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1.
Nat Commun ; 11(1): 5508, 2020 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-33139726

RESUMO

The ubiquitous redox coenzyme nicotinamide adenine dinucleotide (NAD) acts as a non-canonical cap structure on prokaryotic and eukaryotic ribonucleic acids. Here we find that in budding yeast, NAD-RNAs are abundant (>1400 species), short (<170 nt), and mostly correspond to mRNA 5'-ends. The modification percentage of transcripts is low (<5%). NAD incorporation occurs mainly during transcription initiation by RNA polymerase II, which uses distinct promoters with a YAAG core motif for this purpose. Most NAD-RNAs are 3'-truncated. At least three decapping enzymes, Rai1, Dxo1, and Npy1, guard against NAD-RNA at different cellular locations, targeting overlapping transcript populations. NAD-mRNAs are not translatable in vitro. Our work indicates that in budding yeast, most of the NAD incorporation into RNA seems to be disadvantageous to the cell, which has evolved a diverse surveillance machinery to prematurely terminate, decap and reject NAD-RNAs.


Assuntos
Endorribonucleases/metabolismo , NAD/metabolismo , Capuzes de RNA/metabolismo , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/genética , Regiões 5' não Traduzidas , Núcleo Celular/genética , Pirofosfatases/metabolismo , Estabilidade de RNA , Proteínas de Ligação a RNA/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcrição Genética
2.
Nat Commun ; 11(1): 4894, 2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32994400

RESUMO

Identification of the complete set of translated genes of viruses is important to understand viral replication and pathogenesis as well as for therapeutic approaches to control viral infection. Here, we use chemical proteomics, integrating bio-orthogonal non-canonical amino acid tagging and high-resolution mass spectrometry, to characterize the newly synthesized herpes simplex virus 1 (HSV-1) proteome in infected cells. In these infected cells, host cellular protein synthesis is shut-off, increasing the chance to preferentially detect viral proteomes. We identify nine previously cryptic orphan protein coding sequences whose translated products are expressed in HSV-1-infected cells. Functional characterization of one identified protein, designated piUL49, shows that it is critical for HSV-1 neurovirulence in vivo by regulating the activity of virally encoded dUTPase, a key enzyme that maintains accurate DNA replication. Our results demonstrate that cryptic orphan protein coding genes of HSV-1, and probably other large DNA viruses, remain to be identified.


Assuntos
Encefalite por Herpes Simples/virologia , Herpesvirus Humano 1/patogenicidade , Pirofosfatases/metabolismo , Proteínas Virais/metabolismo , Fatores de Virulência/metabolismo , Animais , Encéfalo/patologia , Encéfalo/virologia , Chlorocebus aethiops , Replicação do DNA , Modelos Animais de Doenças , Encefalite por Herpes Simples/patologia , Feminino , Genes Virais/genética , Células HEK293 , Células HeLa , Herpesvirus Humano 1/genética , Humanos , Camundongos , Biossíntese de Proteínas , Proteômica/métodos , Células Vero , Proteínas Virais/genética , Fatores de Virulência/genética , Replicação Viral
3.
Mem Inst Oswaldo Cruz ; 115: e190469, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32638832

RESUMO

BACKGROUND Oxidative stress is responsible for generating DNA lesions and the 8-oxoguanine (8-oxoG) is the most commonly lesion found in DNA damage. When this base is incorporated during DNA replication, it could generate double-strand DNA breaks and cellular death. MutT enzyme hydrolyzes the 8-oxoG from the nucleotide pool, preventing its incorporation during DNA replication. OBJECTIVES To investigate the importance of 8-oxoG in Leishmania infantum and L. braziliensis, in this study we analysed the impact of heterologous expression of Escherichia coli MutT (EcMutT) enzyme in drug-resistance phenotype and defense against oxidative stress. METHODS Comparative analysis of L. braziliensis and L. infantum H2O2 tolerance and cell cycle profile were performed. Lines of L. braziliensis and L. infantum expressing EcMutT were generated and evaluated using susceptibility tests to H2O2 and SbIII, cell cycle analysis, γH2A western blotting, and BrdU native detection assay. FINDINGS Comparative analysis of tolerance to oxidative stress generated by H2O2 showed that L. infantum is more tolerant to exogenous H2O2 than L. braziliensis. In addition, cell cycle analysis showed that L. infantum, after treatment with H2O2, remains in G1 phase, returning to its normal growth rate after 72 h. In contrast, after treatment with H2O2, L. braziliensis parasites continue to move to the next stages of the cell cycle. Expression of the E. coli MutT gene in L. braziliensis and L. infantum does not interfere in parasite growth or in susceptibility to SbIII. Interestingly, we observed that L. braziliensis EcMutT-expressing clones were more tolerant to H2O2 treatment, presented lower activation of γH2A, a biomarker of genotoxic stress, and lower replication stress than its parental non-transfected parasites. In contrast, the EcMutT is not involved in protection against oxidative stress generated by H2O2 in L. infantum. MAIN CONCLUSIONS Our results showed that 8-oxoG clearance in L. braziliensis is important to avoid misincorporation during DNA replication after oxidative stress generated by H2O2.


Assuntos
Antimônio/toxicidade , Proteínas de Escherichia coli/genética , Escherichia coli , Guanina/análogos & derivados , Leishmania braziliensis/efeitos dos fármacos , Leishmania infantum/efeitos dos fármacos , Pirofosfatases , Superóxido Dismutase/metabolismo , Animais , Antiprotozoários/farmacologia , Proteínas de Escherichia coli/metabolismo , Guanina/farmacologia , Humanos , Peróxido de Hidrogênio/toxicidade , Leishmania braziliensis/enzimologia , Leishmania infantum/enzimologia , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Pirofosfatases/genética , Pirofosfatases/metabolismo , Coelhos , Ratos , Superóxido Dismutase/genética
4.
PLoS Pathog ; 16(6): e1008661, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32598377

RESUMO

Mycoplasmas are host-restricted prokaryotes with a nearly minimal genome. To overcome their metabolic limitations, these wall-less bacteria establish intimate interactions with epithelial cells at mucosal surfaces. The alarming rate of antimicrobial resistance among pathogenic species is of particular concern in the medical and veterinary fields. Taking advantage of the reduced mycoplasma genome, random transposon mutagenesis was combined with high-throughput screening in order to identify key determinants of mycoplasma survival in the host-cell environment and potential targets for drug development. With the use of the ruminant pathogen Mycoplasma bovis as a model, three phosphodiesterases of the DHH superfamily were identified as essential for the proliferation of this species under cell culture conditions, while dispensable for axenic growth. Despite a similar domain architecture, recombinant Mbov_0327 and Mbov_0328 products displayed different substrate specificities. While rMbovP328 protein exhibited activity towards cyclic dinucleotides and nanoRNAs, rMbovP327 protein was only able to degrade nanoRNAs. The Mbov_0276 product was identified as a member of the membrane-associated GdpP family of phosphodiesterases that was found to participate in cyclic dinucleotide and nanoRNA degradation, an activity which might therefore be redundant in the genome-reduced M. bovis. Remarkably, all these enzymes were able to convert their substrates into mononucleotides, and medium supplementation with nucleoside monophosphates or nucleosides fully restored the capacity of a Mbov_0328/0327 knock-out mutant to grow under cell culture conditions. Since mycoplasmas are unable to synthesize DNA/RNA precursors de novo, cyclic dinucleotide and nanoRNA degradation are likely contributing to the survival of M. bovis by securing the recycling of purines and pyrimidines. These results point toward proteins of the DHH superfamily as promising targets for the development of new antimicrobials against multidrug-resistant pathogenic mycoplasma species.


Assuntos
Proteínas de Bactérias/metabolismo , Mycoplasma bovis/enzimologia , Pirofosfatases/metabolismo , Ribonucleases/metabolismo , Animais , Proteínas de Bactérias/genética , Linhagem Celular , Camundongos , Camundongos Endogâmicos BALB C , Mycoplasma bovis/genética , Pirofosfatases/genética , Ribonucleases/genética
6.
Proc Natl Acad Sci U S A ; 117(19): 10500-10510, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32345719

RESUMO

Under stressful conditions, bacterial RelA-SpoT Homolog (RSH) enzymes synthesize the alarmone (p)ppGpp, a nucleotide second messenger. (p)ppGpp rewires bacterial transcription and metabolism to cope with stress, and, at high concentrations, inhibits the process of protein synthesis and bacterial growth to save and redirect resources until conditions improve. Single-domain small alarmone synthetases (SASs) are RSH family members that contain the (p)ppGpp synthesis (SYNTH) domain, but lack the hydrolysis (HD) domain and regulatory C-terminal domains of the long RSHs such as Rel, RelA, and SpoT. We asked whether analysis of the genomic context of SASs can indicate possible functional roles. Indeed, multiple SAS subfamilies are encoded in widespread conserved bicistronic operon architectures that are reminiscent of those typically seen in toxin-antitoxin (TA) operons. We have validated five of these SASs as being toxic (toxSASs), with neutralization by the protein products of six neighboring antitoxin genes. The toxicity of Cellulomonas marina toxSAS FaRel is mediated by the accumulation of alarmones ppGpp and ppApp, and an associated depletion of cellular guanosine triphosphate and adenosine triphosphate pools, and is counteracted by its HD domain-containing antitoxin. Thus, the ToxSAS-antiToxSAS system with its multiple different antitoxins exemplifies how ancient nucleotide-based signaling mechanisms can be repurposed as TA modules during evolution, potentially multiple times independently.


Assuntos
Bactérias/crescimento & desenvolvimento , Guanosina Pentafosfato/metabolismo , Sistemas Toxina-Antitoxina/fisiologia , Nucleotídeos de Adenina/metabolismo , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Bases de Dados Genéticas , Regulação Bacteriana da Expressão Gênica/genética , Guanosina Tetrafosfato/metabolismo , Guanosina Trifosfato/metabolismo , Ligases/metabolismo , Pirofosfatases/metabolismo , Transdução de Sinais , Estresse Fisiológico/fisiologia
7.
Nat Commun ; 11(1): 1846, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32296056

RESUMO

Seed size is a key agronomic trait that greatly determines plant yield. Elucidating the molecular mechanism underlying seed size regulation is also an important question in developmental biology. Here, we show that the KIX-PPD-MYC-GIF1 pathway plays a crucial role in seed size control in Arabidopsis thaliana. Disruption of KIX8/9 and PPD1/2 causes large seeds due to increased cell proliferation and cell elongation in the integuments. KIX8/9 and PPD1/2 interact with transcription factors MYC3/4 to form the KIX-PPD-MYC complex in Arabidopsis. The KIX-PPD-MYC complex associates with the typical G-box sequence in the promoter of GRF-INTERACTING FACTOR 1 (GIF1), which promotes seed growth, and represses its expression. Genetic analyses support that KIX8/9, PPD1/2, MYC3/4, and GIF1 function in a common pathway to control seed size. Thus, our results reveal a genetic and molecular mechanism by which the transcription factors MYC3/4 recruit KIX8/9 and PPD1/2 to the promoter of GIF1 and repress its expression, thereby determining seed size in Arabidopsis.


Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Transativadores/genética , Transativadores/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Regiões Promotoras Genéticas , Pirofosfatases/genética , Pirofosfatases/metabolismo
8.
PLoS One ; 15(4): e0231651, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32294118

RESUMO

INTRODUCTION: The nudix hydrolase 15 (NUDT15) gene acts in the metabolism of thiopurine, by catabolizing its active metabolite thioguanosine triphosphate into its inactivated form, thioguanosine monophosphate. The frequency of alternative NUDT15 alleles, in particular those that cause a drastic loss of gene function, varies widely among geographically distinct populations. In the general population of northern Brazilian, high toxicity rates (65%) have been recorded in patients treated with the standard protocol for acute lymphoblastic leukemia, which involves thiopurine-based drugs. The present study characterized the molecular profile of the coding region of the NUDT15 gene in two groups, non-admixed Amerindians and admixed individuals from the Amazon region of northern Brazil. METHODS: The entire NUDT15 gene was sequenced in 64 Amerindians from 12 Amazonian groups and 82 admixed individuals from northern Brazil. The DNA was extracted using phenol-chloroform. The exome libraries were prepared using the Nextera Rapid Capture Exome (Illumina) and SureSelect Human All Exon V6 (Agilent) kits. The allelic variants were annotated in the ViVa® (Viewer of Variants) software. RESULTS: Four NUDT15 variants were identified: rs374594155, rs1272632214, rs147390019, andrs116855232. The variants rs1272632214 and rs116855232 were in complete linkage disequilibrium, and were assigned to the NUDT15*2 genotype. These variants had high frequencies in both our study populations in comparison with other populations catalogued in the 1000 Genomes database. We also identified the NUDT15*4 haplotype in our study populations, at frequencies similar to those reported in other populations from around the world. CONCLUSION: Our findings indicate that Amerindian and admixed populations from northern Brazil have high frequencies of the NUDT15 haplotypes that alter the metabolism profile of thiopurines.


Assuntos
Povos Indígenas/genética , Pirofosfatases/genética , Antimetabólitos Antineoplásicos/farmacologia , Antimetabólitos Antineoplásicos/uso terapêutico , Brasil , Humanos , Mercaptopurina/farmacologia , Mercaptopurina/uso terapêutico , Polimorfismo de Nucleotídeo Único , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Pirofosfatases/metabolismo
9.
Proc Natl Acad Sci U S A ; 117(17): 9356-9364, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32303658

RESUMO

Inositol diphosphates (PP-IPs), also known as inositol pyrophosphates, are high-energy cellular signaling codes involved in nutrient and regulatory responses. We report that the evolutionarily conserved gene product, Vip1, possesses autonomous kinase and pyrophosphatase domains capable of synthesis and destruction of D-1 PP-IPs. Our studies provide atomic-resolution structures of the PP-IP products and unequivocally define that the Vip1 gene product is a highly selective 1-kinase and 1-pyrophosphatase enzyme whose activities arise through distinct active sites. Kinetic analyses of kinase and pyrophosphatase parameters are consistent with Vip1 evolving to modulate levels of 1-IP7 and 1,5-IP8 Individual perturbations in kinase and pyrophosphatase activities in cells result in differential effects on vacuolar morphology and osmotic responses. Analogous to the dual-functional key energy metabolism regulator, phosphofructokinase 2, Vip1 is a kinase and pyrophosphatase switch whose 1-PP-IP products play an important role in a cellular adaptation.


Assuntos
Fosfatos de Inositol/metabolismo , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Difosfatos/metabolismo , Fosfatos de Inositol/fisiologia , Cinética , Fosforilação , Fosfotransferases (Aceptor do Grupo Fosfato)/fisiologia , Pirofosfatases/metabolismo , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais
10.
Sci Rep ; 10(1): 6091, 2020 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-32269250

RESUMO

Genetic circuit-based biosensors have emerged as an effective analytical tool in synthetic biology; these biosensors can be applied to high-throughput screening of new biocatalysts and metabolic pathways. Sigma 54 (σ54)-dependent transcription factor (TF) can be a valuable component of these biosensors owing to its intrinsic silent property compared to most of the housekeeping sigma 70 (σ70) TFs. Here, we show that these unique characteristics of σ54-dependent TFs can be used to control the host cell state to be more appropriate for high-throughput screening. The acclimation of cell state was achieved by using guanosine (penta)tetraphosphate ((p)ppGpp)-related genes (relA, spoT) and nutrient conditions, to link the σ54 TF-based reporter expression with the target enzyme activity. By controlling stringent programmed responses and optimizing assay conditions, catalytically improved tyrosine phenol lyase (TPL) enzymes were successfully obtained using a σ54-dependent DmpR as the TF component, demonstrating the practical feasibility of this biosensor. This combinatorial strategy of biosensors using σ factor-dependent TFs will allow for more effective high-throughput enzyme engineering with broad applicability.


Assuntos
Proteínas de Bactérias/genética , Ensaios de Triagem em Larga Escala/métodos , Engenharia de Proteínas/métodos , Transativadores/genética , Ativação Transcricional , Tirosina Fenol-Liase/genética , Aclimatação , Técnicas Biossensoriais/métodos , Escherichia coli , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , GTP Pirofosfoquinase/genética , GTP Pirofosfoquinase/metabolismo , Regiões Promotoras Genéticas , Pseudomonas putida , Pirofosfatases/genética , Pirofosfatases/metabolismo , Fator sigma/genética , Fator sigma/metabolismo , Tirosina Fenol-Liase/metabolismo
11.
Appl Microbiol Biotechnol ; 104(7): 3061-3079, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32009198

RESUMO

Pseudomonas protegens H78 produces multiple secondary metabolites, including antibiotics and iron carriers. The guanosine pentaphosphate or tetraphosphate ((p)ppGpp)-mediated stringent response is utilized by bacteria to survive during nutritional starvation and other stresses. RelA/SpoT homologues are responsible for the biosynthesis and degradation of the alarmone (p)ppGpp. Here, we investigated the global effect of relA/spoT dual deletion on the transcriptomic profiles, physiology, and metabolism of P. protegens H78 grown to mid- to late log phase. Transcriptomic profiling revealed that relA/spoT deletion globally upregulated the expression of genes involved in DNA replication, transcription, and translation; amino acid metabolism; carbohydrate and energy metabolism; ion transport and metabolism; and secretion systems. Bacterial growth was partially increased, while the cell survival rate was significantly reduced by relA/spoT deletion in H78. The utilization of some nutritional elements (C, P, S, and N) was downregulated due to relA/spoT deletion. In contrast, relA/spoT mutation globally inhibited the expression of secondary metabolic gene clusters (plt, phl, prn, ofa, fit, pch, pvd, and has). Correspondingly, antibiotic and iron carrier biosynthesis, iron utilization, and antibiotic resistance were significantly downregulated by the relA/spoT mutation. This work highlights that the (p)ppGpp-mediated stringent response regulatory system plays an important role in inhibiting primary metabolism and activating secondary metabolism in P. protegens.


Assuntos
Guanosina Pentafosfato/metabolismo , Guanosina Tetrafosfato/metabolismo , Pseudomonas/metabolismo , Metabolismo Secundário/genética , Antibacterianos/biossíntese , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana/genética , Metabolismo Energético/genética , GTP Pirofosfoquinase/genética , GTP Pirofosfoquinase/metabolismo , Deleção de Genes , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Viabilidade Microbiana/genética , Pseudomonas/efeitos dos fármacos , Pseudomonas/genética , Pseudomonas/crescimento & desenvolvimento , Pirofosfatases/genética , Pirofosfatases/metabolismo
12.
J Biosci ; 452020.
Artigo em Inglês | MEDLINE | ID: mdl-32020910

RESUMO

The nucleotide derivatives (p)ppGpp, comprising ppGpp and pppGpp, are important signalling molecules that control various facets of gene regulation and protein synthesis in Escherichia coli. Their synthesis is catalysed by RelA (in response to amino acid limitation) and by SpoT (in response to the limitation of carbon source or fatty acids). SpoT is also a hydrolase for degradation of both ppGpp and pppGpp, while GppA catalyses the conversion of pppGpp to ppGpp. Here we provide evidence to show that pppGpp exerts heightened toxicity compared to that by ppGpp. Thus, gppA spoT double mutants exhibited lethality under conditions in which the single mutants were viable. The extent of RelA-catalysed (p)ppGpp accumulation in the gppA spoT strain was substantially greater than that in its isogenic gppA+ derivative. The data is interpreted in terms of a model in which toxicity of pppGpp in the gppA spoT mutants is mediated by its activation of RelA so as to result in a vicious cycle of (p)ppGpp synthesis.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , GTP Pirofosfoquinase/metabolismo , Guanosina Pentafosfato/análise , Guanosina Pentafosfato/biossíntese , Guanosina Tetrafosfato/análise , Guanosina Tetrafosfato/biossíntese , Pirofosfatases/metabolismo , Mutação , Fenótipo , Plasmídeos
13.
Proc Natl Acad Sci U S A ; 117(8): 4109-4116, 2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32041866

RESUMO

The UDP-2,3-diacylglucosamine pyrophosphate hydrolase LpxH is an essential lipid A biosynthetic enzyme that is conserved in the majority of gram-negative bacteria. It has emerged as an attractive novel antibiotic target due to the recent discovery of an LpxH-targeting sulfonyl piperazine compound (referred to as AZ1) by AstraZeneca. However, the molecular details of AZ1 inhibition have remained unresolved, stymieing further development of this class of antibiotics. Here we report the crystal structure of Klebsiella pneumoniae LpxH in complex with AZ1. We show that AZ1 fits snugly into the L-shaped acyl chain-binding chamber of LpxH with its indoline ring situating adjacent to the active site, its sulfonyl group adopting a sharp kink, and its N-CF3-phenyl substituted piperazine group reaching out to the far side of the LpxH acyl chain-binding chamber. Intriguingly, despite the observation of a single AZ1 conformation in the crystal structure, our solution NMR investigation has revealed the presence of a second ligand conformation invisible in the crystalline state. Together, these distinct ligand conformations delineate a cryptic inhibitor envelope that expands the observed footprint of AZ1 in the LpxH-bound crystal structure and enables the design of AZ1 analogs with enhanced potency in enzymatic assays. These designed compounds display striking improvement in antibiotic activity over AZ1 against wild-type K. pneumoniae, and coadministration with outer membrane permeability enhancers profoundly sensitizes Escherichia coli to designed LpxH inhibitors. Remarkably, none of the sulfonyl piperazine compounds occupies the active site of LpxH, foretelling a straightforward path for rapid optimization of this class of antibiotics.


Assuntos
Aciltransferases/antagonistas & inibidores , Aciltransferases/metabolismo , Antibacterianos/química , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Pirofosfatases/antagonistas & inibidores , Pirofosfatases/metabolismo , Aciltransferases/genética , Proteínas de Bactérias/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , Metabolismo dos Lipídeos , Testes de Sensibilidade Microbiana , Mutação , Piperazinas/química , Piperazinas/farmacologia , Conformação Proteica , Pirofosfatases/genética
14.
mBio ; 11(1)2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32098823

RESUMO

Guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), together named (p)ppGpp, regulate diverse aspects of Salmonella pathogenesis, including synthesis of nutrients, resistance to inflammatory mediators, and expression of secretion systems. In Salmonella, these nucleotide alarmones are generated by the synthetase activities of RelA and SpoT proteins. In addition, the (p)ppGpp hydrolase activity of the bifunctional SpoT protein is essential to preserve cell viability. The contribution of SpoT to physiology and pathogenesis has proven elusive in organisms such as Salmonella, because the hydrolytic activity of this RelA and SpoT homologue (RSH) is vital to prevent inhibitory effects of (p)ppGpp produced by a functional RelA. Here, we describe the biochemical and functional characterization of a spoT-Δctd mutant Salmonella strain encoding a SpoT protein that lacks the C-terminal regulatory elements collectively referred to as "ctd." Salmonella expressing the spoT-Δctd variant hydrolyzes (p)ppGpp with similar kinetics to those of wild-type bacteria, but it is defective at synthesizing (p)ppGpp in response to acidic pH. Salmonella spoT-Δctd mutants have virtually normal adaptations to nutritional, nitrosative, and oxidative stresses, but poorly induce metal cation uptake systems and Salmonella pathogenicity island 2 (SPI-2) genes in response to the acidic pH of the phagosome. Importantly, spoT-Δctd mutant Salmonella replicates poorly intracellularly and is attenuated in a murine model of acute salmonellosis. Collectively, these investigations indicate that (p)ppGpp synthesized by SpoT serves a unique function in the adaptation of Salmonella to the intracellular environment of host phagocytes that cannot be compensated by the presence of a functional RelA.IMPORTANCE Pathogenic bacteria experience nutritional challenges during colonization and infection of mammalian hosts. Binding of the alarmone nucleotide guanosine tetraphosphate (ppGpp) to RNA polymerase coordinates metabolic adaptations and virulence gene transcription, increasing the fitness of diverse Gram-positive and Gram-negative bacteria as well as that of actinomycetes. Gammaproteobacteria such as Salmonella synthesize ppGpp by the combined activities of the closely related RelA and SpoT synthetases. Due to its profound inhibitory effects on growth, ppGpp must be removed; in Salmonella, this process is catalyzed by the vital hydrolytic activity of the bifunctional SpoT protein. Because SpoT hydrolase activity is essential in cells expressing a functional RelA, we have a very limited understanding of unique roles these two synthetases may assume during interactions of bacterial pathogens with their hosts. We describe here a SpoT truncation mutant that lacks ppGpp synthetase activity and all C-terminal regulatory domains but retains excellent hydrolase activity. Our studies of this mutant reveal that SpoT uniquely senses the acidification of phagosomes, inducing virulence programs that increase Salmonella fitness in an acute model of infection. Our investigations indicate that the coexistence of RelA/SpoT homologues in a bacterial cell is driven by the need to mount a stringent response to a myriad of physiological and host-specific signatures.


Assuntos
Proteínas de Bactérias/metabolismo , Ligases/metabolismo , Fagossomos/metabolismo , Pirofosfatases/metabolismo , Salmonella/metabolismo , Animais , Sobrevivência Celular , Modelos Animais de Doenças , Bactérias Gram-Negativas/metabolismo , Bactérias Gram-Positivas/metabolismo , Guanosina Pentafosfato/genética , Guanosina Pentafosfato/metabolismo , Guanosina Tetrafosfato/genética , Guanosina Tetrafosfato/metabolismo , Imunidade Inata , Ligases/genética , Camundongos , Pirofosfatases/genética , Salmonella/genética , Fator de Transcrição RelA/metabolismo , Virulência/genética
15.
J Mol Biol ; 432(4): 1126-1142, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-31954130

RESUMO

Precise regulation of dNTPs within the cellular nucleotide pool is essential for high accuracy of DNA replication and is critical for retaining the genomic integrity. Recently, human dCTPase (deoxycytidine triphosphatase), also known as DCTPP1 (human all-alpha dCTP pyrophosphatase 1), has been revealed to be a key player in the balance of pyrimidine nucleotide concentrations within cells, with DCTPP1 deficiency causing DNA damage and genetic instability in both chromosomal and mitochondrial DNA. DCTPP1 also exhibits an additional "house cleaning" function as it has been shown to be highly active against modified cytidine triphosphates, such as 5-methyl-dCTP, which, if incorrectly incorporated into DNA can introduce undesirable epigenetic marking. To date, structural studies of mammalian dCTPase have been limited to inactive constructs, which do not provide information regarding the catalytic mechanism of this important enzyme. We present here the first structures of an active mammalian dCTPase from M. musculus in complex with the nonhydrolyzable substrate analog dCMPNPP and the products 5-Me-dCMP and dCMP. These structures provide clear insights into substrate binding and catalysis and clearly elucidate why previous structures of mammalian dCTPase were catalytically inactive. The overall structure of M. musculus dCTPase is highly similar to enzymes from the all-alpha NTP phosphohydrolase superfamily. Comparison of M. musculus dCTPase with homologs from a diverse range of mammals, including humans, shows that the residues, which contribute to substrate recognition, are entirely conserved, further supporting the importance of this enzyme in the protection of genomic integrity in mammalian cells.


Assuntos
Monoéster Fosfórico Hidrolases/metabolismo , Pirofosfatases/química , Pirofosfatases/metabolismo , Sequência de Aminoácidos , Animais , Dano ao DNA/genética , Nucleotídeos de Desoxicitosina/metabolismo , Epigenômica , Humanos , Cinética , Camundongos , Dados de Sequência Molecular , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/genética , Ligação Proteica , Estrutura Secundária de Proteína , Pirofosfatases/genética
16.
RNA ; 26(4): 419-438, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31915290

RESUMO

The translation preinitiation complex (PIC) scans the mRNA for an AUG codon in a favorable context. Previous findings suggest that the factor eIF1 discriminates against non-AUG start codons by impeding full accommodation of Met-tRNAi in the P site of the 40S ribosomal subunit, necessitating eIF1 dissociation for start codon selection. Consistent with this, yeast eIF1 substitutions that weaken its binding to the PIC increase initiation at UUG codons on a mutant his4 mRNA and particular synthetic mRNA reporters; and also at the AUG start codon of the mRNA for eIF1 itself owing to its poor Kozak context. It was not known however whether such eIF1 mutants increase initiation at suboptimal start codons genome-wide. By ribosome profiling, we show that the eIF1-L96P variant confers increased translation of numerous upstream open reading frames (uORFs) initiating with either near-cognate codons (NCCs) or AUGs in poor context. The increased uORF translation is frequently associated with the reduced translation of the downstream main coding sequences (CDS). Initiation is also elevated at certain NCCs initiating amino-terminal extensions, including those that direct mitochondrial localization of the GRS1 and ALA1 products, and at a small set of main CDS AUG codons with especially poor context, including that of eIF1 itself. Thus, eIF1 acts throughout the yeast translatome to discriminate against NCC start codons and AUGs in poor context; and impairing this function enhances the repressive effects of uORFs on CDS translation and alters the ratios of protein isoforms translated from near-cognate versus AUG start codons.


Assuntos
Códon de Iniciação , Fator de Iniciação 1 em Eucariotos/metabolismo , Fases de Leitura Aberta , Iniciação Traducional da Cadeia Peptídica , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Aminoidrolases/genética , Aminoidrolases/metabolismo , Regulação Fúngica da Expressão Gênica , Genoma Fúngico , Glicina-tRNA Ligase/genética , Glicina-tRNA Ligase/metabolismo , Pirofosfatases/genética , Pirofosfatases/metabolismo , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
17.
Biochim Biophys Acta Proteins Proteom ; 1868(5): 140376, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31981617

RESUMO

Two groups of metabolically related enzymes, the Group III family of Fe2+-dependent alcohol dehydrogenases (ADHs) and the separate subfamily of nucleoside diphosphates linked to x (nudix) hydrolases that activate Group III ADHs are under-characterized. Here we report the steady-state initial-velocity forward direction (alcohol → aldehyde) reaction of a Group III ADH, namely gamma-hydroxybutyrate dehydrogenase (GHBDH, UniProt: Q59104), cloned from Cupriavidus necator as a fusion protein. We also report the effects of nudix hydrolases on the GHBDH reaction. At optimal pH 9.0, the GHBDH reaction is activated ~2-fold by two different saturating purified nudix hydrolases, namely Bacillus methanolicus activator (ACT, UniProt: I3EA59) and Escherichia coli NudF (UniProt Q93K97) proteins. At physiological pH values of ~7.0, ACT activates by >3.5-fold. Initial-rate characterization at pH 9.0 of the forward direction un-activated and ACT-activated reactions show for both cases competitive inhibition by the product succinic semialdehyde versus GHB, and noncompetitive inhibitions by the three other substrate-product combinations. This pattern is consistent with NAD+ binding first in Mono-Iso Theorell-Chance kinetics. Mutants of some possibly important residues in GHBDH also were characterized. H265, conserved among all Group III ADHs and previously proposed to be a critical general base, is only ~4-fold helpful for GHBDH activity relevant to H265A. The four previously proposed conserved Fe2+ chelators (D193, H197, H261 and H280) each are essential for GHBDH activity. A 2-step explanation for cross-species stimulation by sub-stoichiometric ACT in the forward direction and confirmed lack of ACT stimulation in the reverse direction reaction is proposed.


Assuntos
Proteínas de Bactérias/metabolismo , Hidroxibutirato Desidrogenase/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Domínio Catalítico , Cupriavidus necator/enzimologia , Cupriavidus necator/genética , Hidroxibutirato Desidrogenase/química , Hidroxibutirato Desidrogenase/genética , Cinética , Mutação , NAD/metabolismo , Pirofosfatases/metabolismo
18.
BMC Res Notes ; 13(1): 16, 2020 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-31910901

RESUMO

OBJECTIVE: The nuclear and mitochondrial genomes of Dictyostelium discoideum, a unicellular eukaryote, have relatively high A+T-contents of 77.5% and 72.65%, respectively. To begin to investigate how the pyrimidine biosynthetic pathway fulfills the demand for dTTP, we determined the catalytic properties and structure of the key enzyme deoxyuridine triphosphate nucleotidohydrolase (dUTPase) that hydrolyzes dUTP to dUMP, the precursor of dTTP. RESULTS: The annotated genome of D. discoideum identifies a gene encoding a polypeptide containing the five conserved motifs of homotrimeric dUTPases. Recombinant proteins, comprised of either full-length or core polypeptides with all conserved motifs but lacking residues 1-37 of the N-terminus, were active dUTPases. Crystallographic analyses of the core enzyme indicated that the C-termini, normally flexible, were constrained by interactions with the shortened N-termini that arose from the loss of residues 1-37. This allowed greater access of dUTP to active sites, resulting in enhanced catalytic parameters. A tagged protein comprised of the N-terminal forty amino acids of dUTPase fused to green fluorescent protein (GFP) was expressed in D. discoideum cells. Supporting a prediction of mitochondrial targeting information within the N-terminus, localization and subcellular fractionation studies showed GFP to be in mitochondria. N-terminal sequencing of immunoprecipitated GFP revealed the loss of the dUTPase sequence upon import into the organelle.


Assuntos
Dictyostelium/enzimologia , Mitocôndrias/enzimologia , Pirofosfatases/química , Pirofosfatases/metabolismo , Sequência de Aminoácidos , Cinética , Família Multigênica , Multimerização Proteica , Transporte Proteico , Pirofosfatases/genética , Relação Estrutura-Atividade
19.
Anal Chim Acta ; 1097: 222-229, 2020 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-31910963

RESUMO

A long-term limitation of low color resolution in the monocolorimetric sensors seriously restricts its extending application, especially for the traditional organic dyes-based monocolorimetric sensors. Herein, we explore a simple target-mediated surface chemistry-based modulation for regulating the surface of gold nanorods (AuNRs), resulting in a successful multicolorimetric sensor for inorganic pyrophosphatase (PPase) activity. It operates on the principle that PPase modulates the release of Fe3+ free from the ferric-pyrophosphate complex (Fe3+-PPi) through a specific target-catalyzed hydrolysis process, leading to a better mediation for the unique chemical redox reaction between Fe3+ and I-. Subsequently, I- is oxidized into I2, which as a moderate oxidant further regulates the surface of AuNRs due to the rapidly etching reaction between I2 and AuNRs. As a result, the resultant AuNRs surface change further leads to a blue-shift longitudinal LSPR effect of AuNRs, along with a naked-eye-detectable rainbow-like multicolor signal readout. Under optimal conditions, the proposed multicolorimetric sensor exhibits an affordable sensing performance for PPase activity in the range from 1.5 U L-1 to 9 U L-1 with a detection limit of 0.8 U L-1 (S/N = 3). To this point, our strategy not only provides a promising way for breaking the low color resolution limitation of the traditional monocolorimetric sensors, but also broadens the applicability of AuNRs etching-based multicolorimetric sensors.


Assuntos
Cor , Colorimetria , Ouro/química , Nanotubos/química , Ouro/metabolismo , Tamanho da Partícula , Pirofosfatases/química , Pirofosfatases/metabolismo , Propriedades de Superfície
20.
Cancer Res ; 80(5): 999-1010, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31911551

RESUMO

53BP1 controls two downstream subpathways, one mediated by PTIP and Artemis and the other by RIF1 and MAD2L2/Shieldin, to coordinate DNA repair pathway choices. However, the upstream regulator(s) of 53BP1 function in DNA repair remain unknown. We and others recently reported that TIRR associates with 53BP1 to stabilize it and prevents 53BP1 localization to DNA damage sites by blocking 53BP1 Tudor domain binding to H4K20me2 sites. Here, we report that the Nudix hydrolase NUDT16, a TIRR homolog, regulates 53BP1 stability. We identified a novel posttranslational modification of 53BP1 by ADP-ribosylation that is targeted by a PAR-binding E3 ubiquitin ligase, RNF146, leading to 53BP1 polyubiquitination and degradation. In response to DNA damage, ADP-ribosylated 53BP1 increased significantly, resulting in its ubiquitination and degradation. These data suggest that NUDT16 plays a major role in controlling 53BP1 levels under both normal growth conditions and during DNA damage. Notably, overexpression of a NUDT16 catalytically inactive mutant blocked 53BP1 localization to double-strand breaks because (i) the mutant binding to TIRR increased after IR; (ii) the mutant enhanced 53BP1 Tudor domain binding to TIRR, and (iii) the mutant impaired the interaction of 53BP1 Tudor domain with H4K20me2. Moreover, NUDT16's catalytic hydrolase activity was required for 53BP1 de-ADP-ribosylation, 53BP1 protein stability, and its function in cell survival. In summary, we demonstrate that NUDT16 regulates 53BP1 stability and 53BP1 recruitment at double-strand breaks, providing yet another mechanism of 53BP1 regulation.Significance: This study provides a novel mechanism of 53BP1 regulation by demonstrating that NUDT16 has hydrolase activities that remove ADP-ribosylation of 53BP1 to regulate 53BP1 stability and 53BP1 localization at DSBs.


Assuntos
ADP-Ribosilação , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Pirofosfatases/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Células HEK293 , Histonas/metabolismo , Humanos , Mutação , Ligação Proteica , Estabilidade Proteica , Pirofosfatases/genética , Ubiquitina-Proteína Ligases/metabolismo
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