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The catalytic mechanism of cyclic GMP-AMP synthase (cGAS) and implications for innate immunity and inhibition.

ABSTRACT: Cyclic GMP-AMP synthase (cGAS) is activated by ds-DNA binding to produce the secondary messenger 2',3'-cGAMP. cGAS is an important control point in the innate immune response; dysregulation of the cGAS pathway is linked to autoimmune diseases while targeted stimulation may be of benefit in immunoncology. We report here the structure of cGAS with dinucleotides and small molecule inhibitors, and kinetic studies of the cGAS mechanism. Our structural work supports the understanding of how ds-DNA activates cGAS, suggesting a site for small molecule binders that may cause cGAS activation at physiological ATP concentrations, and an apparent hotspot for inhibitor binding. Mechanistic studies of cGAS provide the first kinetic constants for 2',3'-cGAMP formation, and interestingly, describe a catalytic mechanism where 2',3'-cGAMP may be a minor product of cGAS compared with linear nucleotides.

SUBMITTER: Hall J

PROVIDER: S-EPMC5699495 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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The catalytic mechanism of cyclic GMP-AMP synthase (cGAS) and implications for innate immunity and inhibition.

Hall Justin J Ralph Erik C EC Shanker Suman S Wang Hong H Byrnes Laura J LJ Horst Reto R Wong Jimson J Brault Amy A Dumlao Darren D Smith James F JF Dakin Leslie A LA Schmitt Daniel C DC Trujillo John J Vincent Fabien F Griffor Matt M Aulabaugh Ann E AE

Protein science : a publication of the Protein Society 20171025 12

Cyclic GMP-AMP synthase (cGAS) is activated by ds-DNA binding to produce the secondary messenger 2',3'-cGAMP. cGAS is an important control point in the innate immune response; dysregulation of the cGAS pathway is linked to autoimmune diseases while targeted stimulation may be of benefit in immunoncology. We report here the structure of cGAS with dinucleotides and small molecule inhibitors, and kinetic studies of the cGAS mechanism. Our structural work supports the understanding of how ds-DNA act ...[more]

PMID: 28940468

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Project description:HIV-1 replicates in cells that express a wide array of innate immune sensors and may do so simultaneously with other pathogens. How a coexisting innate immune stimulus influences the outcome of HIV-1 sensing, however, remains poorly understood. Here, we demonstrate that the activation of a second signaling pathway enables a cyclic GMP-AMP synthase (cGAS)-dependent type I interferon (IFN-I) response to HIV-1 infection. We used RNA sequencing to determine that HIV-1 alone induced few or no signs of an IFN-I response in THP-1 cells. In contrast, when supplemented with suboptimal levels of bacterial lipopolysaccharide (LPS), HIV-1 infection triggered the production of elevated levels of IFN-I and significant upregulation of interferon-stimulated genes. LPS-mediated enhancement of IFN-I production upon HIV-1 infection, which was observed in primary macrophages, was lost by blocking reverse transcription and with a hyperstable capsid, pointing to viral DNA being an essential immunostimulatory molecule. LPS also synergistically enhanced IFN-I production by cyclic GMP-AMP (cGAMP), a second messenger of cGAS. These observations suggest that the DNA sensor cGAS is responsible for a type I IFN response to HIV-1 in concert with LPS receptor Toll-like receptor 4 (TLR4). Small amounts of a TLR2 agonist also cooperate with HIV-1 to induce type I IFN production. These results demonstrate how subtle immunomodulatory activity renders HIV-1 capable of eliciting an IFN-I response through positive cross talk between cGAS and TLR sensing pathways. IMPORTANCE Innate immune activation is a hallmark of HIV-1 pathogenesis. Thus, it is critical to understand how HIV-1 infection elicits innate immune responses. In this work, we show that HIV-1 infection of macrophages leads to a robust type I interferon (IFN) production only when a second signaling event is initiated by a coexisting immunostimulatory molecule. Our results show that HIV-1 infection alone is not sufficient for triggering a strong IFN response. We find that bacterial membrane components, which are recognized by endosomal innate sensors, enable production of elevated levels of IFNs and significant upregulation of interferon-stimulated genes upon HIV-1 infection. This IFN response is dependent on viral DNA synthesis and prevented by a stable capsid, pointing to an essential role for a DNA sensing molecule. These observations provide new insights into how different innate immune recognition pathways synergize during HIV-1 infection and determine the outcome of innate responses.

Project description:The cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway play a significant role in the production of inflammatory cytokines and type I interferons. This study aims to develop a cGAS-STING pathway-related genes (CSRs) prediction model to predict prognosis in gastric cancer (GC). In the present study, we used The Cancer Genome Atlas (TCGA), Gene Expression Omnibus databases (GEO), CIBERSORT and Tumor Immune Estimation Resource databases (TIMER). The risk model based on five hub genes (IFNB1, IFNA4, IL6, NFKB2, and TRIM25) was constructed to predict the overall survival (OS) of GC. Further univariate Cox regression (URC) and multivariate Cox regression (MCR) analyses revealed that this risk scoring model was an independent factor. The results were verified by GEO external validation set. Multiple immune pathways were assessed by Gene Set Enrichment Analysis (GSEA). TIMER analysis demonstrated that risk score strongly correlated with Macrophage, B cells and CD8 + T cells infiltration. In addition, through 'CIBERSORT' package, the higher levels of infiltration of T cell follicular assistance (P = 0.011), NK cells-activated (P = 0.034), and Dendritic cells resting (P = 0.033) exhibited in high-risk group. Kaplan-Meier (K-M) survival analysis illustrated T cells CD4 memory resting and T cells follicular helper infiltration correlated with overall survival (OS) of GC patients in TCGA and GEO databases. Altogether, the risk score model can be conveniently used to predict prognosis. The immunocyte infiltration analysis provided a novel horizon for monitoring the status of the GC immune microenvironment.Abbreviations:TCGA: The Cancer Genome Atlas databases; GEO: Gene Expression Omnibus databases; GC: Gastric cancer; CSRs: cGAS-STING pathway-related genes; DECSRs: Differential expressed cGAS-STING pathway-related genes; PCSRs: Prognosis related cGAS-STING pathway genes; URC: Univariate Cox regression analyses; MCR: Multivariate Cox regression analyses GSEA: Gene set enrichment analysis; TIIC: Tumor-infiltrating immune cell.

Dataset Information

The catalytic mechanism of cyclic GMP-AMP synthase (cGAS) and implications for innate immunity and inhibition.

Publications

The catalytic mechanism of cyclic GMP-AMP synthase (cGAS) and implications for innate immunity and inhibition.

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