title: The structure function of the death domain of human IRAK-M.
abstract: BACKGROUND
  IRAK-M is an inhibitor of Toll-like receptor signaling that acts by re-directing IRAK-4 activity to TAK1 independent NF-κB 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-κB 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-κB 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.
  
  
  CONCLUSIONS
  IRAK-M employs alternate residues of its DD to inhibit the different inflammatory mediators induced by varying TLRs and cells.
authors.forename: Jiangfeng
authors.initials: J
authors.lastname: Du
authors.forename: Gerry Af
authors.initials: GA
authors.lastname: Nicolaes
authors.forename: Danielle
authors.initials: D
authors.lastname: Kruijswijk
authors.forename: Miranda
authors.initials: M
authors.lastname: Versloot
authors.forename: Tom
authors.initials: T
authors.lastname: van der Poll
authors.forename: Cornelis
authors.initials: C
authors.lastname: van 't Veer
mesh_terms: D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics; D006801:Humans; D053592:Interleukin-1 Receptor-Associated Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015850:Interleukin-6 / Q000378:metabolism; D016209:Interleukin-8 / Q000235:genetics / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics; D008958:Models, Molecular; D016328:NF-kappa B / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics; D054730:Protein Interaction Domains and Motifs; D012333:RNA, Messenger / Q000378:metabolism; D000071223:Suppressor of Cytokine Signaling 3 Protein; D050826:Suppressor of Cytokine Signaling Proteins / Q000235:genetics; D000072598:Tumor Necrosis Factor alpha-Induced Protein 3; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: C508606:CXCL8 protein, human; D004268:DNA-Binding Proteins; C508600:IL6 protein, human; D015850:Interleukin-6; D016209:Interleukin-8; D047908:Intracellular Signaling Peptides and Proteins; D016328:NF-kappa B; D009687:Nuclear Proteins; D012333:RNA, Messenger; C106644:SOCS3 protein, human; D000071223:Suppressor of Cytokine Signaling 3 Protein; D050826:Suppressor of Cytokine Signaling Proteins; D014409:Tumor Necrosis Factor-alpha; C507771:IRAK3 protein, human; D053592:Interleukin-1 Receptor-Associated Kinases; C076529:TNFAIP3 protein, human; D000072598:Tumor Necrosis Factor alpha-Induced Protein 3