Background: Mal/TIRAP is an integral protein of several Toll-like receptor (TLR) pathways, the activation of which results in NF-kB upregulation and leads to cytokine production. Mal, TLR4, and MyD88 all share a conserved C-terminus containing 3 distinct regions, or boxes, of amino acid (aa) sequence similarity which is collectively referred to as the Toll-Interleukin 1 Receptor (TIR) domain. Mal is unique in that it does not share aa sequence similarity of box 3 within the TIR domain with other TIR proteins. Mal acts as a bridging adaptor between TLR4 and MyD88 amplifying the signaling response pathway. We hypothesize that the TIR domain of Mal specifies the binding and activation proclivities of Mal when activated through TLR4 by LPS stimulation. Methods: Mal and truncated Mal TIR constructs containing various combinations of the 3 boxes and a potential dominant negative mutation (P145H) within the TIR domain were cloned into the expression vector pcDNATM4HisMax and cotransfected into RAW 264.7 cells with an NF-kB luciferase reporter vector and a control vector. Transfected RAW 264.7 cells were then stimulated with lipopolysaccharide (LPS). NF-kB upregulation was measured by luciferase assay. The P145H mutation mimics the box 2 TIR domain mutation found in the dominant negative form of TLR4 identified in C3H-HeJ mice, known to be resistant to the effects of lipopolysaccharide.

Results: Mal P145H was found to significantly abrogate the upregulation of NF-kB in RAW 264.7 cells in response to LPS stimulation, whereas Mal 1,2,3 had increased activity over that of the full length native protein but does not significantly increase the stimulation with LPS. The dominant negative form (Mal 1,2,3 P145H), while having baseline diminished activity compared to Mal 1,2,3 was able to be stimulated by LPS. Mal 1,2 and Mal 2,3 have activity far exceeding that of the full length protein with and without the P145H mutation in box 2 and show increased upregulation of NF-kB in the presence of LPS.

Conclusion: These structure function studies of Mal suggest that although the individual contribution of the N-terminal portion of the protein may be important for MyD88/TLR4 coupling based on computer models, the C-terminal TIR domain is critical for downstream NF-kB and cytokine activation independent of the polymorphic P/H mutation. Identification of the role of each conserved box within the TIR will lead to further understanding of the molecular mechanisms regulating the proinflammatory response.