cGAS produces a 2′-5′-linked cyclic dinucleotide second messenger that activates STING

Abstract

Detection of cytoplasmic DNA represents one of the most fundamental mechanisms of the innate immune system to sense the presence of microbial pathogens1. Moreover, erroneous detection of endogenous DNA by the same sensing mechanisms has an important pathophysiological role in certain sterile inflammatory conditions2, 3. The endoplasmic-reticulum-resident protein STING is critically required for the initiation of type I interferon signalling upon detection of cytosolic DNA of both exogenous and endogenous origin4, 5, 6, 7, 8. Next to its pivotal role in DNA sensing, STING also serves as a direct receptor for the detection of cyclic dinucleotides, which function as second messenger molecules in bacteria9, 10, 11, 12, 13. DNA recognition, however, is triggered in an indirect fashion that depends on a recently characterized cytoplasmic nucleotidyl transferase, termed cGAMP synthase (cGAS), which upon interaction with DNA synthesizes a dinucleotide molecule that in turn binds to and activates STING14, 15. We here show in vivo and in vitro that the cGAS-catalysed reaction product is distinct from previously characterized cyclic dinucleotides. Using a combinatorial approach based on mass spectrometry, enzymatic digestion, NMR analysis and chemical synthesis we demonstrate that cGAS produces a cyclic GMP-AMP dinucleotide, which comprises a 2′-5′ and a 3′-5′ phosphodiester linkage >Gp(2′-5′)Ap(3′-5′)>. We found that the presence of this 2′-5′ linkage was required to exert potent activation of human STING. Moreover, we show that cGAS first catalyses the synthesis of a linear 2′-5′-linked dinucleotide, which is then subject to cGAS-dependent cyclization in a second step through a 3′-5′ phosphodiester linkage. This 13-membered ring structure defines a novel class of second messenger molecules, extending the family of 2′-5′-linked antiviral biomolecules.

TREX1 Deficiency Triggers Cell-Autonomous Immunity in a cGAS-Dependent Manner

“…Cyclic GMP-AMP synthase (cGAS) has been identified as a cytosolic DNA receptor responsible for the induction of an antiviral and proinflammatory gene expression profile (2)(3)(4). Upon dsDNA binding, cGAS catalyzes the production of the second messenger molecule cGAMP(29-59), a unique cyclic dinucleotide molecule containing one 29-59 phosphodiester linkage in addition to a canonical 39-59 linkage (5)(6)(7)(8). Cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) subsequently stimulates the endoplasmic reticulum localized protein STING to induce cytokine expression through activation of the transcription factors IRF3 and NF-kB via the protein kinase TBK1 (9).…”

Interfering with Immunity: Detrimental Role of Type I IFNs during Infection

“…23), the retinoic acid-inducible gene (RIG)-like helicases (reviewed in Ref. 24), and the more recently discovered cytoplasmic DNA sensors (25)(26)(27)(28)(29).…”

“…In addition, cytoplasmic DNA sensors, such as IFN-g-inducible protein 16 (IFI16) (27), DNA-dependent activator of IRFs (26), and cyclic GMP-AMP synthase (cGAS) (25,28), can bind dsDNA and trigger endoplasmic reticulum-associated adaptor molecule stimulator of IFN genes (STING) (32), TBK1, and IRF3-dependent type I IFN induction (31).…”

“…Interestingly, a recent study demonstrated that, in human cells, Listeria-derived DNA, rather than cyclic diadenosine monophosphate, triggers IFN-b production via cytosolic DNA sensors IFI16 and cGAS (55). This occurs, in part, because murine cells are more responsive to bacterial cyclic dinucleotides than human cells (28,56).…”

Innate immune activation of NFκB and its antagonism by poxviruses

“…cGAMP is a direct ligand for STING, which is initially localized in the endoplasmic reticulum, but on binding cGAMP translocates to TBK1-containing membrane-bound compartments leading to IRF3 activation. Interestingly, a recent study demonstrated that, after infection of cells with MVA, cGAMP can diffuse through cellular gap junctions to activate the TI-IFN response in adjacent, uninfected cells, implying that the cGAS/STING system may directly prepare bystander cells for resistance to incoming poxviral infection [84,85]. The cGAS/STING system was also shown to sense MVA DNA in the cytoplasm of conventional DCs during infection [86].…”