Aicardi-Goutières syndrome (AGS) is a rare inherited autoimmune disease caused by mutations in genes encoding the RNase H2 subunits A, B, and C; the DNase three prime repair exonuclease 1 (TREX1); and sterile alpha motif (SAM) domain and HD domain-containing protein 1 (SAMHD1). Using unbiased affinity purification coupled to protein mass spectrometry, we identify SAMHD1 as a nucleic-acid-binding protein displaying a preference for RNA over DNA. In contrast to TREX1 and the RNase H2 complex, SAMHD1 has no obvious nuclease activity. In addition, interrogating truncation mutants of SAMHD1 observed in AGS patients, we map the nucleic-acid-binding domain to residues 164-442, thus overlapping with the HD domain. Furthermore, we show that although wild-type SAMHD1 displays almost exclusive nuclear localization, 11 of 12 SAMHD1 mutants show at least partial mislocalization to the cytosol. Overall, 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.

TANK-binding kinase 1 (TBK1) and inducible IκB-kinase (IKK-i) are central regulators of type-I interferon induction. They are associated with three adaptor proteins called TANK, Sintbad (or TBKBP1) and NAP1 (or TBKBP2, AZI2) whose functional relationship to TBK1 and IKK-i is poorly understood. We performed a systematic affinity purification–mass spectrometry approach to derive a comprehensive TBK1/IKK-i molecular network. The most salient feature of the network is the mutual exclusive interaction of the adaptors with the kinases, suggesting distinct alternative complexes. Immunofluorescence data indicated that the individual adaptors reside in different subcellular locations. TANK, Sintbad and NAP1 competed for binding of TBK1. The binding site for all three adaptors was mapped to the C-terminal coiled-coil 2 region of TBK1. Point mutants that affect binding of individual adaptors were used to reconstitute TBK1/IKK-i-deficient cells and dissect the functional relevance of the individual kinase-adaptor edges within the network. Using a microarray-derived gene expression signature of TBK1 in response virus infection or poly(I∶C) stimulation, we found that TBK1 activation was strictly dependent on the integrity of the TBK1/TANK interaction.

Recognition of foreign DNA by cytosolic innate immune receptors triggers the production of IFN-b. However, it is unclear whether different types of DNA ligands are recognized by similar receptors and whether the resulting response is distinct from the endosomal TLR response. To address these questions, we compared the two most commonly used types of DNA ligands (IFN-stimulatory DNA (ISD) and poly(dAdT)) and assessed the minimal structural requirements for stimulatory capacity in RAW264.7 cells. Gene expression signatures and competition experiments suggest that ISD and poly(dAdT) are qualitatively indistinguishable and differ from the CpG-containing oligonucleotides triggering the TLR9 pathway. Structure -activity relationship analyses revealed that a minimal length of two helical turns is sufficient for ISD-mediated IFN-b induction, while phosphorylation at the 5 0 -end is dispensable. Altogether, our data suggest that, in murine macrophages, only one major cytosolic DNA recognition pathway is operational. IntroductionInnate immunity represents the first line of defense against invading pathogens. PRR recognize molecular features that are conserved among many pathogens, so called PAMP. PRR are localized on the plasma membrane, the endosome or the cytosol and belong to distinct classes, most notably the TLR, the NOD-like receptors and the RIG-I-like helicases (RLH) [1,2]. Engagement of PRR by specific ligands triggers intracellular signaling cascades that culminate in the production and secretion of type-I IFN, cytokines and chemokines.An invading virus can either be sensed by endosomal TLR (TLR3, 7/8 or 9) or by cytosolic RLH [3], whereas, TLR3 and TLR7/8 recognize viral RNA, TLR9 senses the presence of viral DNA containing CpG motifs [4]. The RLH detect viral RNA in the cytosol [5]. This raises the question of how the host cell Eur. J. Immunol. 2009. 39: 1929-1936 DOI 10.1002 Innate immunity 1929distinguishes between viral and cellular RNA that are simultaneously present in the same subcellular compartment. It has recently been shown that many viral RNA, unlike cellular RNA, contain a triphosphate moiety at the 5 0 -end, which is recognized by RIG-I, the founding member of the RLH family [6,7]. Despite ''thousands of man-years worth of DNA transfections'', it has only recently been noticed that introduction of dsDNA into the cytosol triggers a potent innate immune response, leading to the production of IL-1b, IFN-b and other cytokines [8][9][10]. IL-1b secretion is governed by the DNA sensor AIM2 that has recently been identified [11][12][13][14]. IFN-b production is regulated by the DNA-dependent activator of IFN-regulatory factors (DAI, formerly known as DLM-1/ZBP1) [15] and as-yet unknown DNA sensors [16,17]. With regard to the IFN pathway, it has been suggested that the B-conformation of DNA is stimulatory, whereas Z-DNA is not [9]. Another report suggests that the exchange of the phosphate backbone for a phosphorothioate backbone renders the DNA inert, implying that the DNA backbone is the site of recog...