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

Cell Death Dis. 2016 Jul 28;7(7):e2313. doi: 10.1038/cddis.2016.226.


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.

MeSH terms

  • Adaptor Proteins, Signal Transducing / metabolism
  • Animals
  • Cytokines / metabolism
  • Gene Expression Regulation / drug effects
  • Gene Knockdown Techniques
  • HEK293 Cells
  • Humans
  • Inflammation Mediators / metabolism
  • Lipopolysaccharides / pharmacology
  • MAP Kinase Kinase Kinases / metabolism
  • Mice
  • Models, Biological
  • NF-kappa B / metabolism
  • Nerve Tissue Proteins / metabolism
  • Peptide Hydrolases / metabolism*
  • Protein Binding / drug effects
  • Sepsis / genetics
  • Sepsis / pathology
  • Signal Transduction* / drug effects
  • TNF Receptor-Associated Factor 6 / metabolism*
  • Toll-Like Receptor 4 / metabolism
  • Ubiquitin-Protein Ligases
  • Ubiquitination* / drug effects


  • Adaptor Proteins, Signal Transducing
  • CRBN protein, human
  • Crbn protein, mouse
  • Cytokines
  • Inflammation Mediators
  • Lipopolysaccharides
  • NF-kappa B
  • Nerve Tissue Proteins
  • TAB1 protein, human
  • TAB2 protein, human
  • TNF Receptor-Associated Factor 6
  • Toll-Like Receptor 4
  • Ubiquitin-Protein Ligases
  • MAP Kinase Kinase Kinases
  • MAP kinase kinase kinase 7
  • Peptide Hydrolases