RESUMEN

Bacterial small molecule metabolites such as adenosine-diphosphate-d-glycero-ß-d-manno-heptose (ADP-heptose) and their derivatives act as effective innate immune agonists in mammals. We show that functional nucleotide-diphosphate-heptose biosynthetic enzymes (HBEs) are distributed widely in bacteria, archaea, eukaryotes, and viruses. We identified a conserved STTR5 motif as a hallmark of heptose nucleotidyltransferases that can synthesize not only ADP-heptose but also cytidine-diphosphate (CDP)- and uridine-diphosphate (UDP)-heptose. Both CDP- and UDP-heptoses are agonists that trigger stronger alpha-protein kinase 1 (ALPK1)-dependent immune responses than ADP-heptose in human and mouse cells and mice. We also produced ADP-heptose in archaea and verified its innate immune agonist functions. Hence, the ß-d-manno-heptoses are cross-kingdom, small-molecule, pathogen-associated molecular patterns that activate the ALPK1-dependent innate immune signaling cascade.

Asunto(s)

Heptosas , Nucleotidiltransferasas , Moléculas de Patrón Molecular Asociado a Patógenos , Animales , Humanos , Ratones , Secuencias de Aminoácidos , Archaea/enzimología , Bacterias/enzimología , Bacterias/metabolismo , Heptosas/biosíntesis , Heptosas/inmunología , Inmunidad Innata , Nucleotidiltransferasas/química , Nucleotidiltransferasas/clasificación , Nucleotidiltransferasas/genética , Moléculas de Patrón Molecular Asociado a Patógenos/inmunología , Moléculas de Patrón Molecular Asociado a Patógenos/metabolismo , Proteínas Quinasas/metabolismo , Virus/enzimología

RESUMEN

UDP-N-acetylglucosamine pyrophosphorylase (UAP) catalyzes the last step in the hexosamine biosynthesis pathway to directly produce UDP-N-acetylglucosamine (UDP-GlcNAc). Because UAPs play important physiological and pathological roles in organisms, they are considered potential targets for drug and pesticide development. However, the lack of efficient and selective inhibitors is a bottleneck that must be overcome. This study reports the first crystal structure of the insect UAP from Spodoptera frugiperda (SfUAP) in complex with UDP-GlcNAc. SfUAP has two insect-specific structural characteristics in the active pocket, namely, a free Cys (Cys334) and a Mg2+ binding site, which differentiate it from human UAP (HsAGX1) and fungal UAP (AfUAP) in terms of substrate and inhibitor binding. N-(4-Nitrophenyl)maleimide (pNPMI) and myricetin are discovered as potent covalent and noncovalent inhibitors of SfUAP, respectively. Moreover, myricetin can significantly reduce the level of cellular O-GlcNAcylation by inhibiting both UAP and O-GlcNAc transferase. These findings provide novel insights into the development of UAP-based drugs and pesticides.

Asunto(s)

Inhibidores Enzimáticos , Hexosaminas , Proteínas de Insectos , Spodoptera , Animales , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Proteínas de Insectos/metabolismo , Proteínas de Insectos/química , Proteínas de Insectos/genética , Proteínas de Insectos/antagonistas & inhibidores , Hexosaminas/química , Hexosaminas/metabolismo , Hexosaminas/biosíntesis , Nucleotidiltransferasas/química , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/antagonistas & inhibidores , Nucleotidiltransferasas/genética , Vías Biosintéticas , Sitios de Unión

RESUMEN

Infections that are acquired due to a prolonged hospital stay and manifest 2 days following the admission of a patient to a health-care institution can be classified as hospital-acquired infections. Klebsiella pneumoniae (K. pneumoniae) has become a critical pathogen, posing serious concern globally due to the rising incidences of hypervirulent and carbapenem-resistant strains. Glutaredoxin is a redox protein that protects cells from oxidative stress as it associates with glutathione to reduce mixed disulfides. Protein adenylyltransferase (PrAT) is a pseudokinase with a proposed mechanism of transferring an AMP group from ATP to glutaredoxin. Inducing oxidative stress to the bacterium by inhibiting the activity of PrAT is a promising approach to combating its contribution to hospital-acquired infections. Thus, this study aims to overexpress, purify, and analyse the effects of ATP and Mg2+ binding to Klebsiella pneumoniae PrAT (KpPrAT). The pET expression system and nickel affinity chromatography were effective in expressing and purifying KpPrAT. Far-UV CD spectroscopy demonstrates that the protein is predominantly α-helical, even in the presence of Mg2+. Extrinsic fluorescence spectroscopy with ANS indicates the presence of a hydrophobic pocket in the presence of ATP and Mg2+, while mant-ATP studies allude to the potential nucleotide binding ability of KpPrAT. The presence of Mg2+ increases the thermostability of the protein. Isothermal titration calorimetry provides insight into the binding affinity and thermodynamic parameters associated with the binding of ATP to KpPrAT, with or without Mg2+. Conclusively, the presence of Mg2+ induces a conformation in KpPrAT that favours nucleotide binding.

Asunto(s)

Proteínas Bacterianas , Klebsiella pneumoniae , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/enzimología , Klebsiella pneumoniae/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/biosíntesis , Adenosina Trifosfato/metabolismo , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/química , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/aislamiento & purificación , Expresión Génica , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Magnesio/metabolismo , Magnesio/química , Magnesio/farmacología

RESUMEN

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.

Asunto(s)

Escherichia coli , Proteínas Recombinantes , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/aislamiento & purificación , UTP-Hexosa-1-Fosfato Uridililtransferasa/genética , UTP-Hexosa-1-Fosfato Uridililtransferasa/metabolismo , UTP-Hexosa-1-Fosfato Uridililtransferasa/química , Expresión Génica , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/química , Clonación Molecular , Galactosafosfatos/metabolismo , Galactosafosfatos/genética , Galactosiltransferasas/genética , Galactosiltransferasas/metabolismo , Galactosiltransferasas/química

RESUMEN

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).

Asunto(s)

Nucleotidiltransferasas , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/química , Nucleotidiltransferasas/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Cristalografía por Rayos X , Modelos Moleculares , Conformación Proteica , Hidrólisis

RESUMEN

Tumor-suppressor let-7 pre-microRNAs (miRNAs) are regulated by terminal uridylyltransferases TUT7 and TUT4 that either promote let-7 maturation by adding a single uridine nucleotide to the pre-miRNA 3' end or mark them for degradation by the addition of multiple uridines. Oligo-uridylation is increased in cells by enhanced TUT7/4 expression and especially by the RNA-binding pluripotency factor LIN28A. Using cryogenic electron microscopy, we captured high-resolution structures of active forms of TUT7 alone, of TUT7 plus pre-miRNA and of both TUT7 and TUT4 bound with pre-miRNA and LIN28A. Our structures reveal that pre-miRNAs engage the enzymes in fundamentally different ways depending on the presence of LIN28A, which clamps them onto the TUTs to enable processive 3' oligo-uridylation. This study reveals the molecular basis for mono- versus oligo-uridylation by TUT7/4, as determined by the presence of LIN28A, and thus their mechanism of action in the regulation of cell fate and in cancer.

Asunto(s)

Microscopía por Crioelectrón , MicroARNs , Proteínas de Unión al ARN , Humanos , MicroARNs/metabolismo , MicroARNs/genética , MicroARNs/química , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/química , Modelos Moleculares , ARN Nucleotidiltransferasas/metabolismo , ARN Nucleotidiltransferasas/química , ARN Nucleotidiltransferasas/genética , Precursores del ARN/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/química , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/química , Conformación Proteica

RESUMEN

Cytidine diphosphate diacylglycerol (CDP-DAG) is a critical intermediate that is converted to multiple phospholipids in prokaryotes and eukaryotes. In budding yeast, CDP-DAG synthesis from cytidine triphosphate (CTP) and phosphatidic acid (PA) is catalyzed by the membrane-integrated protein Cds1 in the endoplasmic reticulum and the peripheral membrane-bound protein Tam41 in mitochondria. Although a recent study revealed that the fission yeast SpTam41 consists of a nucleotidyltransferase domain and a winged helix domain, forming an active-site pocket for CTP binding between the two domains together with a C-terminal amphipathic helix for membrane association, how CTP and Mg 2+, a most-favoured divalent cation, are accommodated with PA remains obscure. A more recent report by Kimura et al. (J. Biochem. 2022; 171:429-441) solved the crystal structure of FbTam41, a functional ortholog from a Firmicutes bacterium, with CTP-Mg 2+, successfully providing a detailed molecular view of CDP-DAG synthesis. In this commentary, our current understanding of Tam41-mediated reaction is discussed.

Asunto(s)

Citidina Difosfato Diglicéridos , Citidina Difosfato Diglicéridos/metabolismo , Schizosaccharomyces/enzimología , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Citidina Trifosfato/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/química , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/química , Nucleotidiltransferasas/genética

RESUMEN

Rev1-Polζ-dependent translesion synthesis (TLS) of DNA is crucial for maintaining genome integrity. To elucidate the mechanism by which the two polymerases cooperate in TLS, we determined the cryogenic electron microscopic structure of the Saccharomyces cerevisiae Rev1-Polζ holocomplex in the act of DNA synthesis (3.53 Å). We discovered that a composite N-helix-BRCT module in Rev1 is the keystone of Rev1-Polζ cooperativity, interacting directly with the DNA template-primer and with the Rev3 catalytic subunit of Polζ. The module is positioned akin to the polymerase-associated domain in Y-family TLS polymerases and is set ideally to interact with PCNA. We delineate the full extent of interactions that the carboxy-terminal domain of Rev1 makes with Polζ and identify potential new druggable sites to suppress chemoresistance from first-line chemotherapeutics. Collectively, our results provide fundamental new insights into the mechanism of cooperativity between Rev1 and Polζ in TLS.

Asunto(s)

Microscopía por Crioelectrón , ADN Polimerasa Dirigida por ADN , Modelos Moleculares , Nucleotidiltransferasas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/química , Nucleotidiltransferasas/ultraestructura , Nucleotidiltransferasas/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/genética , ADN Polimerasa Dirigida por ADN/metabolismo , ADN Polimerasa Dirigida por ADN/química , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Replicación del ADN , Proteínas Nucleares/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/ultraestructura , ADN/metabolismo , ADN/química , ADN de Hongos/metabolismo , ADN de Hongos/química , ADN de Hongos/genética , Unión Proteica , Síntesis Translesional de ADN

RESUMEN

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.

Asunto(s)

Células Presentadoras de Antígenos , Inmunidad Innata , Péptidos , Animales , Inmunidad Innata/efectos de los fármacos , Péptidos/química , Péptidos/farmacología , Ratones , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/efectos de los fármacos , Humanos , Femenino , Cationes/química , Ratones Endogámicos C57BL , Línea Celular Tumoral , Receptor Toll-Like 9/metabolismo , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/tratamiento farmacológico , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/química

RESUMEN

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.

Asunto(s)

Nucleotidiltransferasas , Enzimas Ubiquitina-Conjugadoras , Ubiquitinación , Enzimas Ubiquitina-Conjugadoras/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/química , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/química , Transducción de Señal , Nucleótidos Cíclicos/metabolismo , Bacteriófagos/genética , Bacteriófagos/enzimología , Ubiquitina/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Humanos , Microscopía por Crioelectrón , Inmunidad Innata

RESUMEN

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.

Asunto(s)

Dominio Catalítico , Flavina-Adenina Dinucleótido , Modelos Moleculares , Unión Proteica , Humanos , Flavina-Adenina Dinucleótido/metabolismo , Flavina-Adenina Dinucleótido/química , Cristalografía por Rayos X , Nucleotidiltransferasas/química , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/genética , Multimerización de Proteína , Sitios de Unión , Dominios Proteicos , Secuencia de Aminoácidos

RESUMEN

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.

Asunto(s)

Proteínas Nucleares , Nucleosomas , Nucleotidiltransferasas , Proteolisis , Ubiquitina-Proteína Ligasas , Animales , Humanos , Ratones , Núcleo Celular/metabolismo , Microscopía por Crioelectrón , Degrones , Infecciones por Virus ADN/inmunología , Virus ADN/inmunología , Virus ADN/metabolismo , ADN Viral/inmunología , ADN Viral/metabolismo , Inmunidad Innata , Reconocimiento de Inmunidad Innata , Interferón Tipo I/inmunología , Proteínas Nucleares/metabolismo , Nucleosomas/química , Nucleosomas/metabolismo , Nucleosomas/ultraestructura , Nucleotidiltransferasas/química , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/ultraestructura , Complejo de la Endopetidasa Proteasomal/metabolismo , Estabilidad Proteica , Especificidad por Sustrato , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/ultraestructura , Ubiquitinación

RESUMEN

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.

Asunto(s)

Aminoglicósidos , Antibacterianos , Klebsiella pneumoniae , Klebsiella pneumoniae/efectos de los fármacos , Klebsiella pneumoniae/genética , Aminoglicósidos/farmacología , Aminoglicósidos/química , Antibacterianos/farmacología , Antibacterianos/química , Humanos , Farmacorresistencia Bacteriana/efectos de los fármacos , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/química , Nucleotidiltransferasas/antagonistas & inhibidores , Infecciones por Klebsiella/tratamiento farmacológico , Infecciones por Klebsiella/microbiología , Pruebas de Sensibilidad Microbiana

RESUMEN

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.

Asunto(s)

Neoplasias , Nucleótidos Cíclicos , Humanos , Nucleótidos Cíclicos/química , Nucleótidos Cíclicos/metabolismo , Nucleotidiltransferasas/química , Inmunidad Innata

RESUMEN

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.

Asunto(s)

Coenzima A , NAD , Coenzima A/metabolismo , Nucleotidiltransferasas/química , Adenosina Trifosfato

RESUMEN

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.

Asunto(s)

Cápside , Proteínas de Unión al ADN , VIH-1 , Humanos , Cápside/química , Proteínas de la Cápside/química , Proteínas de Unión al ADN/química , VIH-1/química , Inmunidad Innata , Nucleotidiltransferasas/química , Unión Proteica , Conformación Proteica

RESUMEN

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.

Asunto(s)

Proteínas de la Membrana , Nucleotidiltransferasas , Separación de Fases , Humanos , Nucleotidiltransferasas/química , Transducción de Señal/fisiología , Proteínas de la Membrana/química

RESUMEN

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.

Asunto(s)

Nucleotidiltransferasas , ARN , Animales , Filogenia , ARN Mensajero/genética , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/química , Metiltransferasas/genética , Guanina , Caperuzas de ARN/genética , Mamíferos/genética

RESUMEN

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.

Asunto(s)

Nucleótidos , Nucleotidiltransferasas , Animales , Nucleotidiltransferasas/química , Azúcares , Retroalimentación , Bacterias/metabolismo , Azúcares de Nucleósido Difosfato , Mamíferos/metabolismo

RESUMEN

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.

Asunto(s)

Simulación de Dinámica Molecular , Nucleotidiltransferasas , Humanos , Animales , Ratones , Nucleotidiltransferasas/química , ADN/química