Human cGAS catalytic domain has an additional DNA-binding interface that enhances enzymatic activity and liquid-phase condensation

Xie, Wei; Lama, Lodoe; Adura, Carolina; Tomita, Daisuke; Glickman, J. Fraser; Tuschl, Thomas; Patel, Dinshaw J.

doi:10.1073/pnas.1905013116

Cited by 152 publications

(115 citation statements)

References 50 publications

(80 reference statements)

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“…Finally, accumulated cGAS-dsDNA complexes can go through a liquid-phase separation and condense into gellike droplets as a reaction unit (Figure 1D). This conformation requires a sufficiently long dsDNA strand to form multivalent interaction positions, also requires the function of the N-terminal tail of cGAS and a recently discovered dsDNA-binding site in the catalytic domain of cGAS (site C) (22,23). Meanwhile, the N-terminal tail of cGAS mediates cGAS localization onto the membrane by binding to phosphatidylinositol 4,5bisphosphate (PI (4, 5) P2) and prevents liberation of cGAS and oligomerization, but can release cGAS during cell stress (24).…”

Section: Cgas Recognizes Cytosolic Dna and Produces Cgampmentioning

confidence: 99%

Research Advances in How the cGAS-STING Pathway Controls the Cellular Inflammatory Response

2020

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Double-stranded DNA (dsDNA) sensor cyclic-GMP-AMP synthase (cGAS) along with the downstream stimulator of interferon genes (STING) acting as essential immunesurveillance mediators have become hot topics of research. The intrinsic function of the cGAS-STING pathway facilitates type-I interferon (IFN) inflammatory signaling responses and other cellular processes such as autophagy, cell survival, senescence. cGAS-STING pathway interplays with other innate immune pathways, by which it participates in regulating infection, inflammatory disease, and cancer. The therapeutic approaches targeting this pathway show promise for future translation into clinical applications. Here, we present a review of the important previous works and recent advances regarding the cGAS-STING pathway, and provide a comprehensive understanding of the modulatory pattern of the cGAS-STING pathway under multifarious pathologic states.

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Section: Cgas Recognizes Cytosolic Dna and Produces Cgampmentioning

confidence: 99%

Research Advances in How the cGAS-STING Pathway Controls the Cellular Inflammatory Response

2020

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“…Compared to m-cGAS, h-cGAS exhibits greater preference to long dsDNA. 18,54 The increased number of basic residues in the so-called site-C, which is a newly identified DNA-binding interface in the catalytic domain of cGAS, of h-cGAS contributes to the multivalence of this protein, facilitating liquid-phase condensation and enhancing enzymatic activity upon the detection of long dsDNA. 54 In addition, substitutions of N172/R180 in m-cGAS to K187/L195 in h-cGAS weaken a portion of the cGAS-DNA-binding surface that is necessary during the recognition of short dsDNA but is dispensable upon detection of long dsDNA.…”

Section: Evolution Of the Cgas-sting Pathwaymentioning

confidence: 99%

The interactions between cGAS-STING pathway and pathogens

Cheng

Dai

et al. 2020

Sig Transduct Target Ther

137

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Cytosolic DNA is an indicator of pathogen invasion or DNA damage. The cytosolic DNA sensor cyclic guanosine monophosphateadenosine monophosphate (cGAMP) synthase (cGAS) detects DNA and then mediates downstream immune responses through the molecule stimulator of interferon genes (STING, also known as MITA, MPYS, ERIS and TMEM173). Recent studies focusing on the roles of the cGAS-STING pathway in evolutionary distant species have partly sketched how the mammalian cGAS-STING pathways are shaped and have revealed its evolutionarily conserved mechanism in combating pathogens. Both this pathway and pathogens have developed sophisticated strategies to counteract each other for their survival. Here, we summarise current knowledge on the interactions between the cGAS-STING pathway and pathogens from both evolutionary and mechanistic perspectives. Deeper insight into these interactions might enable us to clarify the pathogenesis of certain infectious diseases and better harness the cGAS-STING pathway for antimicrobial methods.

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“…Obviously, other amino acids also play a major role for the enzyme's activity. A recent study highlighted another cGAS-DNA interface, which is formed by an extended basic patch of positively charged residues originating from the α-region (Q264, K275, K279, K282, and K285), the KRKR-motif (K299, R300, K301, and R302), and the KKH-loop (K427, K428, and K432) [35]. Whereas the residues in the α-region and in the KRKR-motif are mainly conserved throughout the investigated homologues, the KKH-loop is completely inhomogeneous and could explain varying enzyme activities.…”

Section: Of 15mentioning

confidence: 99%

Screening and Identification of Novel cGAS Homologues Using a Combination of in Vitro and In Vivo Protein Synthesis

Rolf

Siedentop

Lütz

et al. 2019

IJMS

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The cyclic GMP-AMP synthase (cGAS) catalyzes the synthesis of the multifunctional second messenger, cGAMP, in metazoans. Although numerous cGAS homologues are predicted in protein databases, the catalytic activity towards cGAMP synthesis has been proven for only four of them. Therefore, we selected five novel and yet uncharacterized cGAS homologues, which cover a broad range in the field of vertebrates. Cell-free protein synthesis (CFPS) was used for a pre-screening to investigate if the cGAS genes originating from higher organisms can be efficiently expressed in a bacterial expression system. As all tested cGAS variants were expressible, enzymes were synthesized in vivo to supply higher amounts for a subsequent in vitro activity assay. The assays were carried out with purified enzymes and revealed vast differences in the activity of the homologues. For the first time, the cGAS homologues from the Przewalski's horse, naked mole-rat, bald eagle, and zebrafish were proven to catalyze the synthesis of cGAMP. The extension of the list of described cGAS variants enables the acquisition of further knowledge about the structural and molecular mechanism of cGAS, potentially leading to functional improvement of the enzyme.The chemical synthesis of 2 3 -cGAMP contains eight reaction steps and suffers from low yields [8,9], whereas the enzymatic reaction reaches nearly full conversion within a few hours [6]. In recent studies, human cGAS was investigated predominantly with regards to the identification of key residues [10,11] and the kinetic mechanism [12]. Although several homologous enzymes are known, only murine cGAS [6,7], porcine cGAS [10,13], and chicken cGAS [14] received more attention. For the murine and porcine homologue, crystal structures are also available and, in parts, crucial amino acids with relevance for the catalytic function are known. Alignments of amino acid sequences of identified cGAS homologues revealed a low-sequence homology in regions without determined function [10,15]. This high level of variation and the markedly reduced activity of human cGAS in comparison to murine cGAS [11] demonstrate the possibility that other cGAS homologues might be available with enhanced properties or different characteristics and functions. For characterization, cGAS enzymes were synthesized with eukaryotic cell lines or expressed recombinantly in Escherichia coli. Eukaryotic genes tend to be difficult to express in bacterial systems, though fusion-tags, such as maltose-binding protein (MBP, 42.5 kDa) [16], glutathione S-transferase (GST, 26 kDa) [2], and small ubiquitin-like modifier (SUMO, 12 kDa) [1], were used for better solubility and functional expression of cGAS. Challenges remain to find suitable expression systems for eukaryotic protein production with good yield and purity, and under conditions conducive to functional protein studies. To realize higher throughput in heterologous protein synthesis, other methods than traditional recombinant expression can be considered.

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Human cGAS catalytic domain has an additional DNA-binding interface that enhances enzymatic activity and liquid-phase condensation

Cited by 152 publications

References 50 publications

Research Advances in How the cGAS-STING Pathway Controls the Cellular Inflammatory Response

Research Advances in How the cGAS-STING Pathway Controls the Cellular Inflammatory Response

The interactions between cGAS-STING pathway and pathogens

Screening and Identification of Novel cGAS Homologues Using a Combination of in Vitro and In Vivo Protein Synthesis

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