Enzymatic Preparation of 2′–5′,3′–5′-Cyclic Dinucleotides, Their Binding Properties to Stimulator of Interferon Genes Adaptor Protein, and Structure/Activity Correlations

Novotná, Barbora; Vaneková, Lenka; Zavřel, Martin; Budêšínský, Miloś; Dejmek, Milan; Smola, Miroslav; Gutten, Ondrej; Tehrani, Zahra Aliakbar; Polidarová, Markéta Pimková; Brázdová, Andrea; Liboska, Radek; Stĕpánek, I; Vavřina, Zdeněk; Jandušík, Tomáš; Nencka, Radim; Rulı́s̆ek, Lubomı́r; Bouřa, Evžen; Brynda, J.; Páv, Ondřej; Birkuš, Gabriel

doi:10.1021/acs.jmedchem.9b01062

Cited by 50 publications

(119 citation statements)

References 55 publications

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“…It is therefore plausible that human cGAS accepts substrate derivatives with modifications at most of the chosen positions in the nucleobase, ribose or α‐phosphate. Interestingly, our measured conversions do not completely cover recently published results about the synthesis of 2′3′‐cGAMP derivatives using full‐length cGAS from human, mouse and chicken [16] . Full‐length human cGAS did not catalyze the conversion of for example 2′‐F‐ATP or 6‐S‐GTP.…”

Section: Methodscontrasting

confidence: 90%

“…[16] Next to the bisphosphothionate analogue, it turned out that especially cyclic dinucleotides with a fluorine substitution in the 2'-position of the adenosine ribose shows improved STING binding. [16] We were now able to synthesize cyclic GMP-2'-F-AMP using a biocatalyst instead of a complex chemical route. We additionally synthesized for the first time derivatives with modifications at the 8-position of the nucleoside.…”

Section: 8 57mentioning

confidence: 99%

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Catalytic Promiscuity of cGAS: A Facile Enzymatic Synthesis of 2′‐3′‐Linked Cyclic Dinucleotides

et al. 2020

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Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor that catalyzes the synthesis of the cyclic GMP-AMP dinucleotide 2'3'-cGAMP. 2'3'-cGAMP functions as inducer for the production of type I interferons. Derivatives of this important second messenger are highly valuable for pharmaceutical applications. However, the production of these analogues requires complex, multistep syntheses. Herein, human cGAS is shown to react with a series of unnatural nucleotides, thus leading to novel cyclic dinucleotides. Most substrate derivatives with modifications at the nucleobase, ribose, and the α-thio phosphate were accepted. These results demonstrate the catalytic promiscuity of human cGAS and its utility for the biocatalytic synthesis of cyclic dinucleotide derivatives.

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Section: Methodscontrasting

confidence: 90%

Section: 8 57mentioning

confidence: 99%

Catalytic Promiscuity of cGAS: A Facile Enzymatic Synthesis of 2′‐3′‐Linked Cyclic Dinucleotides

et al. 2020

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“…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.…”

Section: Introductionmentioning

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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“…Although formation of diS-bound dimer upon H2O2 and 2'3'-cGAMP stimulation was also impaired, the oxidation of Cys 148 was not directly demonstrated (60,61). Despite the fact that none of the available human ligand-bound STING structures allows to see the linker region containing Cys 148 in electronic density maps (29,(61)(62)(63)(64)(65)(81)(82)(83)(84)(85)(86)(87)(88)(89)(90)(91), it was proposed that Cys 148 is engaged in a ligand-inducible diS bond that stabilizes the uncrossed linker regions in the STING polymer (60,61). Our data, however, argues in favor of a different model in which Cys 148 oxidation is already present at basal levels.…”

Section: Discussionmentioning

confidence: 99%

Pinpointing of cysteine oxidation sites in vivo by high-resolution proteomics reveals mechanism of redox-dependent inhibition of STING

Cuervo

Fortin

Caron

et al. 2020

Preprint

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Brief summary of the main results.The function of proteins is regulated by post-translational modifications, among which reversible oxidation of Cys recently emerged as a key component. Comprehension of redox regulation of cellular responses requires identification of specific oxidation sites in vivo. Using a bioswitch method to specifically label Cys subjected to reversible oxidation coupled to mass spectrometry, we identified thousands of novel oxidation sites. Many are relevant to virus -host interaction pathways. Here, we focused on the oxid ation of STING, an adaptor critical for activating the innate immune type I interferon pathway engaged upon cytosolic DNA sensing. Molecular studies led us to establish a new model in which STING Cys 148 is oxidized at basal levels, while Cys 206 oxidation is induced by oxidative stress and ligand binding. We show that oxidation of Cys 206 has an inhibitory function to prevent STING hyperactivation. This study provides ground for novel research avenues aimed at designing therapeutics that target this pathway. AbstractProtein function is regulated by post-translational modifications, among which reversible oxidation of Cys (Cys ox-PTM) emerged as a key regulatory mechanism of cellular responses. The redox regulation of virus-host interactions is well documented, but in most cases, proteins subjected to Cys ox-PTM remain unknown. The identification of Cys ox-PTM sites in vivo is essential to underpin our understanding of the mechanisms of the redox regulation. In this study, we present a proteome-wide identification of reversible Cys ox-PTM sites in vivo during stimulation by oxidants using a maleimide-based bioswitch method coupled to mass spectrometry. We identified 2720 unique Cys ox-PTM sites encompassing 1473 proteins with distinct abundance, location and functions. Label-free quantification (LFQ)-based analysis revealed the enrichment of ox-PTM in numerous pathways, many relevant to virus-host interaction. Here, we focused on the oxidation of STING, the central adaptor of the innate immune type I interferon 2 pathway induced upon detection of cytosolic DNA. We provide the first in vivo demonstration of reversible oxidation of Cys 148 and Cys 206 of STING. Molecular analyses led us to establish a new model in which Cys 148 oxidation is constitutive, while Cys 206 oxidation is inducible by oxidative stress or by the natural ligand 2'3'-cGAMP. We show that oxidation of Cys 206 has an inhibitory function to prevent STING hyperactivation through the constraint of a conformational change associated with the formation of inactive polymers containing intermolecular disulfide bonds. This provides new ground for the design of therapies targeting STING relevant to autoinflammatory disorders, immunotherapies and vaccines.

show abstract

Enzymatic Preparation of 2′–5′,3′–5′-Cyclic Dinucleotides, Their Binding Properties to Stimulator of Interferon Genes Adaptor Protein, and Structure/Activity Correlations

Cited by 50 publications

References 55 publications

Catalytic Promiscuity of cGAS: A Facile Enzymatic Synthesis of 2′‐3′‐Linked Cyclic Dinucleotides

Catalytic Promiscuity of cGAS: A Facile Enzymatic Synthesis of 2′‐3′‐Linked Cyclic Dinucleotides

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

Pinpointing of cysteine oxidation sites in vivo by high-resolution proteomics reveals mechanism of redox-dependent inhibition of STING

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