Innate Immunity relies on the ability of the molecular machinery of the cell to orchestrate precise molecular interactions for the detection of invading pathogens and the ensuing intracellular and intercellular signaling. Protein kinases, such as TANK-binding kinase 1 (TBK1) and the highly related inducible IB-kinase (IKK-i), are central components of innate immunity intracellular signaling but are poorly understood in terms of wiring logic. We set out to untangle their molecular interactions as well as those of their known partners TANK, Sintbad and NAP1. We show that the adaptor proteins bind to TBK1 and IKK-i in a mutually exclusive manner suggesting distinct alternative complexes. Binding of each adaptor protein to TBK1 was disrupted by single point mutations on the C-terminal coiled-coil 2 region of TBK1.
Using point mutants that affect binding of individual adaptors we found that TBK1 activation in response to double stranded RNA or viral infection was strictly dependent on the integrity of the TBK1/TANK interaction. TBK1 activation occurs upon recognition of foreign nucleic acids by specific innate immunity receptors. As a complementary approach to identify proteins involved in innate immunity we used immobilized nucleic acids and analyzed binding proteins by mass spectrometry. We identify SAMHD1, a protein associated with Aicardi Goutières Syndrome (AGS), as a nucleic acid binding protein displaying a preference for RNA over DNA. We map a region containing the HD domain as the nucleic acid binding domain. Contrary to wild type SAMHD1, mutants of SAMHD1 observed in AGS patients localize to the cytosol. These data suggest that SAMHD1 has a role in the nucleus that, if disrupted by mutation, leads to cytosolic accumulation of SAMHD1 and autoimmune disease. Overall, the proteomics approach used in my thesis enabled us to identify new players in innate immunity from two different angles of the pathway: TBK1 and IKKi protein complexes; and SAMHD1 as a nucleic-acid interactor. Thus, my thesis provides for an improved understanding of their physical and functional relationship in innate immunity and, potentially, in autoimmune disease.