 ligase substrate receptor cereblon

“…the AMP-activated protein kinase ␣1 subunit (40), TAK1-TRAF6 (41), and CD147-MCT1 complex (42), where CRBN plays ubiquitin-independent roles in pathway regulation. The mechanistic underpinnings for induced limb deformities in chickens and zebrafish, but not in mice, appears dependent on sequence differences in mouse CRBN (20), which brings into question whether these differences in CRBN render it functionally inactive.…”

Cereblon suppresses the lipopolysaccharide-induced inflammatory response by promoting the ubiquitination and degradation of c-Jun

“…Immunomodulatory drugs (IMiDs), including thalidomide and its structural analogs lenalidomide and pomalidomide, are a class of compounds that can be used to alleviate the inflammatory response. However, how CRBN regulates inflammation has only begun to unfold (41). It has been reported that CRBN binds to the ubiquitination domain of TRAF6 and attenuates the ubiquitination of TRAF6 and TAK1-binding protein 2 (TAB2), leading to suppression of NF-B activation by negatively regulating the Toll-like receptor 4 (TLR4) signaling pathway (41).…”

“…However, how CRBN regulates inflammation has only begun to unfold (41). It has been reported that CRBN binds to the ubiquitination domain of TRAF6 and attenuates the ubiquitination of TRAF6 and TAK1-binding protein 2 (TAB2), leading to suppression of NF-B activation by negatively regulating the Toll-like receptor 4 (TLR4) signaling pathway (41). This discovery revealed the important nonenzymatic function of CRBN in the regulation of inflammation.…”