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1.
Nat Commun ; 12(1): 2426, 2021 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-33893288

RESUMO

To adapt to fluctuating protein folding loads in the endoplasmic reticulum (ER), the Hsp70 chaperone BiP is reversibly modified with adenosine monophosphate (AMP) by the ER-resident Fic-enzyme FICD/HYPE. The structural basis for BiP binding and AMPylation by FICD has remained elusive due to the transient nature of the enzyme-substrate-complex. Here, we use thiol-reactive derivatives of the cosubstrate adenosine triphosphate (ATP) to covalently stabilize the transient FICD:BiP complex and determine its crystal structure. The complex reveals that the TPR-motifs of FICD bind specifically to the conserved hydrophobic linker of BiP and thus mediate specificity for the domain-docked conformation of BiP. Furthermore, we show that both AMPylation and deAMPylation of BiP are not directly regulated by the presence of unfolded proteins. Together, combining chemical biology, crystallography and biochemistry, our study provides structural insights into a key regulatory mechanism that safeguards ER homeostasis.


Assuntos
Proteínas de Choque Térmico/metabolismo , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/metabolismo , Processamento de Proteína Pós-Traducional , Repetições de Tetratricopeptídeos , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Retículo Endoplasmático/metabolismo , Células HEK293 , Proteínas de Choque Térmico/química , Homeostase , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Simulação de Dinâmica Molecular , Nucleotidiltransferases/química , Nucleotidiltransferases/genética , Ligação Proteica , Conformação Proteica , Especificidade por Substrato
2.
Science ; 371(6535)2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33542149

RESUMO

The cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase (cGAS) detects microbial and self-DNA in the cytosol to activate immune and inflammatory programs. cGAS also associates with chromatin, especially after nuclear envelope breakdown when cells enter mitosis. How cGAS is regulated during cell cycle transition is not clear. Here, we found direct biochemical evidence that cGAS activity was selectively suppressed during mitosis in human cell lines and uncovered two parallel mechanisms underlying this suppression. First, cGAS was hyperphosphorylated at the N terminus by mitotic kinases, including Aurora kinase B. The N terminus of cGAS was critical for sensing nuclear chromatin but not mitochondrial DNA. Chromatin sensing was blocked by hyperphosphorylation. Second, oligomerization of chromatin-bound cGAS, which is required for its activation, was prevented. Together, these mechanisms ensure that cGAS is inactive when associated with chromatin during mitosis, which may help to prevent autoimmune reaction.


Assuntos
Cromatina/metabolismo , Mitose , Nucleotidiltransferases/metabolismo , Aurora Quinase B/metabolismo , Ciclo Celular , Linhagem Celular , DNA/metabolismo , DNA Mitocondrial/metabolismo , Ativação Enzimática , Humanos , Nucleotídeos Cíclicos/metabolismo , Nucleotidiltransferases/química , Fosforilação , Multimerização Proteica
3.
Nucleic Acids Res ; 49(4): 2065-2084, 2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33555350

RESUMO

We previously reported that human Rev1 (hRev1) bound to a parallel-stranded G-quadruplex (G4) from the c-MYC promoter with high affinity. We have extended those results to include other G4 motifs, finding that hRev1 exhibited stronger affinity for parallel-stranded G4 than either anti-parallel or hybrid folds. Amino acids in the αE helix of insert-2 were identified as being important for G4 binding. Mutating E466 and Y470 to alanine selectively perturbed G4 binding affinity. The E466K mutant restored wild-type G4 binding properties. Using a forward mutagenesis assay, we discovered that loss of hRev1 increased G4 mutation frequency >200-fold compared to the control sequence. Base substitutions and deletions occurred around and within the G4 motif. Pyridostatin (PDS) exacerbated this effect, as the mutation frequency increased >700-fold over control and deletions upstream of the G4 site more than doubled. Mutagenic replication of G4 DNA (±PDS) was partially rescued by wild-type and E466K hRev1. The E466A or Y470A mutants failed to suppress the PDS-induced increase in G4 mutation frequency. These findings have implications for the role of insert-2, a motif conserved in vertebrates but not yeast or plants, in Rev1-mediated suppression of mutagenesis during G4 replication.


Assuntos
Replicação do DNA , DNA/química , DNA/metabolismo , Quadruplex G , Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Linhagem Celular , DNA Polimerase Dirigida por DNA/metabolismo , Genes myc , Humanos , Modelos Moleculares , Mutação , Motivos de Nucleotídeos , Nucleotidiltransferases/genética , Ligação Proteica
4.
Int J Mol Sci ; 22(3)2021 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-33503880

RESUMO

Recent studies have identified cyclic GMP-AMP synthase (cGAS) as an important target for treating autoimmune diseases, and several inhibitors of human cGAS (hcGAS) and their structures in complexation with hcGAS have been reported. However, the mechanisms via which these inhibitors interact with hcGAS are not completely understood. Here, we aimed to assess the performance of molecular mechanics/Poisson-Boltzmann solvent-accessible surface area (MM/PBSA) in evaluating the binding affinity of various hcGAS inhibitors and to elucidate their detailed interactions with hcGAS from an energetic viewpoint. Using molecular dynamics (MD) simulation and MM/PBSA approaches, the estimated free energies were in good agreement with the experimental ones, with a Pearson's correlation coefficient and Spearman's rank coefficient of 0.67 and 0.46, respectively. In per-residue energy decomposition analysis, four residues, K362, R376, Y436, and K439 in hcGAS were found to contribute significantly to the binding with inhibitors via hydrogen bonding, salt bridges, and various π interactions, such as π· · ·π stacking, cation· · ·π, hydroxyl· · ·π, and alkyl· · ·π interactions. In addition, we discussed other key interactions between specific residues and ligands, in particular, between H363 and JUJ, F379 and 9BY, and H437 and 8ZM. The sandwiched structures of the inhibitor bound to the guanidinium group of R376 and the phenyl ring of Y436 were also consistent with the experimental data. The results indicated that MM/PBSA in combination with other virtual screening methods, could be a reliable approach to discover new hcGAS inhibitors and thus is valuable for potential treatments of cGAS-dependent inflammatory diseases.


Assuntos
Inibidores Enzimáticos/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Nucleotidiltransferases/química , Aminoácidos , Sítios de Ligação , Inibidores Enzimáticos/farmacologia , Humanos , Ligação de Hidrogênio , Ligantes , Conformação Molecular , Estrutura Molecular , Nucleotidiltransferases/antagonistas & inibidores , Ligação Proteica , Relação Estrutura-Atividade
5.
Biochem J ; 478(1): 235-245, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33346350

RESUMO

Flavonoids play beneficial roles in various human diseases. In this study, a flavonoid library was employed to probe inhibitors of d-glycero-ß-d-manno-heptose-1-phosphate adenylyltransferase from Burkholderia pseudomallei (BpHldC) and two flavonoids, epigallocatechin gallate (EGCG) and myricetin, have been discovered. BpHldC is one of the essential enzymes in the ADP-l-glycero-ß-d-manno-heptose biosynthesis pathway constructing lipopolysaccharide of B. pseudomallei. Enzyme kinetics study showed that two flavonoids work through different mechanisms to block the catalytic activity of BpHldC. Among them, a docking study of EGCG was performed and the binding mode could explain its competitive inhibitory mode for both ATP and ßG1P. Analyses with EGCG homologs could reveal the important functional moieties, too. This study is the first example of uncovering the inhibitory activity of flavonoids against the ADP-l-glycero-ß-d-manno-heptose biosynthesis pathway and especially targeting HldC. Since there are no therapeutic agents and vaccines available against melioidosis, EGCG and myricetin can be used as templates to develop antibiotics over B. pseudomallei.


Assuntos
Burkholderia pseudomallei/enzimologia , Flavonoides/química , Manose/química , Nucleotidiltransferases/química , Piranos/química , Trifosfato de Adenosina/química , Catequina/análogos & derivados , Catequina/química , Cristalografia por Raios X , Escherichia coli/metabolismo , Concentração Inibidora 50 , Cinética , Ligantes , Simulação de Acoplamento Molecular , Nucleotidiltransferases/antagonistas & inibidores , Nucleotidiltransferases/metabolismo
6.
PLoS Comput Biol ; 16(8): e1007898, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32797038

RESUMO

New treatments for diseases caused by antimicrobial-resistant microorganisms can be developed by identifying unexplored therapeutic targets and by designing efficient drug screening protocols. In this study, we have screened a library of compounds to find ligands for the flavin-adenine dinucleotide synthase (FADS) -a potential target for drug design against tuberculosis and pneumonia- by implementing a new and efficient virtual screening protocol. The protocol has been developed for the in silico search of ligands of unexplored therapeutic targets, for which limited information about ligands or ligand-receptor structures is available. It implements an integrative funnel-like strategy with filtering layers that increase in computational accuracy. The protocol starts with a pharmacophore-based virtual screening strategy that uses ligand-free receptor conformations from molecular dynamics (MD) simulations. Then, it performs a molecular docking stage using several docking programs and an exponential consensus ranking strategy. The last filter, samples the conformations of compounds bound to the target using MD simulations. The MD conformations are scored using several traditional scoring functions in combination with a newly-proposed score that takes into account the fluctuations of the molecule with a Morse-based potential. The protocol was optimized and validated using a compound library with known ligands of the Corynebacterium ammoniagenes FADS. Then, it was used to find new FADS ligands from a compound library of 14,000 molecules. A small set of 17 in silico filtered molecules were tested experimentally. We identified five inhibitors of the activity of the flavin adenylyl transferase module of the FADS, and some of them were able to inhibit growth of three bacterial species: C. ammoniagenes, Mycobacterium tuberculosis, and Streptococcus pneumoniae, where the last two are human pathogens. Overall, the results show that the integrative VS protocol is a cost-effective solution for the discovery of ligands of unexplored therapeutic targets.


Assuntos
Antibacterianos , Proteínas de Bactérias , Nucleotidiltransferases , Antibacterianos/química , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Corynebacterium/efeitos dos fármacos , Corynebacterium/enzimologia , Desenho de Fármacos , Farmacorresistência Bacteriana/efeitos dos fármacos , Ligantes , Simulação de Dinâmica Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Nucleotidiltransferases/antagonistas & inibidores , Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo
7.
J Phys Chem Lett ; 11(11): 4430-4435, 2020 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-32392072

RESUMO

The pandemic outbreak of a new coronavirus (CoV), SARS-CoV-2, has captured the world's attention, demonstrating that CoVs represent a continuous global threat. As this is a highly contagious virus, it is imperative to understand RNA-dependent-RNA-polymerase (RdRp), the key component in virus replication. Although the SARS-CoV-2 genome shares 80% sequence identity with severe acute respiratory syndrome SARS-CoV, their RdRps and nucleotidyl-transferases (NiRAN) share 98.1% and 93.2% identity, respectively. Sequence alignment of six coronaviruses demonstrated higher identity among their RdRps (60.9%-98.1%) and lower identity among their Spike proteins (27%-77%). Thus, a 3D structural model of RdRp, NiRAN, non-structural protein 7 (nsp7), and nsp8 of SARS-CoV-2 was generated by modeling starting from the SARS counterpart structures. Furthermore, we demonstrate the binding poses of three viral RdRp inhibitors (Galidesivir, Favipiravir, and Penciclovir), which were recently reported to have clinical significance for SARS-CoV-2. The network of interactions established by these drug molecules affirms their efficacy to inhibit viral RNA replication and provides an insight into their structure-based rational optimization for SARS-CoV-2 inhibition.


Assuntos
Betacoronavirus/enzimologia , Nucleotidiltransferases/química , /química , Adenina/análogos & derivados , Adenina/química , Adenina/metabolismo , Adenosina/análogos & derivados , Amidas/química , Amidas/metabolismo , Antivirais/química , Antivirais/metabolismo , Betacoronavirus/isolamento & purificação , Sítios de Ligação , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Humanos , Simulação de Acoplamento Molecular , Nucleotidiltransferases/metabolismo , Pandemias , Pneumonia Viral/epidemiologia , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Estrutura Terciária de Proteína , Pirazinas/química , Pirazinas/metabolismo , Pirrolidinas/química , Pirrolidinas/metabolismo , /metabolismo
8.
Nucleic Acids Res ; 48(9): 5157-5168, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32266935

RESUMO

The N6-methyladenosine modification at position 43 (m6A43) of U6 snRNA is catalyzed by METTL16, and is important for the 5'-splice site recognition by U6 snRNA during pre-mRNA splicing. Human METTL16 consists of the N-terminal methyltransferase domain (MTD) and the C-terminal vertebrate conserved region (VCR). While the MTD has an intrinsic property to recognize a specific sequence in the distinct structural context of RNA, the VCR functions have remained uncharacterized. Here, we present structural and functional analyses of the human METTL16 VCR. The VCR increases the affinity of METTL16 toward U6 snRNA, and the conserved basic region in VCR is important for the METTL16-U6 snRNA interaction. The VCR structure is topologically homologous to the C-terminal RNA binding domain, KA1, in U6 snRNA-specific terminal uridylyl transferase 1 (TUT1). A chimera of the N-terminal MTD of METTL16 and the C-terminal KA1 of TUT1 methylated U6 snRNA more efficiently than the MTD, indicating the functional conservation of the VCR and KA1 for U6 snRNA biogenesis. The VCR interacts with the internal stem-loop (ISL) within U6 snRNA, and this interaction would induce the conformational rearrangement of the A43-containing region of U6 snRNA, thereby modifying the RNA structure to become suitable for productive catalysis by the MTD. Therefore, the MTD and VCR in METTL16 cooperatively facilitate the m6A43 U6 snRNA modification.


Assuntos
Metiltransferases/química , RNA Nuclear Pequeno/química , Adenosina/análogos & derivados , Adenosina/metabolismo , Sequência de Aminoácidos , Sequência Conservada , Humanos , Metilação , Metiltransferases/metabolismo , Conformação de Ácido Nucleico , Nucleotidiltransferases/química , Ligação Proteica , RNA Nuclear Pequeno/metabolismo
9.
Biochem Biophys Res Commun ; 525(3): 780-785, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32147095

RESUMO

Sugar Nucleotidyl Transferases (SNTs) constitute a large family of enzymes that play important metabolic roles. Earlier, for one such SNT, termed N-acetylglucosamine-1-phosphate uridyltransferase- GlmU, we had established that two magnesium ions - Mg2+A and Mg2+B - catalyze the sugar-nucleotidyl transfer reaction. Despite a common structural framework that SNTs share, we recognized key differences around the active-site based on the analysis of available structures. Based on these differences, we had classified SNTs into two major groups, Group - I & II; and further, variation in 'Mg2+A-stabilizing motifs' led us to sub-classify them into five distinct sub-groups. Since group specific conservation of 'Mg2+A-stabilizing motifs' was based only for 45 available structures, here we validate this via an exhaustive analysis of 1,42,025 protein sequences. Previously, we had hypothesized that a metal-ion-catalyzed mechanism would be operative in all SNTs. Here, we validate it biochemically and establish that Mg2+ is a strict requirement for nucleotidyl transfer reactions in every group or sub-group and that a common metal ion dependent mechanism operates in SNTs. Further, mutating Mg2+A coordinating residue in each sub-group led to abolished catalysis, indicating an important role for both of these residues and suggest that SNTs employ variations over 'a conserved catalytic mechanism mediated by Mg2+ ion(s)', to bring about functional diversity. This would constitute a comprehensive study to establish the catalytic mechanism across the family of SNTs.


Assuntos
Família Multigênica , Nucleotidiltransferases/química , Nucleotidiltransferases/classificação , Açúcares/metabolismo , Sequência de Aminoácidos , Sequência Conservada , Estabilidade Enzimática , Glucosamina/análogos & derivados , Glucosamina/química , Glucosamina/metabolismo , Magnésio/metabolismo , Modelos Moleculares
10.
Nucleic Acids Res ; 48(8): 4435-4447, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32170294

RESUMO

Cyclic-G/AMP (cGAMP) synthase (cGAS) triggers host innate immune responses against cytosolic double-stranded (ds)DNA arising from genotoxic stress and pathogen invasion. The canonical activation mechanism of cGAS entails dsDNA-binding and dimerization. Here, we report an unexpected activation mechanism of cGAS in which Mn2+ activates monomeric cGAS without dsDNA. Importantly, the Mn2+-mediated activation positively couples with dsDNA-dependent activation in a concerted manner. Moreover, the positive coupling between Mn2+ and dsDNA length-dependent activation requires the cognate ATP/GTP substrate pair, while negative-cooperativity suppresses Mn2+ utilization by either ATP or GTP alone. Additionally, while Mn2+ accelerates the overall catalytic activity, dsDNA length-dependent dimerization specifically accelerates the cyclization of cGAMP. Together, we demonstrate how the intrinsic allostery of cGAS efficiently yet precisely tunes its activity.


Assuntos
DNA/metabolismo , Manganês , Nucleotidiltransferases/metabolismo , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Biocatálise , Linhagem Celular , DNA/química , Ativação Enzimática , Humanos , Nucleotidiltransferases/química , Especificidade por Substrato
11.
Nucleic Acids Res ; 48(5): 2733-2748, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-32009146

RESUMO

Family with sequence similarity (FAM46) proteins are newly identified metazoan-specific poly(A) polymerases (PAPs). Although predicted as Gld-2-like eukaryotic non-canonical PAPs, the detailed architecture of FAM46 proteins is still unclear. Exact biological functions for most of FAM46 proteins also remain largely unknown. Here, we report the first crystal structure of a FAM46 protein, FAM46B. FAM46B is composed of a prominently larger N-terminal catalytic domain as compared to known eukaryotic PAPs, and a C-terminal helical domain. FAM46B resembles prokaryotic PAP/CCA-adding enzymes in overall folding as well as certain inter-domain connections, which distinguishes FAM46B from other eukaryotic non-canonical PAPs. Biochemical analysis reveals that FAM46B is an active PAP, and prefers adenosine-rich substrate RNAs. FAM46B is uniquely and highly expressed in human pre-implantation embryos and pluripotent stem cells, but sharply down-regulated following differentiation. FAM46B is localized to both cell nucleus and cytosol, and is indispensable for the viability of human embryonic stem cells. Knock-out of FAM46B is lethal. Knock-down of FAM46B induces apoptosis and restricts protein synthesis. The identification of the bacterial-like FAM46B, as a pluripotent stem cell-specific PAP involved in the maintenance of translational efficiency, provides important clues for further functional studies of this PAP in the early embryonic development of high eukaryotes.


Assuntos
Células-Tronco Embrionárias Humanas/metabolismo , Nucleotidiltransferases/metabolismo , Polinucleotídeo Adenililtransferase/metabolismo , Células Procarióticas/metabolismo , Animais , Biocatálise , Linhagem Celular , Sobrevivência Celular , Desenvolvimento Embrionário , Humanos , Modelos Moleculares , Nucleotidiltransferases/química , Nucleotidiltransferases/genética , Polinucleotídeo Adenililtransferase/química , Ligação Proteica , Domínios Proteicos , RNA/metabolismo , Especificidade por Substrato , Xenopus
12.
Org Biomol Chem ; 18(4): 738-744, 2020 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-31912849

RESUMO

5,7-Di-N-acetyllegionaminic acid (Leg5,7Ac2) is a bacterial nonulosonic acid (NulO) analogue of sialic acids, an important class of monosaccharides in mammals and in some bacteria. To develop efficient one-pot multienzyme (OPME) glycosylation systems for synthesizing Leg5,7Ac2-glycosides, Legionella pneumophila cytidine 5'-monophosphate (CMP)-Leg5,7Ac2 synthetase (LpCLS) was cloned and characterized. It was successfully used in producing Leg5,7Ac2-glycosides from chemoenzymatically synthesized Leg5,7Ac2 using a one-pot two-enzyme system or from its chemically synthesized six-carbon monosaccharide precursor 2,4-diacetamido-2,4,6-trideoxymannose (6deoxyMan2,4diNAc) in a one-pot three-enzyme system. In addition, LpCLS was shown to tolerate Neu5Ac7NAc, a C9-hydroxyl analogue of Leg5,7Ac2 and also a stable analogue of 7-O-acetylneuraminic acid (Neu5,7Ac2), to allow OPME synthesis of the corresponding α2-3-linked sialosides, from chemically synthesized six-carbon monosaccharide precursor 4-N-acetyl-4-deoxy-N-acetylmannosamine (ManNAc7NAc).


Assuntos
Proteínas de Bactérias/química , Glicosídeos/síntese química , Legionella pneumophila/enzimologia , Nucleotidiltransferases/química , Ácidos Siálicos/síntese química , Proteínas de Bactérias/genética , Escherichia coli/genética , Nucleotidiltransferases/genética
13.
Nat Commun ; 11(1): 517, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31980631

RESUMO

Posttranslational modification (PTM) of proteins represents an important cellular mechanism for controlling diverse functions such as signalling, localisation or protein-protein interactions. AMPylation (also termed adenylylation) has recently been discovered as a prevalent PTM for regulating protein activity. In human cells AMPylation has been exclusively studied with the FICD protein. Here we investigate the role of AMPylation in human neurogenesis by introducing a cell-permeable propargyl adenosine pronucleotide probe to infiltrate cellular AMPylation pathways and report distinct modifications in intact cancer cell lines, human-derived stem cells, neural progenitor cells (NPCs), neurons and cerebral organoids (COs) via LC-MS/MS as well as imaging methods. A total of 162 AMP modified proteins were identified. FICD-dependent AMPylation remodelling accelerates differentiation of neural progenitor cells into mature neurons in COs, demonstrating a so far unknown trigger of human neurogenesis.


Assuntos
Monofosfato de Adenosina/metabolismo , Proteínas de Membrana/metabolismo , Neurogênese , Nucleotidiltransferases/metabolismo , Processamento de Proteína Pós-Traducional , Adenosina/metabolismo , Sequência de Aminoácidos , Catepsina B/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Regulação para Baixo , Humanos , Proteínas de Membrana/química , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Nucleotidiltransferases/química , Organoides/metabolismo
14.
Mol Cell ; 77(4): 709-722.e7, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-31932165

RESUMO

Bacteria are continually challenged by foreign invaders, including bacteriophages, and have evolved a variety of defenses against these invaders. Here, we describe the structural and biochemical mechanisms of a bacteriophage immunity pathway found in a broad array of bacteria, including E. coli and Pseudomonas aeruginosa. This pathway uses eukaryotic-like HORMA domain proteins that recognize specific peptides, then bind and activate a cGAS/DncV-like nucleotidyltransferase (CD-NTase) to generate a cyclic triadenylate (cAAA) second messenger; cAAA in turn activates an endonuclease effector, NucC. Signaling is attenuated by a homolog of the AAA+ ATPase Pch2/TRIP13, which binds and disassembles the active HORMA-CD-NTase complex. When expressed in non-pathogenic E. coli, this pathway confers immunity against bacteriophage λ through an abortive infection mechanism. Our findings reveal the molecular mechanisms of a bacterial defense pathway integrating a cGAS-like nucleotidyltransferase with HORMA domain proteins for threat sensing through protein detection and negative regulation by a Trip13 ATPase.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Bactérias/metabolismo , Escherichia coli/virologia , Nucleotidiltransferases/metabolismo , ATPases Associadas a Diversas Atividades Celulares/química , Proteínas de Bactérias/química , Bacteriófago lambda/fisiologia , Desoxirribonuclease I/metabolismo , Escherichia coli/imunologia , Escherichia coli/metabolismo , Nucleotidiltransferases/química , Peptídeos/metabolismo , Sistemas do Segundo Mensageiro
15.
J Mol Biol ; 432(2): 552-568, 2020 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-31786265

RESUMO

Nucleic acids derived from microorganisms are powerful triggers for innate immune responses. Proteins called RNA and DNA sensors detect foreign nucleic acids and, in mammalian cells, include RIG-I, cGAS, and AIM2. On binding to nucleic acids, these proteins initiate signaling cascades that activate host defense responses. An important aspect of this defense program is the production of cytokines such as type I interferons and IL-1ß. Studies conducted over recent years have revealed that nucleic acid sensors also activate programmed cell death pathways as an innate immune response to infection. Indeed, RNA and DNA sensors induce apoptosis, pyroptosis, and necroptosis. Cell death via these pathways prevents replication of pathogens by eliminating the infected cell and additionally contributes to the release of cytokines and inflammatory mediators. Interestingly, recent evidence suggests that programmed cell death triggered by nucleic acid sensors plays an important role in a number of noninfectious pathologies. In addition to nonself DNA and RNA from microorganisms, nucleic acid sensors also recognize endogenous nucleic acids, for example when cells are damaged by genotoxic agents and in certain autoinflammatory diseases. This review article summarizes current knowledge on the links between nucleic acid sensing and cell death and explores important open questions for future studies in this area.


Assuntos
Morte Celular/genética , Imunidade Inata/genética , Ácidos Nucleicos/genética , Proteína DEAD-box 58/química , Proteína DEAD-box 58/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Humanos , Interferon Tipo I/genética , Interleucina-1beta/genética , Ácidos Nucleicos/química , Nucleotidiltransferases/química , Nucleotidiltransferases/genética , Proteínas de Ligação a RNA/genética
16.
Int J Biol Macromol ; 142: 181-190, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31525415

RESUMO

Phosphopantetheine adenylyl transferase catalyzes a rate limiting penultimate step of the multistep reaction which produces coenzyme A (CoA) as a final product. CoA is required as an essential cofactor in a number of metabolic reactions. Therefore inhibiting the function of this enzyme will lead to cell death in bacteria. Acinetobacter baumannii is multi drug resistant pathogen and causes infections in immunocompromised patients. AbPPAT has been cloned, expressed, purified and crystallized and structures of two complexes of AbPPAT with dephospho coenzyme A (dPCoA) and coenzyme A (CoA) have been determined. Both dPCoA and CoA molecules are observed in the substrate binding site of AbPPAT. A comparison with the structures of the complexes of PPAT from other species shows that the orientations of dPCoA are identical in all the structures. On the other hand, as observed from the structures of the complexes of CoA with PPAT, the orientations of CoA are found to differ considerably. This shows that the substrates occupy identical positions in the substrate binding sites of enzymes whereas the positions of inhibitors may differ. The binding studies carried out using fluorescence method and surface plasmon resonance techniques showed that binding affinity of CoA towards AbPPAT is nearly three times higher than that of dPCoA.


Assuntos
Acinetobacter baumannii/enzimologia , Coenzima A/química , Nucleotidiltransferases/química , Acinetobacter baumannii/genética , Sítios de Ligação , Fenômenos Bioquímicos , Clonagem Molecular , Coenzima A/biossíntese , Cristalografia por Raios X , Modelos Moleculares , Nucleotidiltransferases/genética , Conformação Proteica
17.
Cell ; 179(7): 1525-1536.e12, 2019 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-31835031

RESUMO

Poxviruses use virus-encoded multisubunit RNA polymerases (vRNAPs) and RNA-processing factors to generate m7G-capped mRNAs in the host cytoplasm. In the accompanying paper, we report structures of core and complete vRNAP complexes of the prototypic Vaccinia poxvirus (Grimm et al., 2019; in this issue of Cell). Here, we present the cryo-electron microscopy (cryo-EM) structures of Vaccinia vRNAP in the form of a transcribing elongation complex and in the form of a co-transcriptional capping complex that contains the viral capping enzyme (CE). The trifunctional CE forms two mobile modules that bind the polymerase surface around the RNA exit tunnel. RNA extends from the vRNAP active site through this tunnel and into the active site of the CE triphosphatase. Structural comparisons suggest that growing RNA triggers large-scale rearrangements on the surface of the transcription machinery during the transition from transcription initiation to RNA capping and elongation. Our structures unravel the basis for synthesis and co-transcriptional modification of poxvirus RNA.


Assuntos
RNA Polimerases Dirigidas por DNA/química , Metiltransferases/química , Complexos Multienzimáticos/química , Nucleotidiltransferases/química , Monoéster Fosfórico Hidrolases/química , Vírus Vaccinia/ultraestrutura , Proteínas Virais/química , Microscopia Crioeletrônica , Complexos Multienzimáticos/ultraestrutura , RNA Mensageiro/química , Imagem Individual de Molécula , Transcrição Genética , Vírus Vaccinia/genética , Vírus Vaccinia/metabolismo
18.
Int J Mol Sci ; 20(24)2019 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-31835305

RESUMO

FAD synthase (FADS, or FMN:ATP adenylyl transferase) coded by the FLAD1 gene is the last enzyme in the pathway of FAD synthesis. The mitochondrial isoform 1 and the cytosolic isoform 2 are characterized by the following two domains: the C-terminal PAPS domain (FADSy) performing FAD synthesis and pyrophosphorolysis; the N-terminal molybdopterin-binding domain (FADHy) performing a Co++/K+-dependent FAD hydrolysis. Mutations in FLAD1 gene are responsible for riboflavin responsive and non-responsive multiple acyl-CoA dehydrogenases and combined respiratory chain deficiency. In patients harboring frameshift mutations, a shorter isoform (hFADS6) containing the sole FADSy domain is produced representing an emergency protein. With the aim to ameliorate its function we planned to obtain an engineered more efficient hFADS6. Thus, the D238A mutant, resembling the D181A FMNAT "supermutant" of C. glabrata, was overproduced and purified. Kinetic analysis of this enzyme highlighted a general increase of Km, while the kcat was two-fold higher than that of WT. The data suggest that the FAD synthesis rate can be increased. Additional modifications could be performed to further improve the synthesis of FAD. These results correlate with previous data produced in our laboratory, and point towards the following proposals (i) FAD release is the rate limiting step of the catalytic cycle and (ii) ATP and FMN binding sites are synergistically connected.


Assuntos
Flavina-Adenina Dinucleotídeo/química , Mutação de Sentido Incorreto , Nucleotidiltransferases/química , Substituição de Aminoácidos , Ácido Aspártico/química , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Flavina-Adenina Dinucleotídeo/genética , Flavina-Adenina Dinucleotídeo/metabolismo , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo
19.
Genes (Basel) ; 11(1)2019 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-31861576

RESUMO

The FANCJ helicase unfolds G-quadruplexes (G4s) in human cells to support DNA replication. This action is coupled to the recruitment of REV1 polymerase to synthesize DNA across from a guanine template. The precise mechanisms of these reactions remain unclear. While FANCJ binds to G4s with an AKKQ motif, it is not known whether this site recognizes damaged G4 structures. FANCJ also has a PIP-like (PCNA Interacting Protein) region that may recruit REV1 to G4s either directly or through interactions mediated by PCNA protein. In this work, we measured the affinities of a FANCJ AKKQ peptide for G4s formed by (TTAGGG)4 and (GGGT)4 using fluorescence spectroscopy and biolayer interferometry (BLI). The effects of 8-oxoguanine (8oxoG) on these interactions were tested at different positions. BLI assays were then performed with a FANCJ PIP to examine its recruitment of REV1 and PCNA. FANCJ AKKQ bound tightly to a TTA loop and was sequestered away from the 8oxoG. Reducing the loop length between guanine tetrads increased the affinity of the peptide for 8oxoG4s. FANCJ PIP targeted both REV1 and PCNA but favored interactions with the REV1 polymerase. The impact of these results on the remodeling of damaged G4 DNA is discussed herein.


Assuntos
Proteínas de Grupos de Complementação da Anemia de Fanconi/química , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Guanina/análogos & derivados , Nucleotidiltransferases/genética , Antígeno Nuclear de Célula em Proliferação/genética , RNA Helicases/química , RNA Helicases/metabolismo , Motivos de Aminoácidos , Sítios de Ligação/efeitos dos fármacos , Linhagem Celular , Dicroísmo Circular , Quadruplex G , Guanina/química , Humanos , Modelos Moleculares , Nucleotidiltransferases/química , Antígeno Nuclear de Célula em Proliferação/química , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Domínios Proteicos , Espectrometria de Fluorescência
20.
Fish Shellfish Immunol ; 94: 871-879, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31597087

RESUMO

Mammalian cyclic GMP-AMP synthase (cGAS) senses double-stranded (ds) DNA in the cytosol to activate the innate antiviral response. In the present study, a cGAS-like gene, namely cGASL, was cloned from grass carp Ctenopharyngodon idellus, and its role as a negative regulator of the IFN response was revealed. Phylogenetic analysis indicated that cGASL was evolutionarily closest to cGAS, but was not a true ortholog of cGAS. Overexpression of cGASL inhibited poly I:C-stimulated grass carp (gc)IFN1pro and ISRE activities. In addition, MITA-, but not TBK1-mediated activation of gcIFN1pro was impaired by cGASL. Co-immunoprecipitation and Western blot experiments indicated that cGASL interacted with MITA and TBK1, resulting in a reduction in the phosphorylation of MITA. Lastly, overexpression of cGASL reduced the transcriptional levels of several IFN-stimulated genes activated by MITA. Collectively, these data suggest that cGASL is a negative regulator of IFN response by targeting MITA in fish.


Assuntos
Carpas/genética , Carpas/imunologia , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Nucleotidiltransferases/genética , Nucleotidiltransferases/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Interferons/metabolismo , Nucleotidiltransferases/química , Filogenia , Poli I-C/farmacologia , Alinhamento de Sequência/veterinária
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