Structural and functional characterization of the bacterial Cap3 enzyme in deconjugation and regulation of the cyclic dinucleotide transferase CD-NTase.

The Cyclic GMP-AMP synthase (cGAS) and cGAS/DncV-like nucleotidyltransferase (CD-NTase) enzymes belong to the key components of the innate immune sensor system that generates cyclic dinucleotide molecules in response to danger signals. Recently, it was discovered that CD-NTase in bacteria can undergo conjugation to protein substrates via an E1/E2 enzyme-mediated process, resembling ubiquitin modification system. Subsequently, these CD-NTase conjugated molecules will be hydrolyzed by the Cap3 enzyme in the same gene cluster. However, the experimental structure of bacterial CD-NTase recognized by Cap3 is unknown. Here, we first determined the crystal structure of the Cap3 enzyme in complex with the C-terminal tail of CD-NTase. Our structural and enzymatic analysis revealed that the C-terminal tail of CD-NTase is both necessary and sufficient for the Cap3-mediated hydrolysis of CD-NTase from its substrates. Interestingly, we further observed that after the hydrolysis reaction, the terminal glycine residue of the CD-NTase C-terminal tail was sequentially removed by Cap3, indicating that Cap3 might play a role in quenching the CD-NTase conjugation reaction. Our work provides experimental evidence elucidating the interaction between Cap3 and CD-NTase, and suggests a potential role for Cap3 in the bacterial Cyclic-oligonucleotide-based anti-phage signaling system (CBASS).

Heterologous expression of a novel galactose-1-phosphate uridylyltransferase from Thermodesulfatator indicus and its application for bioproduction of Gal-ß-1,4-GlcNAc-X.

Nucleotide sugars (UDP-Sugars) are essential for the production of polysaccharides and glycoconjugates utilized in medicines, cosmetics, and food industries. The enzyme Galactose-1-phosphate uridylyltransferase (GalU; EC 2.7.7.12) is responsible for the synthesis of UDP-galactose from α-d-galactose-1-phosphate (Gal-1P) and UTP. A novel bacterial GalU (TiGalU) encoded from a thermophilic bacterium, Thermodesulfatator indicus, was successfully purified using the Ni-NTA column after being expressed in Escherichia coli. The optimal pH for recombinant TiGalU was determined to be 5.5. The optimum temperature of the enzyme was 45 °C. The activity of TiGalU was not dependent on Mg2+ and was strongly inhibited by SDS. When coupled with galactose kinase (GALK1) and ß-1,4-galactosyltransferase 1 (B4GALT1), the enzyme enabled the one-pot synthesis of Gal-ß-1,4-GlcNAc-X by utilizing galactose and UTP as substrates. This study reported the in vitro biosynthesis of Gal-ß-1,4-GlcNAc-X for the first time, providing an environmentally friendly way to biosynthesis glycosides and other polysaccharides.

Synthetic cationic helical polypeptides for the stimulation of antitumour innate immune pathways in antigen-presenting cells.

Intracellular DNA sensors regulate innate immunity and can provide a bridge to adaptive immunogenicity. However, the activation of the sensors in antigen-presenting cells (APCs) by natural agonists such as double-stranded DNAs or cyclic nucleotides is impeded by poor intracellular delivery, serum stability, enzymatic degradation and rapid systemic clearance. Here we show that the hydrophobicity, electrostatic charge and secondary conformation of helical polypeptides can be optimized to stimulate innate immune pathways via endoplasmic reticulum stress in APCs. One of the three polypeptides that we engineered activated two major intracellular DNA-sensing pathways (cGAS-STING (for cyclic guanosine monophosphate-adenosine monophosphate synthase-stimulator of interferon genes) and Toll-like receptor 9) preferentially in APCs by promoting the release of mitochondrial DNA, which led to the efficient priming of effector T cells. In syngeneic mouse models of locally advanced and metastatic breast cancers, the polypeptides led to potent DNA-sensor-mediated antitumour responses when intravenously given as monotherapy or with immune checkpoint inhibitors. The activation of multiple innate immune pathways via engineered cationic polypeptides may offer therapeutic advantages in the generation of antitumour immune responses.

Phage defence system CBASS is regulated by a prokaryotic E2 enzyme that imitates the ubiquitin pathway.

The cyclic-oligonucleotide-based anti-phage signalling system (CBASS) is a type of innate prokaryotic immune system. Composed of a cyclic GMP-AMP synthase (cGAS) and CBASS-associated proteins, CBASS uses cyclic oligonucleotides to activate antiviral immunity. One major class of CBASS contains a homologue of eukaryotic ubiquitin-conjugating enzymes, which is either an E1-E2 fusion or a single E2. However, the functions of single E2s in CBASS remain elusive. Here, using biochemical, genetic, cryo-electron microscopy and mass spectrometry investigations, we discover that the E2 enzyme from Serratia marcescens regulates cGAS by imitating the ubiquitination cascade. This includes the processing of the cGAS C terminus, conjugation of cGAS to a cysteine residue, ligation of cGAS to a lysine residue, cleavage of the isopeptide bond and poly-cGASylation. The poly-cGASylation activates cGAS to produce cGAMP, which acts as an antiviral signal and leads to cell death. Thus, our findings reveal a unique regulatory role of E2 in CBASS.

Structural insights into the bifunctional enzyme human FAD synthase.

Human flavin adenine dinucleotide synthase (hFADS) is a bifunctional, multi-domain enzyme that exhibits both flavin mononucleotide adenylyltransferase and pyrophosphatase activities. Here we report the crystal structure of full-length hFADS2 and its C-terminal PAPS domain in complex with flavin adenine dinucleotide (FAD), and dissect the structural determinants underlying the contribution of each individual domain, within isoforms 1 and 2, to each of the two enzymatic activities. Structural and functional characterization performed on complete or truncated constructs confirmed that the C-terminal domain tightly binds FAD and catalyzes its synthesis, while the combination of the N-terminal molybdopterin-binding and KH domains is the minimal essential substructure required for the hydrolysis of FAD and other ADP-containing dinucleotides. hFADS2 associates in a stable C2-symmetric dimer, in which the packing of the KH domain of one protomer against the N-terminal domain of the other creates the adenosine-specific active site responsible for the hydrolytic activity.

Combating Aminoglycoside Resistance: From Structural and Functional Characterisation to Therapeutic Challenges with RKAAT.

A comprehensive knowledge of aminoglycoside-modifying enzymes (AMEs) and their role in bacterial resistance mechanisms is urgently required due to the rising incidence of antibiotic resistance, particularly in Klebsiella pneumoniae infections. This study explores the essential features of AMEs, including their structural and functional properties, the processes by which they contribute to antibiotic resistance, and the therapeutic importance of aminoglycosides. The study primarily examines the Recombinant Klebsiella pneumoniae Aminoglycoside Adenylyl Transferase (RKAAT), particularly emphasizing its biophysical characteristics and the sorts of resistance it imparts. Furthermore, this study examines the challenges presented by RKAAT-mediated resistance, an evaluation of treatment methods and constraints, and options for controlling infection. The analysis provides a prospective outlook on strategies to address and reduce antibiotic resistance. This extensive investigation seeks to provide vital insights into the continuing fight against bacterial resistance, directing future research efforts and medicinal approaches.

Fluorinated cGAMP analogs, which act as STING agonists and are not cleavable by poxins: Structural basis of their function.

The cGAS-STING pathway is a crucial part of innate immunity; it serves to detect DNA in the cytoplasm and to defend against certain cancers, viruses, and bacteria. We designed and synthesized fluorinated carbocyclic cGAMP analogs, MD1203 and MD1202D (MDs), to enhance their stability and their affinity for STING. These compounds demonstrated exceptional activity against STING. Despite their distinct chemical modifications relative to the canonical cyclic dinucleotides (CDNs), crystallographic analysis revealed a binding mode with STING that was consistent with the canonical CDNs. Importantly, MDs were resistant to cleavage by viral poxin nucleases and MDs-bound poxin adopted an unliganded-like conformation. Moreover, MDs complexed with poxin showed a conformation distinct from cGAMP bound to poxin, closely resembling their conformation when bound to STING. In conclusion, the development of MD1203 and MD1202D showcases their potential as potent STING activators with remarkable stability against poxin-mediated degradation-a crucial characteristic for future development of antivirals.

The CRL5-SPSB3 ubiquitin ligase targets nuclear cGAS for degradation.

Cyclic GMP-AMP synthase (cGAS) senses aberrant DNA during infection, cancer and inflammatory disease, and initiates potent innate immune responses through the synthesis of 2'3'-cyclic GMP-AMP (cGAMP)1-7. The indiscriminate activity of cGAS towards DNA demands tight regulatory mechanisms that are necessary to maintain cell and tissue homeostasis under normal conditions. Inside the cell nucleus, anchoring to nucleosomes and competition with chromatin architectural proteins jointly prohibit cGAS activation by genomic DNA8-15. However, the fate of nuclear cGAS and its role in cell physiology remains unclear. Here we show that the ubiquitin proteasomal system (UPS) degrades nuclear cGAS in cycling cells. We identify SPSB3 as the cGAS-targeting substrate receptor that associates with the cullin-RING ubiquitin ligase 5 (CRL5) complex to ligate ubiquitin onto nuclear cGAS. A cryo-electron microscopy structure of nucleosome-bound cGAS in a complex with SPSB3 reveals a highly conserved Asn-Asn (NN) minimal degron motif at the C terminus of cGAS that directs SPSB3 recruitment, ubiquitylation and cGAS protein stability. Interference with SPSB3-regulated nuclear cGAS degradation primes cells for type I interferon signalling, conferring heightened protection against infection by DNA viruses. Our research defines protein degradation as a determinant of cGAS regulation in the nucleus and provides structural insights into an element of cGAS that is amenable to therapeutic exploitation.

Molecular Determinants of PQBP1 Binding to the HIV-1 Capsid Lattice.

Human immunodeficiency virus type 1 (HIV-1) stimulates innate immune responses upon infection, including cyclic GMP-AMP synthase (cGAS) signaling that results in type I interferon production. HIV-1-induced activation of cGAS requires the host cell factor polyglutamine binding protein 1 (PQBP1), an intrinsically disordered protein that bridges capsid recognition and cGAS recruitment. However, the molecular details of PQBP1 interactions with the HIV-1 capsid and their functional implications remain poorly understood. Here, we show that PQBP1 binds to HIV-1 capsids through charge complementing contacts between acidic residues in the N-terminal region of PQBP1 and an arginine ring in the central channel of the HIV-1 CA hexamer that makes up the viral capsid. These studies reveal the molecular details of PQBP1's primary interaction with the HIV-1 capsid and suggest that additional elements are likely to contribute to stable capsid binding.

Post-transcriptional capping generates coenzyme A-linked RNA.

NAD can be inserted co-transcriptionally via non-canonical initiation to form NAD-RNA. However, that mechanism is unlikely for CoA-linked RNAs due to low intracellular concentration of the required initiator nucleotide, 3'-dephospho-CoA (dpCoA). We report here that phosphopantetheine adenylyltransferase (PPAT), an enzyme of CoA biosynthetic pathway, accepts RNA transcripts as its acceptor substrate and transfers 4'-phosphopantetheine to yield CoA-RNA post-transcriptionally. Synthetic natural (RNAI) and small artificial RNAs were used to identify the features of RNA that are needed for it to serve as PPAT substrate. RNAs with 4-10 unpaired nucleotides at the 5' terminus served as PPAT substrates, but RNAs having <4 unpaired nucleotides did not undergo capping. No capping was observed when the +1A was changed to G or when 5' triphosphate was removed by RNA pyrophosphohydrolase (RppH), suggesting the enzyme recognizes pppA-RNA as an ATP analog. PPAT binding affinities were equivalent for transcripts with +1A, +1 G, or 5'OH (+1A), indicating that productive enzymatic recognition is driven more by local positioning effects than by overall binding affinity. Capping rates were independent of the number of unpaired nucleotides in the range of 4-10 nucleotides. Capping was strongly inhibited by ATP, reducing CoA-RNA production ~70% when equimolar ATP and substrate RNA were present. Dual bacterial expression of candidate RNAs with different 5' structures followed by CoA-RNA CaptureSeq revealed 12-fold enrichment of the better PPAT substrate, consistent with in vivo CoA-capping of RNA transcripts by PPAT. These results suggest post-transcriptional RNA capping as a possible mechanism for the biogenesis of CoA-RNAs in bacteria.

Phase separation in cGAS-STING signaling.

Biomolecular condensates formed by phase separation are widespread and play critical roles in many physiological and pathological processes. cGAS-STING signaling functions to detect aberrant DNA signals to initiate anti-infection defense and antitumor immunity. At the same time, cGAS-STING signaling must be carefully regulated to maintain immune homeostasis. Interestingly, exciting recent studies have reported that biomolecular phase separation exists and plays important roles in different steps of cGAS-STING signaling, including cGAS condensates, STING condensates, and IRF3 condensates. In addition, several intracellular and extracellular factors have been proposed to modulate the condensates in cGAS-STING signaling. These studies reveal novel activation and regulation mechanisms of cGAS-STING signaling and provide new opportunities for drug discovery. Here, we summarize recent advances in the phase separation of cGAS-STING signaling and the development of potential drugs targeting these innate immune condensates.

Biochemical characterization of mRNA capping enzyme from Faustovirus.

The mammalian mRNA 5' cap structures play important roles in cellular processes such as nuclear export, efficient translation, and evading cellular innate immune surveillance and regulating 5'-mediated mRNA turnover. Hence, installation of the proper 5' cap is crucial in therapeutic applications of synthetic mRNA. The core 5' cap structure, Cap-0, is generated by three sequential enzymatic activities: RNA 5' triphosphatase, RNA guanylyltransferase, and cap N7-guanine methyltransferase. Vaccinia virus RNA capping enzyme (VCE) is a heterodimeric enzyme that has been widely used in synthetic mRNA research and manufacturing. The large subunit of VCE D1R exhibits a modular structure where each of the three structural domains possesses one of the three enzyme activities, whereas the small subunit D12L is required to activate the N7-guanine methyltransferase activity. Here, we report the characterization of a single-subunit RNA capping enzyme from an amoeba giant virus. Faustovirus RNA capping enzyme (FCE) exhibits a modular array of catalytic domains in common with VCE and is highly efficient in generating the Cap-0 structure without an activation subunit. Phylogenetic analysis suggests that FCE and VCE are descended from a common ancestral capping enzyme. We found that compared to VCE, FCE exhibits higher specific activity, higher activity toward RNA containing secondary structures and a free 5' end, and a broader temperature range, properties favorable for synthetic mRNA manufacturing workflows.

Feedback Inhibition of Bacterial Nucleotidyltransferases by Rare Nucleotide l-Sugars Restricts Substrate Promiscuity.

Bacterial glycomes are rich in prokaryote-specific or "rare" sugars that are absent in mammals. Like common sugars found across organisms, rare sugars are typically activated as nucleoside diphosphate sugars (NDP-sugars) by nucleotidyltransferases. In bacteria, the nucleotidyltransferase RmlA initiates the production of several rare NDP-sugars, which in turn regulate downstream glycan assembly through feedback inhibition of RmlA via binding to an allosteric site. In vitro, RmlA activates a range of common sugar-1-phosphates to produce NDP-sugars for biochemical and synthetic applications. However, our ability to probe bacterial glycan biosynthesis is hindered by limited chemoenzymatic access to rare NDP-sugars. We postulate that natural feedback mechanisms impact nucleotidyltransferase utility. Here, we use synthetic rare NDP-sugars to identify structural features required for regulation of RmlA from diverse bacterial species. We find that mutation of RmlA to eliminate allosteric binding of an abundant rare NDP-sugar facilitates the activation of noncanonical rare sugar-1-phosphate substrates, as products no longer affect turnover. In addition to promoting an understanding of nucleotidyltransferase regulation by metabolites, this work provides new routes to access rare sugar substrates for the study of important bacteria-specific glycan pathways.

Investigating the Activation Mechanism Differences between Human and Mouse cGAS by Molecular Dynamics Simulations.

Cyclic GMP-AMP synthase (cGAS) has been widely investigated as a drug target for its crucial role in innate immunity. However, the inhibitors designed using mouse model were often shown to be ineffective for humans. This outcome indicates that the activation mechanisms of human and mouse cGAS (mcGAS) are different. The cGAS activation is achieved by dimerization via binding to DNA, the detailed mechanism of which, however, is not entirely clear. To investigate these mechanisms, molecular dynamics (MD) simulations were performed on several states of four types of cGAS, namely, the mcGAS, the wild-type and A- and C-type mutations of human cGAS (hcGAS). We find that sequence differences between hcGAS and mcGAS can directly affect the protein structure stability, especially that of the siteB domain. The sequence and structural differences also contribute to DNA-binding differences. In addition, the conformational fluctuations of cGAS are found to correlate with the regulation of catalytic capacity. More importantly, we illustrate that dimerization enhances the correlation among distant residues and significantly reinforces the allosteric signal transmission among the DNA-binding interfaces and the catalytic pocket, which facilitates rapid immune response to cytosolic DNA. We conclude that siteB domain plays a prominent role in mcGAS activation, while siteA domain is key to hcGAS activation.

Versatile strategy using vaccinia virus-capping enzyme to synthesize functional 5' cap-modified mRNAs.

The potential of synthetic mRNA as a genetic carrier has increased its application in scientific fields. Because the 5' cap regulates the stability and translational activity of mRNAs, there are concerted efforts to search for and synthesize chemically-modified 5' caps that improve the functionality of mRNA. Here, we report an easy and efficient method to synthesize functional mRNAs by modifying multiple 5' cap analogs using a vaccinia virus-capping enzyme. We show that this enzyme can introduce a variety of GTP analogs to the 5' end of RNA to generate 5' cap-modified mRNAs that exhibit different translation levels. Notably, some of these modified mRNAs improve translation efficiency and can be conjugated to chemical structures, further increasing their functionality. Our versatile method to generate 5' cap-modified mRNAs will provide useful tools for RNA therapeutics and biological research.

Ubiquitin-like conjugation by bacterial cGAS enhances anti-phage defence.

cGAS is an evolutionarily conserved enzyme that has a pivotal role in immune defence against infection1-3. In vertebrate animals, cGAS is activated by DNA to produce cyclic GMP-AMP (cGAMP)4,5, which leads to the expression of antimicrobial genes6,7. In bacteria, cyclic dinucleotide (CDN)-based anti-phage signalling systems (CBASS) have been discovered8-11. These systems are composed of cGAS-like enzymes and various effector proteins that kill bacteria on phage infection, thereby stopping phage spread. Of the CBASS systems reported, approximately 39% contain Cap2 and Cap3, which encode proteins with homology to ubiquitin conjugating (E1/E2) and deconjugating enzymes, respectively8,12. Although these proteins are required to prevent infection of some bacteriophages8, the mechanism by which the enzymatic activities exert an anti-phage effect is unknown. Here we show that Cap2 forms a thioester bond with the C-terminal glycine of cGAS and promotes conjugation of cGAS to target proteins in a process that resembles ubiquitin conjugation. The covalent conjugation of cGAS increases the production of cGAMP. Using a genetic screen, we found that the phage protein Vs.4 antagonized cGAS signalling by binding tightly to cGAMP (dissociation constant of approximately 30 nM) and sequestering it. A crystal structure of Vs.4 bound to cGAMP showed that Vs.4 formed a hexamer that was bound to three molecules of cGAMP. These results reveal a ubiquitin-like conjugation mechanism that regulates cGAS activity in bacteria and illustrates an arms race between bacteria and viruses through controlling CDN levels.

Species-specific self-DNA detection mechanisms by mammalian cyclic GMP-AMP synthases.

The mechanisms by which innate immune receptors mediate self-nonself discrimination are unclear. In this study, we found species-specific molecular determinants of self-DNA reactivity by cyclic guanosine monophosphate-adenosine monophosphate (GMP-AMP) synthase (cGAS). Human cGAS contained a catalytic domain that was intrinsically self-DNA reactive and stimulated interferon responses in diverse cell types. This reactivity was prevented by an upstream amino (N)-terminal domain. The cGAS proteins from several nonhuman primate species exhibited a similar pattern of self-DNA reactivity in cells, but chimpanzee cGAS was inactive even when its amino-terminal domain was deleted. In contrast, the N terminus of mouse cGAS promoted self-DNA reactivity. When expressed within tumors, only self-DNA-reactive cGAS proteins protected mice from tumor-induced lethality. In vitro studies of DNA- or chromatin-induced cGAS activation did not reveal species-specific activities that correlate with self-DNA reactivity observed in macrophages. Cell biological analysis revealed that self-DNA reactivity by human cGAS, but not mouse cGAS, correlated with localization to mitochondria. We found that epitope tag positions affected self-DNA reactivity in cells and that DNA present in cell lysates undermines the reliability of cGAS biochemical fractionations. These studies reveal species-specific diversity of cGAS functions, even within the primate lineage, and highlight experimental considerations for the study of this innate immune receptor.

Current understanding of the cGAS-STING signaling pathway: Structure, regulatory mechanisms, and related diseases.

The innate immune system protects the host from external pathogens and internal damage in various ways. The cGAS-STING signaling pathway, comprised of cyclic GMP-AMP synthase (cGAS), stimulator of interferon genes (STING), and downstream signaling adaptors, plays an essential role in protective immune defense against microbial DNA and internal damaged-associated DNA and is responsible for various immune-related diseases. After binding with DNA, cytosolic cGAS undergoes conformational change and DNA-linked liquid-liquid phase separation to produce 2'3'-cGAMP for the activation of endoplasmic reticulum (ER)-localized STING. However, further studies revealed that cGAS is predominantly expressed in the nucleus and strictly tethered to chromatin to prevent binding with nuclear DNA, and functions differently from cytosolic-localized cGAS. Detailed delineation of this pathway, including its structure, signaling, and regulatory mechanisms, is of great significance to fully understand the diversity of cGAS-STING activation and signaling and will be of benefit for the treatment of inflammatory diseases and cancer. Here, we review recent progress on the above-mentioned perspectives of the cGAS-STING signaling pathway and discuss new avenues for further study.

Phase separation in cGAS-STING signaling / 医学前沿

Biomolecular condensates formed by phase separation are widespread and play critical roles in many physiological and pathological processes. cGAS-STING signaling functions to detect aberrant DNA signals to initiate anti-infection defense and antitumor immunity. At the same time, cGAS-STING signaling must be carefully regulated to maintain immune homeostasis. Interestingly, exciting recent studies have reported that biomolecular phase separation exists and plays important roles in different steps of cGAS-STING signaling, including cGAS condensates, STING condensates, and IRF3 condensates. In addition, several intracellular and extracellular factors have been proposed to modulate the condensates in cGAS-STING signaling. These studies reveal novel activation and regulation mechanisms of cGAS-STING signaling and provide new opportunities for drug discovery. Here, we summarize recent advances in the phase separation of cGAS-STING signaling and the development of potential drugs targeting these innate immune condensates.

The mechanism of RNA capping by SARS-CoV-2.

The RNA genome of SARS-CoV-2 contains a 5' cap that facilitates the translation of viral proteins, protection from exonucleases and evasion of the host immune response1-4. How this cap is made in SARS-CoV-2 is not completely understood. Here we reconstitute the N7- and 2'-O-methylated SARS-CoV-2 RNA cap (7MeGpppA2'-O-Me) using virally encoded non-structural proteins (nsps). We show that the kinase-like nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain5 of nsp12 transfers the RNA to the amino terminus of nsp9, forming a covalent RNA-protein intermediate (a process termed RNAylation). Subsequently, the NiRAN domain transfers the RNA to GDP, forming the core cap structure GpppA-RNA. The nsp146 and nsp167 methyltransferases then add methyl groups to form functional cap structures. Structural analyses of the replication-transcription complex bound to nsp9 identified key interactions that mediate the capping reaction. Furthermore, we demonstrate in a reverse genetics system8 that the N terminus of nsp9 and the kinase-like active-site residues in the NiRAN domain are required for successful SARS-CoV-2 replication. Collectively, our results reveal an unconventional mechanism by which SARS-CoV-2 caps its RNA genome, thus exposing a new target in the development of antivirals to treat COVID-19.