The structure function of the death domain of human IRAK-M

Jiangfeng Du, Gerry A. F. Nicolaes, Danielle Kruijswijk, Miranda Versloot, Tom van der Poll, Cornelis van 't Veer*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

Background: IRAK-M is an inhibitor of Toll-like receptor signaling that acts by re-directing IRAK-4 activity to TAK1 independent NF-kappa B activation and by inhibition of IRAK-1/IRAK-2 activity. IRAK-M is expressed in monocytes/macrophages and lung epithelial cells. Lack of IRAK-M in mice greatly improves the resistance to nosocomial pneumonia and lung tumors, which entices IRAK-M as a potential therapeutic target. IRAK-M consists of an N-terminal death domain (DD), a dysfunctional kinase domain and unstructured C-terminal domain. Little is known however on IRAK-M's structure-function relationships. Results: Since death domains provide the important interactions of IRAK-1, IRAK-2 and IRAK-4 molecules, we generated a 3D structure model of the human IRAK-M-DD (residues C5-G119) to guide mutagenesis studies and predict protein-protein interaction points. First we identified the DD residues involved in the endogenous capacity of IRAK-M to activate NF-kappa B that is displayed upon overexpression in 293T cells. W74 and R97, at distinct interfaces of the IRAK-M-DD, were crucial for this endogenous NF-kappa B activating capacity, as well as the C-terminal domain (S445-E596) of IRAK-M. Resulting anti-inflammatory A20 and pro-inflammatory IL-8 transcription in 293T cells was W74 dependent, while IL-8 protein expression was dependent on R97 and the TRAF6 binding motif at P478. The IRAK-M-DD W74 and R97 binding interfaces are predicted to interact with opposite sides of IRAK-4-DD's. Secondly we identified DD residues important for the inhibitory action of IRAK-M by stable overexpression of mutants in THP-1 macrophages and H292 lung epithelial cells. IRAK-M inhibited TLR2/4-mediated cytokine production in macrophages in a manner that is largely dependent on W74. R97 was not involved in inhibition of TNF production but was engaged in IL-6 down-regulation by IRAK-M. Protein-interactive residues D19-A23, located in between W74 and R97, were also observed to be crucial for inhibition of TLR2/4 mediated cytokine induction in macrophages. Remarkably, IRAK-M inhibited TLR5 mediated IL-8 production by lung epithelial cells independent of W74 and R97, but dependent on D19-A23 and R70, two surface-exposed regions that harbor predicted IRAK-2-DD interaction points of IRAK-M. Conclusion: IRAK-M employs alternate residues of its DD to inhibit the different inflammatory mediators induced by varying TLRs and cells.
Original languageEnglish
Article number77
JournalCell Communication and Signaling
Volume12
Issue number1
DOIs
Publication statusPublished - 7 Dec 2014

Keywords

  • IRAK-M
  • Inflammation
  • Death domain
  • Structure-function
  • TLR

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