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1.
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
2.
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
3.
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
4.
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
5.
Int J Mol Sci ; 20(20)2019 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-31614972

RESUMO

Bifunctional FAD synthases (FADSs) catalyze FMN (flavin mononucleotide) and FAD (flavinadenine dinucleotide) biosynthesis at their C-riboflavin kinase (RFK) and N-FMN:adenylyltransferase (FMNAT) modules, respectively. Biophysical properties and requirements for their FMNAT activity differ among species. Here, we evaluate the relevance of the integrity of the binding site of the isoalloxazine of flavinic substrates for FMNAT catalysis in Corynebacterium ammoniagenes FADS (CaFADS). We have substituted P56 and P58, belonging to a conserved motif, as well as L98. These residues shape the isoalloxazine FMNAT site, although they are not expected to directly contact it. All substitutions override enzyme ability to transform substrates at the FMNAT site, although most variants are able to bind them. Spectroscopic properties and thermodynamic parameters for the binding of ligands indicate that mutations alter their interaction modes. Substitutions also modulate binding and kinetic properties at the RFK site, evidencing the crosstalk of different protomers within CaFADS assemblies during catalysis. In conclusion, despite the FMNAT site for the binding of substrates in CaFADS appearing as a wide open cavity, it is finely tuned to provide the competent binding conformation of substrates. In particular, P56, P58 and L98 shape the isoalloxazine site to place the FMN- and FAD-reacting phosphates in optimal geometry for catalysis.


Assuntos
Corynebacterium/enzimologia , Óxido Nítrico Sintase/química , Nucleotidiltransferases/química , Termodinâmica , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Domínio Catalítico/genética , Corynebacterium/genética , Flavina-Adenina Dinucleotídeo/metabolismo , Cinética , Ligantes , Modelos Moleculares , Óxido Nítrico Sintase/genética , Nucleotidiltransferases/genética , Especificidade por Substrato
6.
EMBO J ; 38(21): e102177, 2019 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-31531998

RESUMO

AMPylation is an inactivating modification that alters the activity of the major endoplasmic reticulum (ER) chaperone BiP to match the burden of unfolded proteins. A single ER-localised Fic protein, FICD (HYPE), catalyses both AMPylation and deAMPylation of BiP. However, the basis for the switch in FICD's activity is unknown. We report on the transition of FICD from a dimeric enzyme, that deAMPylates BiP, to a monomer with potent AMPylation activity. Mutations in the dimer interface, or of residues along an inhibitory pathway linking the dimer interface to the enzyme's active site, favour BiP AMPylation in vitro and in cells. Mechanistically, monomerisation relieves a repressive effect allosterically propagated from the dimer interface to the inhibitory Glu234, thereby permitting AMPylation-competent binding of MgATP. Moreover, a reciprocal signal, propagated from the nucleotide-binding site, provides a mechanism for coupling the oligomeric state and enzymatic activity of FICD to the energy status of the ER.


Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Multimerização Proteica , Processamento de Proteína Pós-Traducional , Células HEK293 , Humanos , Conformação Proteica
7.
Biotechnol Appl Biochem ; 66(5): 880-899, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31397000

RESUMO

The present study was aimed to explore the molecular and structural features of UDP-N-acetylglucosamine pyrophosphorylase of Bombyx mori (BmUAP), an essential enzyme for chitin synthesis in insects. The BmUAP cDNA sequence was cloned and expression profiles were monitored during the molting and feeding stages of silkworm larvae. The effect of 20-hydroxyecdysone (20E) on BmUAP expression, and on silkworm molting was studied, which revealed that 20E regulates its expression. Multiple sequence alignment of various pyrophosphorylases revealed that the residues N223, G290, N327, and K407 of human UAP (PDB ID: 1JV1) were found to be highly conserved in BmUAP and all other eukaryotic UAPs considered for the study. Phylogenetic analysis inferred that the UAPs possess discrete variations in primary structure among different insect Orders while sharing good identity between species of the Order. The structure of BmUAP was predicted and its interactions with uridine triphosphate, N-acetylglucosamine-1-phosphate, and UDP-N-acetylglucosamine were analyzed. Virtual screening with a library of natural compounds resulted in five potential hits with good binding affinities. On further analysis, these five hits were found to be mimicking substrate and product, in inducing conformational changes in the active site. This work provides crucial information on molecular interactions and structural dynamics of insect UAPs.


Assuntos
Bombyx/enzimologia , Bombyx/genética , Clonagem Molecular , Simulação por Computador , Regulação Enzimológica da Expressão Gênica/genética , Simulação de Acoplamento Molecular , Nucleotidiltransferases/química , Nucleotidiltransferases/genética , Animais , Humanos , Nucleotidiltransferases/metabolismo , Conformação Proteica
8.
Yakugaku Zasshi ; 139(7): 969-973, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31257254

RESUMO

Translesion DNA synthesis (TLS) is an emergency system activated to inhibit cell death caused by DNA damage-induced replication arrest. Thus, TLS enables cancer cells to acquire resistance to alkylate anticancer drugs. REV7 functions as the hub protein that interacts with both the inserter DNA polymerase REV1 and the extender DNA polymerase REV3 in TLS. REV7-mediated protein-protein interactions (PPIs) are essential for the activation of TLS, and are therefore attractive targets for anticancer drug development. To clarify the REV7-REV3 and REV7-REV1 PPIs, we determined the structures of REV7-REV3 and REV7-REV3-REV1 complexes. In the structures of REV7-REV3 and REV7-REV3-REV1 complexes, REV7 wraps around the REV3 fragment, and the REV1-binding interface is distinct from the REV3-binding site of REV7. We also identified a novel REV7 binding protein, transcription factor II-I (TFII-I), which is required for TLS. Of note, TFII-I binds the REV7-REV3-REV1 complex, suggesting that REV7-TFII-I PPIs are independent of other REV7-mediated PPIs. Furthermore, we found a small-molecule compound that inhibits TLS by targeting the REV7-REV3 PPIs. Lastly, we determined the structure of REV7 in complex with chromosome alignment maintaining phosphoprotein (CAMP), a known kinetochore-microtubule attachment protein. The overall structure of the REV7-CAMP complex is similar to that of the REV7-REV3 complex, but the REV7-CAMP PPIs are markedly different from the REV7-REV3 PPIs. These findings improve our understanding of multifunctional hub proteins, and are helpful for designing small-molecule compounds for novel anticancer drug development.


Assuntos
Antineoplásicos , Descoberta de Drogas , Proteína Semelhante a ELAV 2/química , Cristalografia por Raios X , DNA/biossíntese , Dano ao DNA , Proteínas de Ligação a DNA/química , DNA Polimerase Dirigida por DNA/química , Humanos , Proteínas Mad2/química , Peso Molecular , Proteínas Nucleares/química , Nucleotidiltransferases/química , Ligação Proteica , Mapas de Interação de Proteínas , Estrutura Terciária de Proteína
9.
Nat Commun ; 10(1): 1558, 2019 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-30952857

RESUMO

Cofactor F420 plays critical roles in primary and secondary metabolism in a range of bacteria and archaea as a low-potential hydride transfer agent. It mediates a variety of important redox transformations involved in bacterial persistence, antibiotic biosynthesis, pro-drug activation and methanogenesis. However, the biosynthetic pathway for F420 has not been fully elucidated: neither the enzyme that generates the putative intermediate 2-phospho-L-lactate, nor the function of the FMN-binding C-terminal domain of the γ-glutamyl ligase (FbiB) in bacteria are known. Here we present the structure of the guanylyltransferase FbiD and show that, along with its archaeal homolog CofC, it accepts phosphoenolpyruvate, rather than 2-phospho-L-lactate, as the substrate, leading to the formation of the previously uncharacterized intermediate dehydro-F420-0. The C-terminal domain of FbiB then utilizes FMNH2 to reduce dehydro-F420-0, which produces mature F420 species when combined with the γ-glutamyl ligase activity of the N-terminal domain. These new insights have allowed the heterologous production of F420 from a recombinant F420 biosynthetic pathway in Escherichia coli.


Assuntos
Vias Biossintéticas , Escherichia coli/metabolismo , Riboflavina/análogos & derivados , Modelos Moleculares , Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Fosfoenolpiruvato/química , Fosfoenolpiruvato/metabolismo , Células Procarióticas/metabolismo , Riboflavina/biossíntese
10.
Biochim Biophys Acta Proteins Proteom ; 1867(6): 616-626, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30959222

RESUMO

The I326T mutation in the TRNT1 gene encoding human tRNA nucleotidyltransferase (tRNA-NT) is linked to a relatively mild form of SIFD. Previous work indicated that the I326T variant was unable to incorporate AMP into tRNAs in vitro, however, expression of the mutant allele from a strong heterologous promoter supported in vivo CCA addition to both cytosolic and mitochondrial tRNAs in a yeast strain lacking tRNA-NT. To address this discrepancy, we determined the biochemical and biophysical characteristics of the I326T variant enzyme and the related variant, I326A. Our in vitro analysis revealed that the I326T substitution decreases the thermal stability of the enzyme and causes a ten-fold reduction in enzyme activity. We propose that the structural changes in the I326T variant that lead to these altered parameters result from a rearrangement of helices within the body domain of the protein which can be probed by the inability of the monomeric enzyme to form a covalent dimer in vitro mediated by C373. In addition, we confirm that the effects of the I326T or I326A substitutions are relatively mild in vivo by demonstrating that the mutant alleles support both mitochondrial and cytosolic CCA-addition in yeast.


Assuntos
Substituição de Aminoácidos , Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Anemia Sideroblástica/genética , Domínio Catalítico , Estabilidade Enzimática , Humanos , Modelos Moleculares , Nucleotidiltransferases/genética , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Termodinâmica
11.
Biochim Biophys Acta Proteins Proteom ; 1867(6): 537-547, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30885618

RESUMO

Phosphopantetheine adenylyltransferase (PPAT, EC. 2.7.7.3) catalyzes an essential step in the reaction that transfers an adenylyl group from adenosine tri phosphate (ATP) to 4'-phosphopantetheine (pPant) yielding 3'- dephospho-coenzyme A (dPCoA) and pyrophosphate (PP) in the coenzyme A (CoA) biosynthesis pathway. The enzyme PPAT from Acinetobacter baumannii (AbPPAT) was cloned, expressed and purified. The binding studies of AbPPAT were carried out with two compounds, tri­sodium citrate (TSC) and l-ascorbic acid (LAA, vitamin-C) using fluorescence spectroscopic (FS) and surface Plasmon resonance (SPR) methods. Both methods provided similar values of dissociation constants for TSC and LAA which were of the order of 10-8 M and 10-5 M respectively. The computer aided docking studies indicated fewer interactions of LAA with AbPPAT as compared to those of TSC. The freshly purified samples of AbPPAT were crystallized. The crystals of AbPPAT were soaked in the solutions containing TSC and LAA. However, the crystals of the complex of AbPPAT with LAA did not diffract well and hence the structure of the complex of AbPPAT with LAA could not be determined. On the other hand, the crystals of the complex of AbPPAT with TSC diffracted well and the structure was determined at 1.76 Šresolution. It showed that TSC bound to AbPPAT at the ATP binding site and formed several intermolecular contacts including 12 hydrogen bonds. The results of binding studies for both TSC and LAA and the structure of the complex of AbPPAT with TSC clearly indicated a potential role of TSC and LAA as antibacterial agents.


Assuntos
Acinetobacter baumannii/enzimologia , Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Acinetobacter baumannii/química , Acinetobacter baumannii/genética , Ácido Ascórbico/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Clonagem Molecular , Ligação de Hidrogênio , Simulação de Acoplamento Molecular , Nucleotidiltransferases/genética , Ligação Proteica , Citrato de Sódio/metabolismo , Espectrometria de Fluorescência , Ressonância de Plasmônio de Superfície
12.
Nucleic Acids Res ; 47(7): 3631-3639, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30828718

RESUMO

Correct synthesis and maintenance of functional tRNA 3'-CCA-ends is a crucial prerequisite for aminoacylation and must be achieved by the phylogenetically diverse group of tRNA nucleotidyltransferases. While numerous reports on the in vitro characterization exist, robust analysis under in vivo conditions is lacking. Here, we utilize Escherichia coli RNase T, a tRNA-processing enzyme responsible for the tRNA-CCA-end turnover, to generate an in vivo system for the evaluation of A-adding activity. Expression of RNase T results in a prominent growth phenotype that renders the presence of a CCA- or A-adding enzyme essential for cell survival in an E. coli Δcca background. The distinct growth fitness allows for both complementation and selection of enzyme variants in a natural environment. We demonstrate the potential of our system via detection of altered catalytic efficiency and temperature sensitivity. Furthermore, we select functional enzyme variants out of a sequence pool carrying a randomized codon for a highly conserved position essential for catalysis. The presented E. coli-based approach opens up a wide field of future studies including the investigation of tRNA nucleotidyltransferases from all domains of life and the biological relevance of in vitro data concerning their functionality and mode of operation.


Assuntos
Escherichia coli/genética , Exorribonucleases/genética , Nucleotidiltransferases/genética , RNA Nucleotidiltransferases/genética , Aminoacilação/genética , Escherichia coli/crescimento & desenvolvimento , Exorribonucleases/química , Cinética , Conformação de Ácido Nucleico , Nucleotidiltransferases/química , RNA Nucleotidiltransferases/química , Processamento Pós-Transcricional do RNA/genética
13.
Nature ; 567(7747): 194-199, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30787435

RESUMO

Cyclic dinucleotides (CDNs) have central roles in bacterial homeostasis and virulence by acting as nucleotide second messengers. Bacterial CDNs also elicit immune responses during infection when they are detected by pattern-recognition receptors in animal cells. Here we perform a systematic biochemical screen for bacterial signalling nucleotides and discover a large family of cGAS/DncV-like nucleotidyltransferases (CD-NTases) that use both purine and pyrimidine nucleotides to synthesize a diverse range of CDNs. A series of crystal structures establish CD-NTases as a structurally conserved family and reveal key contacts in the enzyme active-site lid that direct purine or pyrimidine selection. CD-NTase products are not restricted to CDNs and also include an unexpected class of cyclic trinucleotide compounds. Biochemical and cellular analyses of CD-NTase signalling nucleotides demonstrate that these cyclic di- and trinucleotides activate distinct host receptors and thus may modulate the interaction of both pathogens and commensal microbiota with their animal and plant hosts.


Assuntos
Proteínas de Bactérias/metabolismo , Nucleotídeos/biossíntese , Nucleotídeos/metabolismo , Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Animais , Cristalografia por Raios X , Fosfatos de Dinucleosídeos/biossíntese , Fosfatos de Dinucleosídeos/metabolismo , Células HEK293 , Humanos , Camundongos , Nucleotídeos/química , Nucleotidiltransferases/genética , Óperon/genética , Simbiose
14.
Cell ; 176(6): 1447-1460.e14, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30799039

RESUMO

The presence of DNA in the cytoplasm is normally a sign of microbial infections and is quickly detected by cyclic GMP-AMP synthase (cGAS) to elicit anti-infection immune responses. However, chronic activation of cGAS by self-DNA leads to severe autoimmune diseases for which no effective treatment is available yet. Here we report that acetylation inhibits cGAS activation and that the enforced acetylation of cGAS by aspirin robustly suppresses self-DNA-induced autoimmunity. We find that cGAS acetylation on either Lys384, Lys394, or Lys414 contributes to keeping cGAS inactive. cGAS is deacetylated in response to DNA challenges. Importantly, we show that aspirin can directly acetylate cGAS and efficiently inhibit cGAS-mediated immune responses. Finally, we demonstrate that aspirin can effectively suppress self-DNA-induced autoimmunity in Aicardi-Goutières syndrome (AGS) patient cells and in an AGS mouse model. Thus, our study reveals that acetylation contributes to cGAS activity regulation and provides a potential therapy for treating DNA-mediated autoimmune diseases.


Assuntos
DNA/imunologia , Nucleotidiltransferases/metabolismo , Tolerância a Antígenos Próprios/imunologia , Acetilação , Sequência de Aminoácidos , Animais , Aspirina/farmacologia , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Doenças Autoimunes do Sistema Nervoso/genética , Doenças Autoimunes do Sistema Nervoso/imunologia , Doenças Autoimunes do Sistema Nervoso/metabolismo , Autoimunidade , Linhagem Celular , DNA/genética , DNA/metabolismo , Modelos Animais de Doenças , Exodesoxirribonucleases/metabolismo , Células HEK293 , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Mutação , Malformações do Sistema Nervoso/genética , Malformações do Sistema Nervoso/imunologia , Malformações do Sistema Nervoso/metabolismo , Nucleotidiltransferases/antagonistas & inibidores , Nucleotidiltransferases/química , Nucleotidiltransferases/genética , Células THP-1
15.
Biochemistry ; 58(7): 951-964, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30640434

RESUMO

Cobamides are coenzymes used by cells from all domains of life but made de novo by only some bacteria and archaea. The last steps of the cobamide biosynthetic pathway activate the corrin ring and the lower ligand base, condense the activated intermediates, and dephosphorylate the product prior to the release of the biologically active coenzyme. In bacteria, a phosphoribosyltransferase (PRTase) enyzme activates the base into its α-mononucleotide. The enzyme from Salmonella enterica ( SeCobT) has been extensively biochemically and structurally characterized. The crystal structure of the putative PRTase from the archaeum Methanocaldococcus jannaschii ( MjCobT) is known, but its function has not been validated. Here we report the in vivo and in vitro characterization of MjCobT. In vivo, in vitro, and phylogenetic data reported here show that MjCobT belongs to a new class of NaMN-dependent PRTases. We also show that the Synechococcus sp. WH7803 CobT protein has PRTase activity in vivo. Lastly, results of isothermal titration calorimetry and analytical ultracentrifugation analysis show that the biologically active form of MjCobT is a dimer, not a trimer, as suggested by its crystal structure.


Assuntos
Proteínas Arqueais/metabolismo , Proteínas de Bactérias/metabolismo , Cobamidas/biossíntese , Archaea/metabolismo , Proteínas Arqueais/química , Proteínas Arqueais/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cianobactérias/metabolismo , Concentração de Íons de Hidrogênio , Mathanococcus/enzimologia , Mathanococcus/genética , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Nucleotidiltransferases/química , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Concentração Osmolar , Pentosiltransferases/química , Pentosiltransferases/genética , Pentosiltransferases/metabolismo , Fosfatos/química , Fosfatos/metabolismo , Filogenia , Compostos de Potássio/química , Compostos de Potássio/metabolismo , Salmonella enterica/genética , Salmonella enterica/metabolismo , Especificidade por Substrato
17.
Nucleic Acids Res ; 47(1): 495-508, 2019 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-30407553

RESUMO

Terminal uridylyl transferase (TUTase) is one type of enzyme that modifies RNA molecules by facilitating the post-transcriptional addition of uridyl ribonucleotides to their 3' ends. Recent researches have reported that Drosophila TUTase, Tailor, exhibits an intrinsic preference for RNA substrates ending in 3'G, distinguishing it from any other known TUTases. Through this unique feature, Tailor plays a crucial role as the repressor in the biogenesis pathway of splicing-derived mirtron pre-miRNAs. Here we describe crystal structures of core catalytic domain of Tailor and its complexes with RNA stretches 5'-AGU-3' and 5'-AGUU-3'. We demonstrate that R327 and N347 are two key residues contributing cooperatively to Tailor's preference for 3'G, and R327 may play an extra role in facilitating the extension of polyuridylation chain. We also demonstrate that conformational stability of the exit of RNA-binding groove also contributes significantly to Tailor's activity. Overall, our work reveals useful insights to explain why Drosophila Tailor can preferentially select RNA substrates ending in 3'G and provides important values for further understanding the biological significances of biogenesis pathway of mirtron in flies.


Assuntos
Proteínas de Drosophila/genética , Drosophila/enzimologia , Nucleotidiltransferases/genética , RNA Nucleotidiltransferases/química , RNA/biossíntese , Regiões 3' não Traduzidas/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , Domínio Catalítico/genética , Drosophila/genética , Proteínas de Drosophila/química , Guanina/química , MicroRNAs/genética , Nucleotidiltransferases/química , RNA/genética , RNA Nucleotidiltransferases/genética , Processamento Pós-Transcricional do RNA/genética , Processamento de RNA/genética , Especificidade por Substrato
18.
Nucleic Acids Res ; 47(2): 1030-1042, 2019 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-30462292

RESUMO

Non-templated 3'-uridylation of RNAs has emerged as an important mechanism for regulating the processing, stability and biological function of eukaryotic transcripts. In Drosophila, oligouridine tailing by the terminal uridylyl transferase (TUTase) Tailor of numerous RNAs induces their degradation by the exonuclease Dis3L2, which serves functional roles in RNA surveillance and mirtron RNA biogenesis. Tailor preferentially uridylates RNAs terminating in guanosine or uridine nucleotides but the structural basis underpinning its RNA substrate selectivity is unknown. Here, we report crystal structures of Tailor bound to a donor substrate analog or mono- and oligouridylated RNA products. These structures reveal specific amino acid residues involved in donor and acceptor substrate recognition, and complementary biochemical assays confirm the critical role of an active site arginine in conferring selectivity toward 3'-guanosine terminated RNAs. Notably, conservation of these active site features suggests that other eukaryotic TUTases, including mammalian TUT4 and TUT7, might exhibit similar, hitherto unknown, substrate selectivity. Together, these studies provide critical insights into the specificity of 3'-uridylation in eukaryotic post-transcriptional gene regulation.


Assuntos
Proteínas de Drosophila/química , Nucleotidiltransferases/química , RNA Nucleotidiltransferases/química , Animais , Domínio Catalítico , Cristalografia por Raios X , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Modelos Moleculares , Nucleotidiltransferases/metabolismo , RNA Nucleotidiltransferases/metabolismo , Especificidade por Substrato
19.
Int J Mol Sci ; 21(1)2019 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-31906195

RESUMO

d-glycero-α-d-manno-heptose-1-phosphate guanylyltransferase (HddC) is the fourth enzyme synthesizing a building component of lipopolysaccharide (LPS) of Gram-negative bacteria. Since HddC is a potential new target to develop antibiotics, the analysis of the structural and functional relationship of the complex structure will lead to a better idea to design inhibitory compounds. X-ray crystallography and biochemical experiments to elucidate the guanine preference were performed based on the multiple sequence alignment. The crystal structure of HddC from Yersinia pseudotuberculosis (YPT) complexed with guanosine 5'-(ß-amino)-diphosphate (GMPPN) has been determined at 1.55 Å resolution. Meanwhile, the mutants revealed their reduced guanine affinity, instead of acquiring noticeable pyrimidine affinity. The complex crystal structure revealed that GMPPN is docked in the catalytic site with the aid of Glu80 positioning on the conserved motif EXXPLGTGGA. In the HddC family, this motif is expected to recruit nucleotides through interacting with bases. The crystal structure shows that oxygen atoms of Glu80 forming two hydrogen bonds play a critical role in interaction with two nitrogen atoms of the guanine base of GMPPN. Interestingly, the binding of GMPPN induced the formation of an oxyanion hole-like conformation on the L(S/A/G)X(S/G) motif and consequently influenced on inducing a conformational shift of the region around Ser55.


Assuntos
Proteínas de Bactérias/química , Guanosina Trifosfato/química , Nucleotidiltransferases/química , Yersinia pseudotuberculosis/enzimologia , Cristalografia por Raios X , Especificidade por Substrato
20.
Elife ; 72018 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-30295605

RESUMO

Cyclic G/AMP synthase (cGAS) initiates type-1 interferon responses against cytosolic double-stranded (ds)DNA, which range from antiviral gene expression to apoptosis. The mechanism by which cGAS shapes this diverse signaling landscape remains poorly defined. We find that substrate-binding and dsDNA length-dependent binding are coupled to the intrinsic dimerization equilibrium of cGAS, with its N-terminal domain potentiating dimerization. Notably, increasing the dimeric fraction by raising cGAS and substrate concentrations diminishes duplex length-dependent activation, but does not negate the requirement for dsDNA. These results demonstrate that reaction context dictates the duplex length dependence, reconciling competing claims on the role of dsDNA length in cGAS activation. Overall, our study reveals how ligand-mediated allostery positions cGAS in standby, ready to tune its signaling pathway in a switch-like fashion.


Assuntos
Nucleotidiltransferases/metabolismo , Transdução de Sinais , Regulação Alostérica , Fenômenos Biofísicos , DNA/metabolismo , Humanos , Cinética , Nucleotidiltransferases/química , Domínios Proteicos , Multimerização Proteica , Especificidade por Substrato
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