RESUMO
Selenophosphate synthetases use selenium and ATP to synthesize selenophosphate. This is required for biological utilization of selenium, most notably for the synthesis of the non-canonical amino acid selenocysteine (Sec). Therefore, selenophosphate synthetases underlie all functions of selenoproteins, which include redox homeostasis, protein quality control, hormone regulation, metabolism, and many others. This protein family comprises two groups, SelD/SPS2 and SPS1. The SelD/SPS2 group represent true selenophosphate synthetases, enzymes central to selenium metabolism which are present in all Sec-utilizing organisms across the tree of life. Notably, many SelD/SPS2 proteins contain Sec as catalytic residue in their N-terminal flexible selenium-binding loop, while others replace it with cysteine (Cys). The SPS1 group comprises proteins originated through gene duplications of SelD/SPS2 in metazoa in which the Sec/Cys-dependent catalysis was disrupted. SPS1 proteins do not synthesize selenophosphate and are not required for Sec synthesis. They have essential regulatory functions related to redox homeostasis and pyridoxal phosphate, which affect signaling pathways for growth and differentiation. In this review, we summarize the knowledge about the selenophosphate synthetase family acquired through decades of research, encompassing their structure, mechanism, function, and evolution.
Assuntos
Selênio , Selenocisteína , Trifosfato de Adenosina/metabolismo , Cisteína , Hormônios , Ligases , Fosfatos , Fosfotransferases/genética , Fosfato de Piridoxal , Selênio/metabolismo , Compostos de Selênio , Selenocisteína/metabolismo , Selenoproteínas/metabolismoRESUMO
Enzymes with hydroxymethylpyrimidine/phosphomethylpyrimidine kinase activity (HMPPK) are essential in the vitamin B1 (thiamine pyrophosphate) biosynthesis and recycling pathways. In contrast, enzymes with pyridoxal kinase activity (PLK) produce pyridoxal phosphate (vitamin B6), an essential cofactor for various biochemical reactions. In the ATP-dependent vitamin kinases family, the members of PLK/HMPPK-like subfamily have both enzymatic activities. It has been proposed that the promiscuous PLK activity of ancestral HMPPK enzymes could have been the starting point for this activity. In earlier work, we reconstructed the ancestral sequences of this family and characterized the substrate specificity of the common ancestor between PLK/HMPPK-like and HMPPK enzymes (AncC). From these studies, the Gln45Met mutation was proposed as a critical event for the PLK activity emergence. Here, we crystallize and determine the AncC structure by X-ray crystallography and assess the role of the Gln45Met mutation by site-directed mutagenesis. Kinetic characterization of this mutant shows a significant increase in the PL affinity. Through molecular dynamics simulation and MM/PBSA calculations some residues, important for substrate interactions and catalysis, were identified in the wild type and in the mutated ancestor. Interestingly, a strong epistatic interaction responsible for the evolutionary pathway of the PLK activity in PLK/HMPPK-like enzymes was revealed. Also, other putative mutations relevant to PLK activity in modern PLK/HMPPK-like enzymes were identified.
Assuntos
Proteínas de Bactérias/química , Evolução Molecular , Simulação de Dinâmica Molecular , Fosfotransferases/química , Proteínas de Bactérias/genética , Cristalografia por Raios X , Fosfotransferases/genéticaRESUMO
PURPOSE: Expression microarrays are powerful technology that allows large-scale analysis of RNA profiles in a tissue; these platforms include underexploited detection scores outputs. We developed an algorithm using the detection score, to generate a detection profile of shared elements in retinoblastoma as well as to determine its transcriptomic size and structure. METHODS: We analyzed eight briefly cultured primary retinoblastomas with the Human transcriptome array 2.0 (HTA2.0). Transcripts and genes detection scores were determined using the Detection Above Background algorithm (DABG). We used unsupervised and supervised computational tools to analyze detected and undetected elements; WebGestalt was used to explore functions encoded by genes in relevant clusters and performed experimental validation. RESULTS: We found a core cluster with 7,513 genes detected and shared by all samples, 4,321 genes in a cluster that was commonly absent, and 7,681 genes variably detected across the samples accounting for tumor heterogeneity. Relevant pathways identified in the core cluster relate to cell cycle, RNA transport, and DNA replication. We performed a kinome analysis of the core cluster and found 4 potential therapeutic kinase targets. Through analysis of the variably detected genes, we discovered 123 differentially expressed transcripts between bilateral and unilateral cases. CONCLUSIONS: This novel analytical approach allowed determining the retinoblastoma transcriptomic size, a shared active transcriptomic core among the samples, potential therapeutic target kinases shared by all samples, transcripts related to inter tumor heterogeneity, and to determine transcriptomic profiles without the need of control tissues. This approach is useful to analyze other cancer or tissue types.
Assuntos
Neoplasias da Retina/genética , Retinoblastoma/genética , Algoritmos , Pré-Escolar , Éxons , Feminino , Perfilação da Expressão Gênica , Genes do Retinoblastoma , Genoma Humano , Humanos , Lactente , Masculino , Família Multigênica , Fosfotransferases/genética , Fosfotransferases/metabolismo , Neoplasias da Retina/enzimologia , Retinoblastoma/enzimologia , Transcriptoma , Células Tumorais CultivadasRESUMO
This work reports the molecular cloning and heterologous expression of the genes coding for α and ß subunits of pyrophosphate-dependent phosphofructokinase (PPi-PFK) from orange. When expressed individually, both recombinant subunits were produced as highly purified monomeric proteins able to phosphorylate fructose-6-phosphate at the expenses of PPi (specific activity of 0.075 and 0.017 units. mg-1 for α and ß subunits, respectively). On the other hand, co-expression rendered a α3ß3 hexamer with specific activity three orders of magnitude higher than the single subunits. All the conformations of the enzyme were characterized with respect to its kinetic properties and sensitivity to the regulator fructose-2,6-bisphosphate. A thorough review of current knowledge on the matter indicates that this is the first report of the recombinant production of active plant PPi-PFK and the characterization of its different conformations. This is a main contribution for future studies focused to better understand the enzyme properties and how it accomplishes its relevant role in plant metabolism.
Assuntos
Citrus sinensis/enzimologia , Fosfofrutoquinases/metabolismo , Fosfotransferases/metabolismo , Citrus sinensis/genética , Clonagem Molecular , Difosfatos/metabolismo , Frutosedifosfatos/metabolismo , Frutosefosfatos/metabolismo , Expressão Gênica , Cinética , Complexos Multiproteicos , Fosfofrutoquinases/genética , Fosforilação , Fosfotransferases/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas RecombinantesRESUMO
INTRODUCTION: Human cytomegalovirus is one of the causes of opportunist infections in immunocompromised patients, and is triggered by factors such as state of viral latency, weakened immune responses, and development of antiviral resistance to ganciclovir, the only drug offered by the public health system in Brazil to treat the infection. The goal of this study was to identify mutations that may be associated with antiviral resistance in immunocompromised patients. METHODS: Molecular analysis was performed in 82 blood samples and subjected to genomic DNA extraction by a silica-based method. Three sequences of the HCMV UL97 gene, which encodes a phosphotransferase protein required for activation of ganciclovir, were amplified by polymerase chain reaction. Pyrosequencing methods were applied to one external 2096-bp segment DNA and two internal sequences between nucleotides 1087 to 1828 to detect mutations in this gene. RESULTS: Approximately 10% of sequences contained mutations between nucleotides 377 and 594, in conserved regions of the UL97 gene, leading to amino acid changes. Eleven coding mutations were identified, including changes leading to amino acid substitutions, E596K and S604F, which were observed in 100% of samples and are described for the first time in Brazil. In addition, one mutation (A594V) that is associated with ganciclovir resistance was detected in a kidney transplant patient. CONCLUSIONS: Further studies to detect mutations associated with HCMV resistance to antiviral drugs are required to demonstrate the need to increase the variety and availability of drugs used to treat viral infections in the public health care system in Brazil.
Assuntos
Antivirais/uso terapêutico , Infecções por Citomegalovirus/tratamento farmacológico , Citomegalovirus/enzimologia , Farmacorresistência Viral/genética , Hospedeiro Imunocomprometido , Mutação/genética , Fosfotransferases/genética , Antivirais/farmacologia , Estudos de Casos e Controles , Estudos Transversais , Citomegalovirus/efeitos dos fármacos , Citomegalovirus/genética , Farmacorresistência Viral/efeitos dos fármacos , Genótipo , Humanos , Reação em Cadeia da PolimeraseRESUMO
Abstract INTRODUCTION: Human cytomegalovirus is one of the causes of opportunist infections in immunocompromised patients, and is triggered by factors such as state of viral latency, weakened immune responses, and development of antiviral resistance to ganciclovir, the only drug offered by the public health system in Brazil to treat the infection. The goal of this study was to identify mutations that may be associated with antiviral resistance in immunocompromised patients. METHODS: Molecular analysis was performed in 82 blood samples and subjected to genomic DNA extraction by a silica-based method. Three sequences of the HCMV UL97 gene, which encodes a phosphotransferase protein required for activation of ganciclovir, were amplified by polymerase chain reaction. Pyrosequencing methods were applied to one external 2096-bp segment DNA and two internal sequences between nucleotides 1087 to 1828 to detect mutations in this gene. RESULTS: Approximately 10% of sequences contained mutations between nucleotides 377 and 594, in conserved regions of the UL97 gene, leading to amino acid changes. Eleven coding mutations were identified, including changes leading to amino acid substitutions, E596K and S604F, which were observed in 100% of samples and are described for the first time in Brazil. In addition, one mutation (A594V) that is associated with ganciclovir resistance was detected in a kidney transplant patient. CONCLUSIONS: Further studies to detect mutations associated with HCMV resistance to antiviral drugs are required to demonstrate the need to increase the variety and availability of drugs used to treat viral infections in the public health care system in Brazil.
Assuntos
Humanos , Antivirais/uso terapêutico , Fosfotransferases/genética , Hospedeiro Imunocomprometido , Infecções por Citomegalovirus/tratamento farmacológico , Citomegalovirus/enzimologia , Farmacorresistência Viral/genética , Mutação/genética , Antivirais/farmacologia , Estudos de Casos e Controles , Reação em Cadeia da Polimerase , Estudos Transversais , Citomegalovirus/efeitos dos fármacos , Citomegalovirus/genética , Farmacorresistência Viral/efeitos dos fármacos , GenótipoRESUMO
AIMS: The identification of novel targets to control inflammation in humans is probably the primary challenge that impairs the development of new anti-inflammatory drugs. Therefore, the modulation of intracellular signaling pathways in phagocytes may be an interesting means of achieving this goal. However, this change to signaling can compromise the host's susceptibility to invading pathogens. We investigated whether the antioxidant nitroxide Tempol regulates the activity of kinases associated with the production of oxidants in neutrophils, which affects the fungicidal capability of these cells. MAIN METHODS: The effects of Tempol on PMA- or fMLP-activated neutrophils were examined by oxygen consumption as an index of the oxidative burst, a release of extracellular and total Reactive Oxygen Species (ROS) by chemiluminescence, kinase activities through analysis of ATP consumption during enzyme activities and the dot blot immunoassay and, finally, by neutrophil capacity of killing Candida albicans. KEY FINDINGS: Tempol significantly inhibited the neutrophil oxidative burst in a concentration-dependent manner and decreased oxygen consumption (IC50 = 45 µM) and extracellular/total ROS formation with an increase on the lag period response. In addition, Tempol inhibited neutrophil kinase activities (i.e., a decrease in protein phosphorylation) elicited through different biochemical pathways and consequently impaired the fungicidal activity of these cells. SIGNIFICANCE: Although Tempol has potential anti-inflammatory activity that acts on different intracellular pathways (such as those involving kinases), researchers should be cautious, since this nitroxide down-regulated oxidants production and the fungicidal response of neutrophils.
Assuntos
Candida albicans/fisiologia , Óxidos N-Cíclicos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , NADPH Oxidases/metabolismo , Fagócitos/efeitos dos fármacos , Animais , Óxidos N-Cíclicos/química , Regulação para Baixo/efeitos dos fármacos , Inflamação , Masculino , Camundongos , Estrutura Molecular , Neutrófilos/enzimologia , Consumo de Oxigênio , Fosfotransferases/genética , Fosfotransferases/metabolismo , Marcadores de SpinRESUMO
Ribavirin is a synthetic guanosine analogue with a broad-spectrum of antiviral activity. It is clinically effective against several viruses, such as respiratory syncytial virus, several hemorrhagic fever viruses and HCV when combined with pegylated interferon-α. Phosphopentomutase (PPM) catalyzes the transfer of intramolecular phosphate (from C1 to C5) on ribose, and is involved in pentose phosphate pathway and in purine metabolism. Reactions catalyzed by this enzyme are useful for nucleoside analogues production. However, out of its natural environment PPM is unstable and its stability is affected by parameters such as pH and temperature. Therefore, to irreversibly immobilize this enzyme, it needs to be stabilized. In this work, PPM from Escherichia coli ATCC 4157 was overexpressed, purified, stabilized at alkaline pH and immobilized on several supports. The activity of different additives as stabilizing agents was evaluated, and the best result was found using 10% (v/v) glycerol. Under this condition, PPM maintained 86% of its initial activity at pH 10 after 18h incubation, which allowed further covalent immobilization of this enzyme on glyoxyl-agarose with a high yield. This is the first time that PPM has been immobilized by multipoint covalent attachment on glyoxyl support, this derivative being able to biosynthesize ribavirin from α-d-ribose-5-phosphate.
Assuntos
Antivirais/metabolismo , Enzimas Imobilizadas/metabolismo , Proteínas de Escherichia coli/metabolismo , Fosfotransferases/metabolismo , Ribavirina/metabolismo , Estabilidade Enzimática , Enzimas Imobilizadas/química , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/isolamento & purificação , Excipientes , Concentração de Íons de Hidrogênio , Modelos Moleculares , Fosfotransferases/química , Fosfotransferases/genética , Fosfotransferases/isolamento & purificação , TemperaturaRESUMO
KEY MESSAGE: The role of the tomato receptor-like kinase SlSOBIR1 in antiviral defense was investigated. SlSOBIR1 was transcriptionally modulated by unrelated viruses but its ectopic expression had no effect on virus accumulation. Leucine-rich repeat receptor-like kinases (LRR-RLK) constitute a diverse group of proteins allowing the cell to recognize and respond to the extracellular environment. In the present study we focused on a gene encoding a tomato LRR-RLK (named SlSOBIR1) involved in the host defense against fungal pathogens. Curiously, SlSOBIR1 has been previously reported to be down-regulated by Pepper yellow mosaic virus (PepYMV) infection. Here, we show that SlSOBIR1 is responsive to wounding and differentially modulated by unrelated virus infection, i.e., down-regulated by PepYMV and up-regulated by Tomato chlorotic spot virus (TCSV). Despite these divergent expression profiles, SlSOBIR1 overexpression in transgenic tobacco plants had no evident effect on TCSV and PepYMV accumulation. On the other hand, overexpression of SlSOBIR1 significantly increased the expression of selected defense genes (PR-1a and PR-6) and exacerbated superoxide production in wounded leaves. Our data indicate that the observed modulation of SlSOBIR1 expression is probably triggered by secondary effects of the virus infection process and suggest that SlSOBIR1 is not directly involved in antiviral signaling response.
Assuntos
Regulação da Expressão Gênica de Plantas , Interações Hospedeiro-Patógeno , Nicotiana/enzimologia , Fosfotransferases/metabolismo , Doenças das Plantas/virologia , Solanum lycopersicum/enzimologia , Sequência de Aminoácidos , Expressão Gênica , Solanum lycopersicum/genética , Fosfotransferases/genética , Imunidade Vegetal , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/imunologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Potyvirus/fisiologia , Nicotiana/genética , Nicotiana/imunologia , Tospovirus/fisiologiaRESUMO
BACKGROUND: During the last two decades many efforts have been directed towards obtaining efficient microbial processes for the production of shikimic acid (SA); however, feeding high amounts of substrate to increase the titer of this compound has invariably rendered low conversion yields, leaving room for improvement of the producing strains. In this work we report an alternative platform to overproduce SA in a laboratory-evolved Escherichia coli strain, based on plasmid-driven constitutive expression of six genes selected from the pentose phosphate and aromatic amino acid pathways, artificially arranged as an operon. Production strains also carried inactivated genes coding for phosphotransferase system components (ptsHIcrr), shikimate kinases I and II (aroK and aroL), pyruvate kinase I (pykF) and the lactose operon repressor (lacI). RESULTS: The strong and constitutive expression of the constructed operon permitted SA production from the beginning of the cultures, as evidenced in 1 L batch-mode fermentors starting with high concentrations of glucose and yeast extract. Inactivation of the pykF gene improved SA production under the evaluated conditions by increasing the titer, yield and productivity of this metabolite compared to the isogenic pykF+ strain. The best producing strain accumulated up to 43 g/L of SA in 30 h and relatively low concentrations of acetate and aromatic byproducts were detected, with SA accounting for 80% of the produced aromatic compounds. These results were consistent with high expression levels of the glycolytic pathway and synthetic operon genes from the beginning of fermentations, as revealed by transcriptomic analysis. Despite the consumption of 100 g/L of glucose, the yields on glucose of SA and of total aromatic compounds were about 50% and 60% of the theoretical maximum, respectively. The obtained yields and specific production and consumption rates proved to be constant with three different substrate concentrations. CONCLUSIONS: The developed production system allowed continuous SA accumulation until glucose exhaustion and eliminated the requirement for culture inducers. The obtained SA titers and yields represent the highest reported values for a high-substrate batch process, postulating the strategy described in this report as an interesting alternative to the traditionally employed fed-batch processes for SA production.