The cGAS-STING signalling axis, comprising the synthase for the second messenger cyclic GMP-AMP (cGAS) and the cyclic GMP-AMP receptor stimulator of interferon genes (STING), detects pathogenic DNA ...to trigger an innate immune reaction involving a strong type I interferon response against microbial infections. Notably however, besides sensing microbial DNA, the DNA sensor cGAS can also be activated by endogenous DNA, including extranuclear chromatin resulting from genotoxic stress and DNA released from mitochondria, placing cGAS-STING as an important axis in autoimmunity, sterile inflammatory responses and cellular senescence. Initial models assumed that co-localization of cGAS and DNA in the cytosol defines the specificity of the pathway for non-self, but recent work revealed that cGAS is also present in the nucleus and at the plasma membrane, and such subcellular compartmentalization was linked to signalling specificity of cGAS. Further confounding the simple view of cGAS-STING signalling as a response mechanism to infectious agents, both cGAS and STING were shown to have additional functions, independent of interferon response. These involve non-catalytic roles of cGAS in regulating DNA repair and signalling via STING to NF-κB and MAPK as well as STING-mediated induction of autophagy and lysosome-dependent cell death. We have also learnt that cGAS dimers can multimerize and undergo liquid-liquid phase separation to form biomolecular condensates that could importantly regulate cGAS activation. Here, we review the molecular mechanisms and cellular functions underlying cGAS-STING activation and signalling, particularly highlighting the newly emerging diversity of this signalling pathway and discussing how the specificity towards normal, damage-induced and infection-associated DNA could be achieved.
Extensive research on antiviral small molecules starting in the early 1970s has led to the identification of 10‐carboxymethyl‐9‐acridanone (CMA) as a potent type I interferon (IFN) inducer. Up to ...date, the mode of action of this antiviral molecule has remained elusive. Here we demonstrate that CMA mediates a cell‐intrinsic type I IFN response, depending on the ER‐resident protein STING. CMA directly binds to STING and triggers a strong antiviral response through the TBK1/IRF3 route. Interestingly, while CMA displays extraordinary activity in phosphorylating IRF3 in the murine system, CMA fails to activate human cells that are otherwise responsive to STING ligands. This failure to activate human STING can be ascribed to its inability to bind to the C‐terminal ligand‐binding domain of human STING. Crystallographic studies show that two CMA molecules bind to the central Cyclic diguanylate (c‐diGMP)‐binding pocket of the STING dimer and fold the lid region in a fashion similar, but partially distinct, to c‐diGMP. Altogether, these results provide novel insight into ligand‐sensing properties of STING and, furthermore, unravel unexpected species‐specific differences of this innate sensor.
Induction of type I interferon responses by an antiviral drug occurs through binding to the murine innate immune sensor STING, whose divergent ligand‐binding domain explains the lack of CMA efficacy in human cells.
Mre11-Rad50: the DNA end game Hopfner, Karl-Peter
Biochemical Society transactions,
04/2023, Letnik:
51, Številka:
2
Journal Article
Recenzirano
The Mre11-Rad50-(Nbs1/Xrs2) complex is an evolutionarily conserved factor for the repair of DNA double-strand breaks and other DNA termini in all kingdoms of life. It is an intricate DNA associated ...molecular machine that cuts, among other functions, a large variety of free and obstructed DNA termini for DNA repair by end joining or homologous recombination, yet leaves undamaged DNA intact. Recent years have brought progress in both the structural and functional analyses of Mre11-Rad50 orthologs, revealing mechanisms of DNA end recognition, endo/exonuclease activities, nuclease regulation and DNA scaffolding. Here, I review our current understanding and recent progress on the functional architecture Mre11-Rad50 and how this chromosome associated coiled-coil ABC ATPase acts as DNA topology specific endo-/exonuclease.
Intracellular recognition of non‐self and also self‐nucleic acids can result in the initiation of potent pro‐inflammatory and antiviral cytokine responses. Most recently, cGAS was shown to be ...critical for the recognition of cytoplasmic dsDNA. Binding of dsDNA to cGAS results in the synthesis of cGAMP(2′–5′), which then binds to the endoplasmic reticulum resident protein STING. This initiates a signaling cascade that triggers the induction of an antiviral immune response. While most studies on intracellular nucleic acids have focused on dsRNA or dsDNA, it has remained unexplored whether cytosolic RNA:DNA hybrids are also sensed by the innate immune system. Studying synthetic RNA:DNA hybrids, we indeed observed a strong type I interferon response upon cytosolic delivery of this class of molecule. Studies in THP‐1 knockout cells revealed that the recognition of RNA:DNA hybrids is completely attributable to the cGAS–STING pathway. Moreover, in vitro studies showed that recombinant cGAS produced cGAMP upon RNA:DNA hybrid recognition. Altogether, our results introduce RNA:DNA hybrids as a novel class of intracellular PAMP molecules and describe an alternative cGAS ligand next to dsDNA.
Synopsis
Next to dsDNA, cGAS can bind to cytoplasmic RNA:DNA hybrids. This results in the formation of cGAMP, which subsequently binds to and activates STING, leading to antiviral gene expression.
Cytosolic delivery of RNA:DNA hybrids triggers antiviral gene expression in myeloid cells.
RNA:DNA hybrid recognition depends on the cGAS–STING axis.
Hybrids directly bind to cGAS, resulting in its enzymatic activity.
RNA:DNA hybrids are a novel class of intracellular PAMP molecules that can directly stimulate cGAS to produce cGAMP leading to the induction of antiviral response genes.
RIG-I-like receptors (RLRs) are cytosolic innate immune sensors that detect pathogenic RNA and induce a systemic antiviral response. During the last decade, many studies focused on their molecular ...characterization and the identification of RNA agonists. Therefore, it became more and more clear that RLR activation needs to be carefully regulated, because constitutive signaling or detection of endogenous RNA through loss of specificity is detrimental. Here, we review the current understanding of RLR activation and selectivity. We specifically focus upon recent findings on the function of the helicase domain in discriminating between different RNAs, and whose malfunctioning causes serious autoimmune diseases.
Nuclear cGAS: guard or prisoner? Oliveira Mann, Carina C; Hopfner, Karl‐Peter
The EMBO journal,
16 August 2021, Letnik:
40, Številka:
16
Journal Article
Recenzirano
Odprti dostop
cGAS, an innate immune sensor of cellular stress, recognizes double‐stranded DNA mislocalized in the cytosol upon infection, mitochondrial stress, DNA damage, or malignancy. Early models suggested ...that cytosolic localization of cGAS prevents autoreactivity to nuclear and mitochondrial self‐DNA, but this paradigm has shifted in light of recent findings of cGAS as a predominantly nuclear protein tightly bound to chromatin. This has raised the question how nuclear cGAS is kept inactive while being surrounded by chromatin, and what function nuclear localization of cGAS may serve in the first place? Cryo‐EM structures have revealed that cGAS interacts with nucleosomes, the minimal units of chromatin, mainly via histones H2A/H2B, and that these protein–protein interactions block cGAS from DNA binding and thus prevent autoreactivity. Here, we discuss the biological implications of nuclear cGAS and its interaction with chromatin, including various mechanisms for nuclear cGAS inhibition, release of chromatin‐bound cGAS, regulation of different cGAS pools in the cell, and chromatin structure/chromatin protein effects on cGAS activation leading to cGAS‐induced autoimmunity.
This review summarizes mounting evidence for cGAS presence in the cell nucleus and on chromatin, and discusses its possible regulatory significance and how it can be reconciled with its cytoplasmic DNA sensor roles.
Recent discoveries in the field of innate immunity have highlighted the existence of a family of nucleic acid-sensing proteins that have similar structural and functional properties. These include ...the well-known oligoadenylate synthase (OAS) family proteins and the recently identified OAS homologue cyclic GMP-AMP (cGAMP) synthase (cGAS). The OAS proteins and cGAS are template-independent nucleotidyltransferases that, once activated by double-stranded nucleic acids in the cytosol, produce unique classes of 2'-5'-linked second messenger molecules, which - through distinct mechanisms - have crucial antiviral functions. 2'-5'-linked oligoadenylates limit viral propagation through the activation of the enzyme RNase L, which degrades host and viral RNA, and 2'-5'-linked cGAMP activates downstream signalling pathways to induce de novo antiviral gene expression. In this Progress article, we describe the striking functional and structural similarities between OAS proteins and cGAS, and highlight their roles in antiviral immunity.
The Mre11–Rad50–Nbs1 (MRN) complex is a central factor in the repair of DNA double‐strand breaks (DSBs). The ATP‐dependent mechanisms of how MRN detects and endonucleolytically processes DNA ends for ...the repair by microhomology‐mediated end‐joining or further resection in homologous recombination are still unclear. Here, we report the crystal structures of the ATPγS‐bound dimer of the Rad50NBD (nucleotide‐binding domain) from the thermophilic eukaryote Chaetomium thermophilum (Ct) in complex with either DNA or CtMre11RBD (Rad50‐binding domain) along with small‐angle X‐ray scattering and cross‐linking studies. The structure and DNA binding motifs were validated by DNA binding experiments in vitro and mutational analyses in Saccharomyces cerevisiae in vivo. Our analyses provide a structural framework for the architecture of the eukaryotic Mre11–Rad50 complex. They show that a Rad50 dimer binds approximately 18 base pairs of DNA along the dimer interface in an ATP‐dependent fashion or bridges two DNA ends with a preference for 3′ overhangs. Finally, our results may provide a general framework for the interaction of ABC ATPase domains of the Rad50/SMC/RecN protein family with DNA.
Synopsis
The structure of the eukaryotic DNA double‐strand break repair protein Rad50 in complex with ATPγS and DNA reveals how ATP‐dependent dimerization of Rad50's ABC ATPase domain generates a positively charged platform that binds continuous B‐DNA. The structure provides a framework for the ATP‐dependent interaction of ABC ATPase domains of the Rad50/SMC/RecN family with DNA.
Eukaryotic Mre11 binds to Rad50 via a five‐helix bundle domain.
Rad50:ATPγS binds DNA along the ABC ATPase domain dimer interface.
Rad50's DNA interaction is critical for the repair of topoisomerase‐induced DNA double‐strand breaks.
The Rad50 dimer can bind either continuous B‐DNA or two joined DNA ends.
The structure of the DNA double‐strand break repair protein Rad50 in complex with DNA reveals how ATP‐dependent dimerization of its ABC ATPase domain generates a positively charged platform for binding DNA.
In the eukaryotic nucleus, DNA is packaged in the form of nucleosomes, each of which comprises about 147 base pairs of DNA wrapped around a histone protein octamer. The position and histone ...composition of nucleosomes is governed by ATP-dependent chromatin remodellers
such as the 15-subunit INO80 complex
. INO80 regulates gene expression, DNA repair and replication by sliding nucleosomes, the exchange of histone H2A.Z with H2A, and the positioning of + 1 and -1 nucleosomes at promoter DNA
. The structures and mechanisms of these remodelling reactions are currently unknown. Here we report the cryo-electron microscopy structure of the evolutionarily conserved core of the INO80 complex from the fungus Chaetomium thermophilum bound to a nucleosome, at a global resolution of 4.3 Å and with major parts at 3.7 Å. The INO80 core cradles one entire gyre of the nucleosome through multivalent DNA and histone contacts. An Rvb1/Rvb2 AAA
ATPase heterohexamer is an assembly scaffold for the complex and acts as a 'stator' for the motor and nucleosome-gripping subunits. The Swi2/Snf2 ATPase motor binds to nucleosomal DNA at superhelical location -6, unwraps approximately 15 base pairs, disrupts the H2A-DNA contacts and is poised to pump entry DNA into the nucleosome. Arp5 and Ies6 bind superhelical locations -2 and -3 to act as a counter grip for the motor, on the other side of the H2A-H2B dimer. The Arp5 insertion domain forms a grappler element that binds the nucleosome dyad, connects the Arp5 actin-fold and entry DNA over a distance of about 90 Å and packs against histone H2A-H2B near the 'acidic patch'. Our structure together with biochemical data
suggests a unified mechanism for nucleosome sliding and histone editing by INO80. The motor is part of a macromolecular ratchet, persistently pumping entry DNA across the H2A-H2B dimer against the Arp5 grip until a large nucleosome translocation step occurs. The transient exposure of H2A-H2B by motor activity as well as differential recognition of H2A.Z and H2A may regulate histone exchange.
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