Investigating the regulation and enzymatic function of human ecsit in innate immune signalling.

Toll-like receptor (TLR) signalling represents the first line of defence against infection. TLRs respond to recognition of pathogens by activating transcription factors such as NFκB and the interferon regulatory factors (IRF)s to induce pro-inflammatory cytokines and IFNs. Whilst many of the components of the TLR signal transduction pathways have been identified, a full understanding of these complex regulatory systems remains to be delineated. This thesis probes the molecular, cellular and physiological roles of a protein termed evolutionary conserved signalling intermediate in toll (ECSIT) in innate immune signalling pathways. ECSIT was initially described as a TRAF6 interacting protein that bridged the TLR signalling intermediate TRAF6 to MEKK1 and downstream activation of NFκB. Subsequent data revealed an important developmental role for ECSIT in the BMP signalling pathway based on the embryonic lethal phenotype in the ECSIT knockout mouse. Other studies have indicated an important role for ECSIT in bacterial killing during infection via the production of mitochondrial reactive oxygen species (mROS). To date no information on the human orthologue of ECSIT (hECSIT) has been available. In this study we describe novel functional roles for hECSIT in TLR signalling. Through use of knockdown studies and a humanised animal model we demonstrate that hECSIT negatively regulates innate immune signalling pathways despite its 80% sequence homology to mECSIT. We show that phosphorylation, ubiquitination and processing of hECSIT leads to an inhibitory C-terminal form that contains both E3 ligase and DUB activity. We also demonstrate that hECSIT can independently target and regulate the ubiquitination of TRAF6, TRAF3 and RIP1 in TLR4/IL-1R, TLR3 and TNF-α signalling, respectively. These findings identify hECSIT as a novel regulator of innate immune signalling and highlight a sophisticated evolutionary difference between two highly conserved proteins