•cGAS/DncV-like Nucleotidyltransferase (CD-NTase) enzymes are a large family of signaling proteins in animals and bacteria.•CD-NTases synthesize nucleotide signals that allow cells to respond to ...pathogens and changing environmental conditions.•The authors highlight the latest discovery of new CD-NTase enzymes and diverse noncanonical RNA signaling products.•The authors describe uncharacterized CD-NTase family members in human cells that potentially control new signaling pathways.
Cyclic GMP–AMP synthase (cGAS) is a signaling enzyme in human cells that controls immune-sensing of cytosolic DNA. The recent discoveries of diverse structural homologs of cGAS in animals and bacteria reveal that cGAS-like signaling is surprisingly ancient and widespread in biology. Together with the Vibrio cholerae protein dinucleotide cyclase in Vibrio (DncV), cGAS and DncV homologs comprise a family of cGAS/DncV-like nucleotidyltransferase (CD-NTase) enzymes that synthesize noncanonical RNA signals including cyclic dinucleotides, cyclic trinucleotides, and linear oligonucleotides. Structural and biochemical breakthroughs provide a framework to understand how CD-NTase signaling allows cells to respond to changing environmental conditions. The CD-NTase family also includes uncharacterized human genes like MB21D2 and Mab21L1, highlighting emerging functions of cGAS-like signaling beyond innate immunity.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPUK, ZAGLJ, ZRSKP
Cyclic GMP-AMP synthase (cGAS) recognition of cytosolic DNA is critical for the immune response to cancer and pathogen infection. Here, we discover that cGAS-DNA phase separation is required to ...resist negative regulation and allow efficient sensing of immunostimulatory DNA. We map the molecular determinants of cGAS condensate formation and demonstrate that phase separation functions to limit activity of the cytosolic exonuclease TREX1. Mechanistically, phase separation forms a selective environment that suppresses TREX1 catalytic function and restricts DNA degradation to an outer shell at the droplet periphery. We identify a TREX1 mutation associated with the severe autoimmune disease Aicardi-Goutières syndrome that increases penetration of TREX1 into the repressive droplet interior and specifically impairs degradation of phase-separated DNA. Our results define a critical function of cGAS-DNA phase separation and reveal a molecular mechanism that balances cytosolic DNA degradation and innate immune activation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPUK, ZAGLJ, ZRSKP
Innate immune recognition of foreign nucleic acids induces protective interferon responses. Detection of cytosolic DNA triggers downstream immune signaling through activation of cyclic GMP-AMP ...synthase (cGAS). We report here the crystal structure of human cGAS, revealing an unanticipated zinc-ribbon DNA-binding domain appended to a core enzymatic nucleotidyltransferase scaffold. The catalytic core of cGAS is structurally homologous to the RNA-sensing enzyme, 2′-5′ oligo-adenylate synthase (OAS), and divergent C-terminal domains account for specific ligand-activation requirements of each enzyme. We show that the cGAS zinc ribbon is essential for STING-dependent induction of the interferon response and that conserved amino acids displayed within the intervening loops are required for efficient cytosolic DNA recognition. These results demonstrate that cGAS and OAS define a family of innate immunity sensors and that structural divergence from a core nucleotidyltransferase enables second-messenger responses to distinct foreign nucleic acids.
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•Crystal structure of the human cytosolic DNA sensor•cGAS and OAS form a family of innate immune receptors•Unique zinc-ribbon motif insertion within cGAS confers DNA specificity
The enzyme cyclic GMP-AMP synthase (cGAS) recognizes cytosolic dsDNA and produces a cyclic dinucleotide second messenger to activate innate immunity. Doudna, Berger, and colleagues now present the crystal structure of human cGAS, revealing a Zinc-ribbon insertion essential for dsDNA recognition and a core nucleotidyltransferase domain structurally homologous to the dsRNA sensor oligo-adenylate synthase (OAS). These results show that cGAS and OAS constitute a family of catalytic OAS-like second-messenger receptors (OLRs), forming the front line of immune defense.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPUK, ZAGLJ, ZRSKP
In humans, the cGAS-STING immunity pathway signals in response to cytosolic DNA via 2′,3′ cGAMP, a cyclic dinucleotide (CDN) second messenger containing mixed 2′–5′ and 3′–5′ phosphodiester bonds. ...Prokaryotes also produce CDNs, but these are exclusively 3′ linked, and thus the evolutionary origins of human 2′,3′ cGAMP signaling are unknown. Here we illuminate the ancient origins of human cGAMP signaling by discovery of a functional cGAS-STING pathway in Nematostella vectensis, an anemone species >500 million years diverged from humans. Anemone cGAS appears to produce a 3′,3′ CDN that anemone STING recognizes through nucleobase-specific contacts not observed in human STING. Nevertheless, anemone STING binds mixed-linkage 2′,3′ cGAMP indistinguishably from human STING, trapping a unique structural conformation not induced by 3′,3′ CDNs. These results reveal that human mixed-linkage cGAMP achieves universal signaling by exploiting a deeply conserved STING conformational intermediate, providing critical insight for therapeutic targeting of the STING pathway.
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•Binding of CDNs is an evolutionarily ancient STING function, predating interferons•cGAS-STING function is conserved in anemone, >500 million years diverged from humans•Anemone cGAS produces a canonical 3′,3′ linked CDN similar to those in bacteria•Vertebrate 2′,3′ cGAMP signaling exploits a deeply conserved STING conformation
Kranzusch and Wilson et al. use structural and biochemical approaches to characterize human and anemone cGAS-STING immune pathways, demonstrating that the product of human cGAS is a potent STING activator because it targets an ancient, conserved, intermediate conformation of STING.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPUK, ZAGLJ, ZRSKP
Cyclic oligonucleotide-based antiphage signaling system (CBASS) immunity is a widespread form of antiphage defense in bacteria and archaea. Each CBASS operon encodes a cGAS/DncV-like ...Nucleotidyltransferase (CD-NTase) enzyme that synthesizes a nucleotide second messenger in response to viral infection. An associated Cap effector protein then binds the nucleotide signal and executes cell death to destroy the host cell and block phage propagation. Here we build upon recent advances to establish rules controlling each step of CBASS activation and antiphage defense. Comparative analysis of CBASS, CRISPR, Pycsar, and cGAS-STING immunity provides insight into the evolution of phage defense and animal innate immunity and highlights new questions emerging in the role of nucleotide second messenger signaling in host–virus interactions.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPUK, ZAGLJ, ZRSKP
Gasdermin proteins form large membrane pores in human cells that release immune cytokines and induce lytic cell death. Gasdermin pore formation is triggered by caspase-mediated cleavage during ...inflammasome signaling and is critical for defense against pathogens and cancer. We discovered gasdermin homologs encoded in bacteria that defended against phages and executed cell death. Structures of bacterial gasdermins revealed a conserved pore-forming domain that was stabilized in the inactive state with a buried lipid modification. Bacterial gasdermins were activated by dedicated caspase-like proteases that catalyzed site-specific cleavage and the removal of an inhibitory C-terminal peptide. Release of autoinhibition induced the assembly of large and heterogeneous pores that disrupted membrane integrity. Thus, pyroptosis is an ancient form of regulated cell death shared between bacteria and animals.
The presence of DNA in the cytosol of mammalian cells is an unusual event that is often associated with genotoxic stress or viral infection. The enzyme cGAS is a sensor of cytosolic DNA that induces ...interferon and inflammatory responses that can be protective or pathologic, depending on the context. Along with other cytosolic innate immune receptors, cGAS is thought to diffuse throughout the cytosol in search of its DNA ligand. Herein, we report that cGAS is not a cytosolic protein but rather localizes to the plasma membrane via the actions of an N-terminal phosphoinositide-binding domain. This domain interacts selectively with PI(4,5)P2, and cGAS mutants defective for lipid binding are mislocalized to the cytosolic and nuclear compartments. Mislocalized cGAS induces potent interferon responses to genotoxic stress, but weaker responses to viral infection. These data establish the subcellular positioning of a cytosolic innate immune receptor as a mechanism that governs self-nonself discrimination.
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•Biochemical and microscopic evidence indicates cGAS is a plasma membrane protein•The cGAS N terminus interacts with PI(4,5)P2 to mediate membrane localization•Mislocalized cGAS mutants drive lethal interferon responses to genotoxic stress•Mislocalized cGAS mutants are poorly responsive to DNA virus infection
The innate immune receptor cGAS interacts with PI(4,5)P2 in order to localize to the plasma membrane, which is critical to prevent aberrant interferon responses to self-DNA under conditions of genotoxic stress, as well as to properly sense viral infections.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPUK, ZAGLJ, ZRSKP
Cytosolic DNA triggers innate immune responses through the activation of cyclic GMP-AMP synthase (cGAS) and production of the cyclic dinucleotide second messenger 2',3'-cyclic GMP-AMP (cGAMP)
. ...2',3'-cGAMP is a potent inducer of immune signalling; however, no intracellular nucleases are known to cleave 2',3'-cGAMP and prevent the activation of the receptor stimulator of interferon genes (STING)
. Here we develop a biochemical screen to analyse 24 mammalian viruses, and identify poxvirus immune nucleases (poxins) as a family of 2',3'-cGAMP-degrading enzymes. Poxins cleave 2',3'-cGAMP to restrict STING-dependent signalling and deletion of the poxin gene (B2R) attenuates vaccinia virus replication in vivo. Crystal structures of vaccinia virus poxin in pre- and post-reactive states define the mechanism of selective 2',3'-cGAMP degradation through metal-independent cleavage of the 3'-5' bond, converting 2',3'-cGAMP into linear Gp2'-5'Ap3'. Poxins are conserved in mammalian poxviruses. In addition, we identify functional poxin homologues in the genomes of moths and butterflies and the baculoviruses that infect these insects. Baculovirus and insect host poxin homologues retain selective 2',3'-cGAMP degradation activity, suggesting an ancient role for poxins in cGAS-STING regulation. Our results define poxins as a family of 2',3'-cGAMP-specific nucleases and demonstrate a mechanism for how viruses evade innate immunity.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Cyclic GMP–AMP synthase (cGAS) recognition of cytosolic DNA is critical for immune responses to pathogen replication, cellular stress, and cancer. Existing structures of the mouse cGAS–DNA complex ...provide a model for enzyme activation but do not explain why human cGAS exhibits severely reduced levels of cyclic GMP–AMP (cGAMP) synthesis compared to other mammals. Here, we discover that enhanced DNA-length specificity restrains human cGAS activation. Using reconstitution of cGAMP signaling in bacteria, we mapped the determinant of human cGAS regulation to two amino acid substitutions in the DNA-binding surface. Human-specific substitutions are necessary and sufficient to direct preferential detection of long DNA. Crystal structures reveal why removal of human substitutions relaxes DNA-length specificity and explain how human-specific DNA interactions favor cGAS oligomerization. These results define how DNA-sensing in humans adapted for enhanced specificity and provide a model of the active human cGAS–DNA complex to enable structure-guided design of cGAS therapeutics.
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•cGAS-DNA-sensing in humans is adapted for enhanced specificity•A bacterial genetic assay allows rapid mapping of human cGAS regulatory determinant•Human cGAS–DNA structures reveal altered contacts that favor DNA-length discrimination•cGAS active site variation explains species-specificity of small-molecule inhibitors
The structure of the human cGAS–DNA complex reveals regulatory adaptations that balance enzymatic activity with DNA-length sensitivity, and additional features important for drug design.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPUK, ZAGLJ, ZRSKP
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