RESUMO
Toxoplasma gondii is a protozoan parasite that is widely parasitic in the nucleated cells of warm-blooded animals. Bioinformatic analysis of alkyl hydroperoxide reductase 1 (AHP1) of T. gondii is a member of the Prxs family and exhibits peroxidase activity. Cys166 was certified to be a key enzyme active site of TgAHP1, indicating that the enzyme follows a cysteine-dependent redox process. TgAHP1 was present in a punctate staining pattern anterior to the T. gondii nucleus. Oxidative stress experiments showed that the ∆Ahp1 strain was more sensitive to tert-butyl hydroperoxide (tBOOH) than hydrogen peroxide (H2O2), indicating that tBOOH may be a sensitive substrate for TgAHP1. Under tBOOH culture conditions, the ∆Ahp1 strain was significantly less invasive, proliferative, and pathogenic in mice. This was mainly due to the induction of tBOOH, which increased the level of reactive oxygen species in the parasites and eventually led to apoptosis. This study shows that TgAHP1 is a peroxisomes protein with cysteine-dependent peroxidase activity and sensitive to tBOOH.
Assuntos
Peróxido de Hidrogênio/metabolismo , Peroxirredoxinas/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/enzimologia , terc-Butil Hidroperóxido/metabolismo , Animais , Feminino , Edição de Genes , Peróxido de Hidrogênio/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Estresse Oxidativo/efeitos dos fármacos , Peroxirredoxinas/classificação , Peroxirredoxinas/genética , Filogenia , Proteínas de Protozoários/classificação , Proteínas de Protozoários/genética , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Toxoplasma/patogenicidade , Toxoplasmose Animal/parasitologia , Toxoplasmose Animal/patologia , terc-Butil Hidroperóxido/farmacologiaRESUMO
Antioxidant proteins (AOPs) play important roles in the management and prevention of several human diseases due to their ability to neutralize excess free radicals. However, the identification of AOPs by using wet-lab experimental techniques is often time-consuming and expensive. In this study, we proposed an accurate computational model, called AOP-HMM, to predict AOPs by extracting discriminatory evolutionary features from hidden Markov model (HMM) profiles. First, auto cross-covariance (ACC) variables were applied to transform the HMM profiles into fixed-length feature vectors. Then, we performed the analysis of variance (ANOVA) method to reduce the dimensionality of the raw feature space. Finally, a support vector machine (SVM) classifier was adopted to conduct the prediction of AOPs. To comprehensively evaluate the performance of the proposed AOP-HMM model, the 10-fold cross-validation (CV), the jackknife CV, and the independent test were carried out on two widely used benchmark datasets. The experimental results demonstrated that AOP-HMM outperformed most of the existing methods and could be used to quickly annotate AOPs and guide the experimental process.
Assuntos
Antioxidantes/química , Aprendizado de Máquina , Peroxirredoxinas/química , Proteínas/química , Algoritmos , Aminoácidos/análise , Antioxidantes/classificação , Biologia Computacional , Bases de Dados de Proteínas/estatística & dados numéricos , Evolução Molecular , Humanos , Cadeias de Markov , Peroxirredoxinas/classificação , Proteínas/classificaçãoRESUMO
In the present study we describe the molecular characterization of the two paralogous mitochondrial peroxiredoxins from Trematomus bernacchii, a teleost that plays a pivotal role in the Antarctic food chain. The two putative amino acid sequences were compared with orthologs from other fish, highlighting a high percentage of identity and similarity with the respective variant, in particular for the residues that are essential for the characteristic peroxidase activity of these enzymes. The temporal expression of Prdx3 and Prdx5 mRNAs in response to short-term thermal stress showed a general upregulation of prdx3, suggesting that this isoform is the most affected by temperature increase. These data, together with the peculiar differences between the molecular structures of the two mitochondrial Prdxs in T. bernacchii as well as in the tropical species Stegastes partitus, suggest an adaptation that allowed these poikilothermic aquatic vertebrates to colonize very different environments, characterized by different temperature ranges.
Assuntos
Mitocôndrias/enzimologia , Perciformes/metabolismo , Peroxirredoxinas , Sequência de Aminoácidos , Animais , Regiões Antárticas , Proteínas de Peixes/classificação , Proteínas de Peixes/metabolismo , Expressão Gênica , Aquecimento Global , Peroxirredoxinas/classificação , Peroxirredoxinas/metabolismo , Filogenia , Isoformas de Proteínas , TemperaturaRESUMO
Peroxiredoxins are antioxidant enzymes that use redox active Cys residues to reduce H2O2 and various organic hydroperoxides to less reactive products, and thereby protect cells against oxidative stress. In yeasts and mammals, the Prx1 proteins are sensitive to hyperoxidation and consequent loss of their peroxidase activity whereas in most bacteria they are not. In this paper we report the characterization of the Prx1 family in the non-parasitic protist Tetrahymena thermophila. In this organism, four genes potentially encoding Prx1 have been identified. In particular, we show that the mitochondrial Prx1 protein (Prx1m) from T. thermophila is relatively robust to hyperoxidation. This is surprising given that T. thermophila is a eukaryote like yeasts and mammals. In addition, the proliferation of the T. thermophila cells was relatively robust to inhibition by H2O2, cumene hydroperoxide and plant natural products that are known to promote the production of H2O2. In the presence of these agents, the abundance of the T. thermophila Prx1m protein was shown to increase. This suggested that the Prx1m protein may be protecting the cells against oxidative stress. There was no evidence for any increase in Prx1m gene expression in the stressed cells. Thus, increasing protein stability rather than increasing gene expression may explain the increasing Prx1m protein abundance we observed.
Assuntos
Peroxirredoxinas/metabolismo , Proteínas de Protozoários/metabolismo , Tetrahymena thermophila/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Antioxidantes/metabolismo , Derivados de Benzeno/metabolismo , Derivados de Benzeno/farmacologia , Produtos Biológicos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Peroxirredoxinas/classificação , Peroxirredoxinas/genética , Filogenia , Proteínas de Protozoários/classificação , Proteínas de Protozoários/genética , Alinhamento de Sequência , Tetrahymena thermophila/genética , Tetrahymena thermophila/crescimento & desenvolvimentoRESUMO
The characteristics of parasitic infections are often tied to host behavior. Although most studies have investigated definitive hosts, intermediate hosts can also play a role in shaping the distribution and accumulation of parasites. This is particularly relevant in larval stages, where intermediate host's behavior could potentially interfere in the molecules secreted by the parasite into the next host during infection. To investigate this hypothesis, we used a proteomic approach to analyze excretion/secretion products (ESP) from Fasciola hepatica newly excysted juveniles (NEJ) derived from two intermediate host species, Lymnaea viatrix and Pseudosuccinea columella. The two analyzed proteomes showed differences in identity, abundance, and functional classification of the proteins. This observation could be due to differences in the biological cycle of the parasite in the host, environmental aspects, and/or host-dependent factors. Categories such as protein modification machinery, protease inhibitors, signal transduction, and cysteine-rich proteins showed different abundance between samples. More specifically, differences in abundance of individual proteins such as peptidyl-prolyl cis-trans isomerase, thioredoxin, cathepsin B, cathepsin L, and Kunitz-type inhibitors were identified. Based on the differences identified between NEJ ESP samples, we can conclude that the intermediate host is a factor influencing the proteomic profile of ESP in F. hepatica.
Assuntos
Fasciola hepatica/metabolismo , Proteínas de Helminto/metabolismo , Lymnaea/parasitologia , Proteômica , Caramujos/parasitologia , Animais , Anidrases Carbônicas/classificação , Anidrases Carbônicas/metabolismo , Proteínas de Helminto/classificação , Larva/metabolismo , Peptídeo Hidrolases/classificação , Peptídeo Hidrolases/metabolismo , Peroxirredoxinas/classificação , Peroxirredoxinas/metabolismo , Inibidores de Proteases/classificação , Inibidores de Proteases/metabolismo , Receptores de Superfície Celular/classificação , Receptores de Superfície Celular/metabolismoRESUMO
Peroxiredoxins (PRXs) are a family of antioxidant enzymes present in all domains of life. To date, the diversity and function of peroxiredoxins within animals have only been studied in a few model species. Thus, we sought to characterize peroxiredoxin diversity in cnidarians and to gain insight into their function in one cnidarian-the sea anemone Nematostella vectensis. Phylogenetic analysis using all six known PRX subfamilies (PRX1-4, PRX5, PRX6, PRXQ/AHPE1, TPX, BCP-PRXQ) revealed that like bilaterians, cnidarians contain representatives from three subfamilies (PRX1-4, PRX5, PRX6). Within the PRX1-4 subfamily, cnidarian sequences fall into two clades: PRX4, and a cnidarian-specific clade, which we term CNID-PRX. This phylogenetic analysis demonstrates that the three PRX subfamilies present in Bilateria were also present in the last common ancestor of the Cnidaria and Bilateria, and further that diversification of the PRX1-4 subfamily has occurred within the cnidarian lineage. We next examined the impact of decreased salinity, increased temperature, and peroxide exposure on the expression of four prx genes in N. vectensis (cnid-prx, prx4, prx5, and prx6). These genes exhibited unique expression patterns in response to these environmental stressors. Expression of prx4 decreased with initial exposure to elevated temperature, cnid-prx increased with exposure to elevated temperatures as well as with hydrogen peroxide exposure, and expression of all prxs transiently decreased with reduced salinity. Predicted subcellular localization patterns also varied among PRX proteins. Together these results provide evidence that peroxiredoxins in N. vectensis serve distinct physiological roles and lay a groundwork for understanding how peroxiredoxins mediate cnidarian developmental processes and environmental responses.
Assuntos
Estuários , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Peroxirredoxinas/classificação , Filogenia , Anêmonas-do-Mar/enzimologia , Estresse Fisiológico/genética , Animais , Antioxidantes/metabolismo , Evolução Molecular , Peróxido de Hidrogênio/metabolismo , Estresse Oxidativo , Peroxirredoxinas/química , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Conformação Proteica , Espécies Reativas de Oxigênio/metabolismo , Estresse Salino , Anêmonas-do-Mar/genética , Anêmonas-do-Mar/metabolismo , Anêmonas-do-Mar/fisiologia , Frações Subcelulares/enzimologia , TemperaturaRESUMO
The peroxiredoxins (Prxs) define a novel and evolutionarily conserved superfamily of peroxidases able to protect cells from oxidative damage by catalyzing the reduction of a wide range of cellular peroxides. Prxs have been identified in prokaryotes as well as in eukaryotes, however, the composition and number of Prxs family members vary in different species. In this study, six Prxs were firstly identified from the mud crab Scylla paramamosain by RT-PCR and RACE methods. Six SpPrxs can be subdivided into three classes: (a) three typical 2-Cys enzymes denominated as Prx1/2, 3, 4, (b) two atypical 2-Cys enzymes known as Prx5-1 and Prx5-2, and (c) a 1-Cys isoform named Prx6. The evolutionarily conserved signatures of peroxiredoxin catalytic center were identified in all six SpPrxs. Phylogenetic analysis revealed that SpPrx3, SpPrx4, SpPrx5s and SpPrx6 were clearly classified into Prx3-6 subclasses, respectively. Although SpPrx1/2 could not be grouped into any known Prx subclasses, SpPrx1/2 clustered together with other arthropods Prx1 or unclassified Prx and could be classified into the typical 2-Cys class. The comparative and evolutionary analysis of the Prx gene family in invertebrates and vertebrates were also conducted for the first time. Tissue-specific expression analysis revealed that these six SpPrxs were expressed in different transcription patterns while the highest expression levels were almost all in the hepatopancreas. Quantitative RT-PCR analysis exhibited that the gene expression profiles of six SpPrxs were distinct when crabs suffered biotic and abiotic stresses including the exposures of Vibrio alginolyticus, poly (I:C), cadmium and hypoosmotic salinity, suggesting that the SpPrxs might play different roles in response to various stresses. The recombinant proteins including the SpPrx1/2, SpPrx4, SpPrx5-1 and SpPrx6 were purified and the peroxidase activity assays indicated that all these proteins can reduce H2O2 in a typical DTT-dependent manner. To our knowledge, this is the first study about the comprehensive characterization of Prx gene family in Scylla paramamosain and even in crustaceans. These results would broaden the current knowledge of the whole Prx family as well as be helpful to understand and clarify the evolutionary pattern of Prx family in invertebrate and vertebrate taxa.
Assuntos
Braquiúros/genética , Família Multigênica , Peroxirredoxinas/genética , RNA Mensageiro/genética , Estresse Fisiológico/genética , Sequência de Aminoácidos , Animais , Braquiúros/microbiologia , Cloreto de Cádmio/farmacologia , Regulação da Expressão Gênica , Peróxido de Hidrogênio/metabolismo , Invertebrados/genética , Especificidade de Órgãos , Peroxirredoxinas/classificação , Peroxirredoxinas/isolamento & purificação , Peroxirredoxinas/metabolismo , Filogenia , Poli I-C/farmacologia , Isoformas de Proteínas/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/isolamento & purificação , Distribuição Aleatória , Proteínas Recombinantes/metabolismo , Salinidade , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Vertebrados/genética , Vibrio alginolyticusRESUMO
Peroxiredoxins (Prxs or Prdxs) are a large protein superfamily of antioxidant enzymes that rapidly detoxify damaging peroxides and/or affect signal transduction and, thus, have roles in proliferation, differentiation, and apoptosis. Prx superfamily members are widespread across phylogeny and multiple methods have been developed to classify them. Here we present an updated atlas of the Prx superfamily identified using a novel method called MISST (Multi-level Iterative Sequence Searching Technique). MISST is an iterative search process developed to be both agglomerative, to add sequences containing similar functional site features, and divisive, to split groups when functional site features suggest distinct functionally-relevant clusters. Superfamily members need not be identified initially-MISST begins with a minimal representative set of known structures and searches GenBank iteratively. Further, the method's novelty lies in the manner in which isofunctional groups are selected; rather than use a single or shifting threshold to identify clusters, the groups are deemed isofunctional when they pass a self-identification criterion, such that the group identifies itself and nothing else in a search of GenBank. The method was preliminarily validated on the Prxs, as the Prxs presented challenges of both agglomeration and division. For example, previous sequence analysis clustered the Prx functional families Prx1 and Prx6 into one group. Subsequent expert analysis clearly identified Prx6 as a distinct functionally relevant group. The MISST process distinguishes these two closely related, though functionally distinct, families. Through MISST search iterations, over 38,000 Prx sequences were identified, which the method divided into six isofunctional clusters, consistent with previous expert analysis. The results represent the most complete computational functional analysis of proteins comprising the Prx superfamily. The feasibility of this novel method is demonstrated by the Prx superfamily results, laying the foundation for potential functionally relevant clustering of the universe of protein sequences.
Assuntos
Bases de Dados de Proteínas , Peroxirredoxinas/química , Peroxirredoxinas/classificação , Mapeamento de Interação de Proteínas/métodos , Análise de Sequência de Proteína/métodos , Homologia de Sequência de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Sistemas de Gerenciamento de Base de Dados , Ativação Enzimática , Ensaios de Triagem em Larga Escala/métodos , Dados de Sequência Molecular , Família Multigênica , Peroxirredoxinas/ultraestrutura , Ligação ProteicaRESUMO
As peroxirredoxinas (Prx) são enzimas antioxidantes que se destacam pela capacidade de decompor uma grande variedade de hidroperóxidos com elevada eficiência (106-108M-1s-1), mantendo essas moléculas em níveis adequados à homeostase celular. Entretanto, já foi demonstrado que em diversos tipos tumorais os níveis de Prx são extremamente aumentados e experimentos envolvendo sua inativação resultam na diferenciação ou apoptose de células tumorais. Recentemente, foi descoberto um diterpenóide denominado adenantina que seria o primeiro inibidor para as Prx1 e Prx2 de humanos e foi demonstrada que sua aplicação em células de leucemia mieloide aguda promoveu diferenciação ou apoptose dessas células. Nesse contexto, o presente trabalho apresenta duas vertentes: 1) A caracterização das alterações estruturais e funcionais promovidas pela ligação da adenantina ao sítio ativo das Prx utilizando Tsa1 de Saccharomyces cerevisiae como modelo biológico, em função da sua alta similaridade com Prx2 de humanos; 2) Avaliação da atividade antitumoral dose dependente de adenantina sobre as linhagens celulares REH e MOLT-4 de leucemia linfoide aguda. No que concerne à primeira linha de investigação, nossos resultados revelam que Tsa1 é suscetível à inibição por adenantina, uma vez que o tratamento reduziu em ~66 % a velocidade de decomposição de peróxido de hidrogênio. Adicionalmente, a mutação da Thr44 de Tsa1, pertencente à chamada tríade catalítica, por uma Ser resultou em uma proteína mais suscetível a alterações na estrutura secundária e à inibição da atividade peroxidásica em função da ligação com adenantina, apresentando uma diminuição de ~85% na velocidade de reação. Características semelhantes foram observadas para a proteoforma Tsa2 de S. cerevisiae, que carreia naturalmente a substituição da Thr44 pela Ser. Análises de sequências de Prx em bancos de dados revelaram que majoritariamente proteínas contendo Ser são encontradas em organismos procariotos, muitos deles patogênicos. Finalmente, demonstramos por meio de ensaios citotoxicidade que as bactérias Staphylococcus aureus e Staphylococcus epidermidis, que possuem uma Ser na tríade catalítica, têm seu crescimento inibido pelo tratamento com adenantina (IC50 de 460µM e 77µM, respectivamente), enquanto que para Escherichia coli, que possui Thr nessa posição, a toxicidade da adenantina foi bastante baixa (não foi possível determinar o IC50 nas condições utilizadas). Dessa forma, os dados apresentados neste trabalho demonstram o potencial da utilização da adenantina tanto como antibiótico quanto como antileucêmico
Peroxiredoxins (Prx) are antioxidant enzymes which stand out due the ability to decompose a wide variety of hydroperoxides with high efficiency (106-108M-1s-1) maintaining these molecules at suitable levels to cellular homeostasis and participating in several signaling events. However, it has been shown that, in many tumor types, Prx levels are extremely increased and experiments involving its inactivation have resulted in differentiation or apoptosis of tumor cells. It was recently found a diterpenoid, called adenanthin, that would be the first human Prx1 and Prx2 inhibitor and it was demonstrated that its application in acute myeloid leukemia cells was able to promote differentiation or apoptosis. In this context, this work presents two lines of research: 1) Characterization of structural and functional changes promoted by adenanthin binding to Prx active site using Tsa1 from Saccharomyces cerevisiae as biological model, due to its high similarity to human Prx2. 2) Evaluation of adenanthin dose-dependent antitumor activity over the acute lymphoid leukemia cell lines REH and MOLT-4. As regards the first line of research, our result reveal that Tsa1 is susceptible to inhibition by adenanthin, since the treatment with this binder reduced the hydrogen peroxide decomposition velocity in ~ 66%. In addition, the replacement of Thr44 from Tsa1, aminoacid belonging to the so-called catalytic triad, by a Ser resulted in a protein more susceptible to alterations in secondary structure and to peroxidase activity inhibition in function of adenanthin binding, presenting ~85% of decrease in reaction velocity. Similar characteristics were observed for Tsa2 proteoform from S. cerevisiae, which naturally carries the substitution of Thr44 by Ser. Prx sequences analyzes in databases revealed that mostly Ser-containing proteins are found in prokaryotic organisms, many of them pathogenic ones. Finally, we demonstrate through cytotoxicity assays that the bacteria Staphylococcus aureus and Staphylococcus epidermidis, which have a Ser in catalytic triad, have their growth inhibited by adenanthin treatment (IC50 of 460µM and 77µM, respectively), whereas for Escherichia Coli, which has Thr at that position, the tocyxicity of adenanthin was quite low (it was not possible to determine the IC50 under the used conditions). Regarding the second line of investigation, we found that adenanthin is able to induce the death of leukemic cell lines REH and MOLT-4, and for the last one, there was an unexpected proliferation of cells treated by the longest incubation period (72 hours), characterizing a possible indication of differentiation process. In this sense, the data presented here demonstrate the potential of adenanthin use in both antibiotic and antileukemic treatment
Assuntos
Saccharomyces cerevisiae/metabolismo , Peroxirredoxinas/classificação , Inibidores do Crescimento/análise , Leucemia Mieloide Aguda/classificação , Diterpenos/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/classificação , Antibacterianos/farmacologia , Antioxidantes/farmacologiaRESUMO
Peroxiredoxins are cysteine-dependent peroxide reductases that group into 6 different, structurally discernable classes. In 2011, our research team reported the application of a bioinformatic approach called active site profiling to extract active site-proximal sequence segments from the 29 distinct, structurally-characterized peroxiredoxins available at the time. These extracted sequences were then used to create unique profiles for the six groups which were subsequently used to search GenBank(nr), allowing identification of â¼3500 peroxiredoxin sequences and their respective subgroups. Summarized in this minireview are the features and phylogenetic distributions of each of these peroxiredoxin subgroups; an example is also provided illustrating the use of the web accessible, searchable database known as PREX to identify subfamily-specific peroxiredoxin sequences for the organism Vitis vinifera (grape).
Assuntos
Peroxirredoxinas/química , Peroxirredoxinas/classificação , Vitis/enzimologia , Bases de Dados de Proteínas , Peroxirredoxinas/genética , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/classificação , Proteínas de Plantas/genética , Análise de Sequência de Proteína , Interface Usuário-Computador , Vitis/genéticaRESUMO
The circadian clock is a cellular timekeeping mechanism that helps organisms from bacteria to humans to organize their behaviour and physiology around the solar cycle. Current models for circadian timekeeping incorporate transcriptional/translational feedback loop mechanisms in the predominant model systems. However, recent evidence suggests that non-transcriptional oscillations such as metabolic and redox cycles may play a fundamental role in circadian timekeeping. Peroxiredoxins, an antioxidant protein family, undergo rhythmic oxidation on the circadian time scale in a variety of species, including bacteria, insects and mammals, but also in red blood cells, a naturally occurring, non-transcriptional system. The profound interconnectivity between circadian and redox pathways strongly suggests that a conserved timekeeping mechanism based on redox cycles could be integral to generating circadian rhythms.
Assuntos
Relógios Circadianos/fisiologia , Ritmo Circadiano/fisiologia , Oxirredução , Peroxirredoxinas/fisiologia , Animais , Retroalimentação Fisiológica , Homeostase/fisiologia , Humanos , Peroxirredoxinas/classificação , Transdução de Sinais/fisiologia , Transcrição GênicaRESUMO
Thioredoxin peroxidase (Tpx), also named peroxiredoxin (Prx), is an important peroxidase that can protect organisms against stressful environments. AccTpx4, a 1-Cys thioredoxin peroxidase gene from the Chinese honey bee Apis cerana cerana, was cloned and characterized. The AccTpx4 gene encodes a protein that is predicted to contain the conserved PVCTTE motif from 1-Cys peroxiredoxin. Quantitative real-time PCR (Q-PCR) and Western blotting revealed that AccTpx4 was induced by various oxidative stresses, such as cold, heat, insecticides, H(2)O(2), and HgCl(2). The in vivo peroxidase activity assay showed that recombinant AccTpx4 protein could efficiently degrade H(2)O(2) in the presence of DL-dithiothreitol (DTT). In addition, disc fusion assays revealed that AccTpx4 could function to protect cells against oxidative stresses. These results indicate that AccTpx4 plays an important role in oxidative stress responses and may contribute to the conservation of honeybees.
Assuntos
Abelhas/genética , Proteínas de Insetos/metabolismo , Peroxirredoxinas/metabolismo , Sequência de Aminoácidos , Animais , Clonagem Molecular , Ditiotreitol/química , Peróxido de Hidrogênio/toxicidade , Proteínas de Insetos/classificação , Proteínas de Insetos/genética , Inseticidas/toxicidade , Cloreto de Mercúrio/toxicidade , Dados de Sequência Molecular , Estresse Oxidativo/efeitos dos fármacos , Peroxirredoxinas/classificação , Peroxirredoxinas/genética , Filogenia , Estrutura Terciária de Proteína , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , TemperaturaRESUMO
Peroxiredoxins were not recognized as a family of enzymes until the 1990s but are now known to be the dominant peroxidases in most organisms. Here, the history and fundamental properties of peroxiredoxins are briefly reviewed, with a special focus on describing how an exquisitely tunable balance between fully folded and locally unfolded conformations plays a large role in peroxiredoxin catalytic properties.
Assuntos
Chaperonas Moleculares/metabolismo , Peroxirredoxinas/metabolismo , Compostos de Sulfidrila/química , Animais , Bactérias/enzimologia , Biocatálise , Fungos/enzimologia , História do Século XX , História do Século XXI , Humanos , Cinética , Modelos Moleculares , Chaperonas Moleculares/classificação , Chaperonas Moleculares/genética , Chaperonas Moleculares/história , Família Multigênica , Oxirredução , Peroxirredoxinas/classificação , Peroxirredoxinas/genética , Peroxirredoxinas/história , Conformação Proteica , Dobramento de Proteína , Multimerização Proteica , Compostos de Sulfidrila/metabolismoRESUMO
Peroxiredoxin (Prx) belongs to a peroxidase family of antioxidant enzymes distributed ubiquitously in aerobic organisms such as yeast, fungi, parasites, mammals and humans. It plays an important role in the defense of reactive oxygen species (ROS) and reactive nitrogen species (RNS) produced by aerobic organisms. This article introduces the classification, mechanism of Prx and the progress of research on Prx in invertebrates.
Assuntos
Invertebrados/enzimologia , Peroxirredoxinas/metabolismo , Animais , Oxirredução , Peroxirredoxinas/classificação , Peroxirredoxinas/genética , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Phlebotomine sand flies are the vectors of medically important Leishmania. The Leishmania protozoa reside in the sand fly gut, but the nature of the immune response to the presence of Leishmania is unknown. Reactive oxygen species (ROS) are a major component of insect innate immune pathways regulating gut-microbe homeostasis. Here we show that the concentration of ROS increased in sand fly midguts after they fed on the insect pathogen Serratia marcescens but not after feeding on the Leishmania that uses the sand fly as a vector. Moreover, the Leishmania is sensitive to ROS either by oral administration of ROS to the infected fly or by silencing a gene that expresses a sand fly ROS-scavenging enzyme. Finally, the treatment of sand flies with an exogenous ROS scavenger (uric acid) altered the gut microbial homeostasis, led to an increased commensal gut microbiota, and reduced insect survival after oral infection with S. marcescens. Our study demonstrates a differential response of the sand fly ROS system to gut microbiota, an insect pathogen, and the Leishmania that utilize the sand fly as a vehicle for transmission between mammalian hosts.
Assuntos
Imunidade/imunologia , Leishmania mexicana/imunologia , Psychodidae/imunologia , Espécies Reativas de Oxigênio/imunologia , Serratia marcescens/imunologia , Sequência de Aminoácidos , Animais , Antioxidantes/administração & dosagem , Antioxidantes/farmacologia , Catalase/classificação , Catalase/genética , Catalase/metabolismo , Feminino , Trato Gastrointestinal/metabolismo , Trato Gastrointestinal/microbiologia , Trato Gastrointestinal/parasitologia , Técnicas de Inativação de Genes , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/imunologia , Peróxido de Hidrogênio/metabolismo , Imunidade/efeitos dos fármacos , Proteínas de Insetos/classificação , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Insetos Vetores/imunologia , Insetos Vetores/microbiologia , Insetos Vetores/parasitologia , Leishmania mexicana/fisiologia , Dados de Sequência Molecular , Peroxirredoxinas/classificação , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Filogenia , Psychodidae/enzimologia , Psychodidae/genética , Espécies Reativas de Oxigênio/metabolismo , Homologia de Sequência de Aminoácidos , Serratia marcescens/fisiologia , Superóxido Dismutase/classificação , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Ácido Úrico/administração & dosagem , Ácido Úrico/farmacologiaRESUMO
Thioredoxin peroxidases (Tpxs) play important roles in protecting organisms against the toxicity of reactive oxygen species (ROS) and regulating intracellular signal transduction. In the present study, we cloned the full cDNA of Tpx1 encoding a 195-amino acid protein from Apis cerana cerana (Acc). Based on the genomic DNA sequence, a 1442-bp 5'-flanking region was obtained, and the putative transcription factor binding sites were predicted. Quantitative PCR analysis showed that AccTpx1 was highly expressed in thorax and that the AccTpx1 transcript reached its highest level in two-week-old adult worker honeybees. Moreover, expression of the AccTpx1 transcript was increased by various abiotic stresses, such as ultraviolet light, HgCl(2) , and insecticide treatments. In addition, the recombinant AccTpx1 protein exhibited antioxidant activity; it removed hydrogen peroxide and protected DNA. These results suggest that AccTpx1 plays an important role in protecting honeybees from oxidative injury and may act in extending the lifespan of them.
Assuntos
Abelhas/enzimologia , Abelhas/genética , Peroxirredoxinas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Dados de Sequência Molecular , Peroxirredoxinas/química , Peroxirredoxinas/classificação , Filogenia , Alinhamento de Sequência , Distribuição TecidualRESUMO
Peroxiredoxins (Prxs), some of nature's dominant peroxidases, use a conserved Cys residue to reduce peroxides. They are highly expressed in organisms from all kingdoms, and in eukaryotes they participate in hydrogen peroxide signaling. Seventy-two Prx structures have been determined that cover much of the diversity of the family. We review here the current knowledge and show that Prxs can be effectively classified by a structural/evolutionary organization into six subfamilies followed by specification of a 1-Cys or 2-Cys mechanism, and for 2-Cys Prxs, the structural location of the resolving Cys. We visualize the varied catalytic structural transitions and highlight how they differ depending on the location of the resolving Cys. We also review new insights into the question of how Prxs are such effective catalysts: the enzyme activates not only the conserved Cys thiolate but also the peroxide substrate. Moreover, the hydrogen-bonding network created by the four residues conserved in all Prx active sites stabilizes the transition state of the peroxidatic S(N)2 displacement reaction. Strict conservation of the peroxidatic active site along with the variation in structural transitions provides a fascinating picture of how the diverse Prxs function to break down peroxide substrates rapidly.
Assuntos
Cisteína/química , Cisteína/metabolismo , Peroxirredoxinas/química , Peroxirredoxinas/classificação , Sequência de Aminoácidos , Catálise , Domínio Catalítico , Dissulfetos/química , Evolução Molecular , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/metabolismo , Cinética , Dados de Sequência Molecular , Peróxidos/química , Dobramento de Proteína , Estrutura Quaternária de Proteína , Estrutura Secundária de ProteínaRESUMO
PREX (http://www.csb.wfu.edu/prex/) is a database of currently 3516 peroxiredoxin (Prx or PRDX) protein sequences unambiguously classified into one of six distinct subfamilies. Peroxiredoxins are a diverse and ubiquitous family of highly expressed, cysteine-dependent peroxidases that are important for antioxidant defense and for the regulation of cell signaling pathways in eukaryotes. Subfamily members were identified using the Deacon Active Site Profiler (DASP) bioinformatics tool to focus in on functionally relevant sequence fragments surrounding key residues required for protein activity. Searches of this database can be conducted by protein annotation, accession number, PDB ID, organism name or protein sequence. Output includes the subfamily to which each classified Prx belongs, accession and GI numbers, genus and species and the functional site signature used for classification. The query sequence is also presented aligned with a select group of Prxs for manual evaluation and interpretation by the user. A synopsis of the characteristics of members of each subfamily is also provided along with pertinent references.
Assuntos
Bases de Dados de Proteínas , Peroxirredoxinas/classificação , Peroxirredoxinas/química , Interface Usuário-ComputadorRESUMO
In Fasciola species, peroxiredoxin (Prx) serves as the major antioxidant enzyme to remove hydrogen peroxide that is generated from various metabolic reactions, because the parasites lack catalase, and only express glutathione peroxidases at minimal levels. We have cloned and characterized two genes, FgPrx-1 and FgPrx-2, belonging to the 2-Cys Prx family, by immunoscreening of an expressed adult stage Fasciola gigantica cDNA library using a rabbit anti-serum against its tegumental antigens. Predicted FgPrx-1 and FgPrx-2 consisted of 218 amino acids each with predicted molecular weights at 24.63 kDa and 24.57 kDa, respectively. The two predicted F. gigantica Prx proteins exhibited 98% identity to each other, and 52% identity to Prx from oxen which is the natural host. A phylogenetic analysis revealed that FgPrx-1 and FgPrx-2 appear to be closely related to those of Fasciola hepatica. The nucleotide sequences of FgPrx-2 are 654 bp, which is similar to that cloned from newly excysted juveniles of F. hepatica. The FgPrx genes were found to be constitutively expressed in all developmental stages, and with a similar pattern. In the adult parasite, FgPrx transcripts were located in the gut epithelial cells, tegument cells, and cells of reproductive organs, including prostate gland, vitelline glands, testis and ovary. In 4-week-old juveniles, a similar distribution pattern was observed. Metacercaria and newly excysted juveniles exhibited strongest signals for mRNA transcripts in the gut epithelium, and moderately in the tegumental cells. Because of their key role in protecting the parasite and specificities, these proteins may have immunodiagnostic as well as vaccine potentials.
Assuntos
Fasciola/genética , Peroxirredoxinas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Bovinos , Clonagem Molecular , DNA Complementar/química , DNA Complementar/isolamento & purificação , Fasciola/química , Fasciola/classificação , Regulação Enzimológica da Expressão Gênica/genética , Biblioteca Gênica , Hibridização In Situ , Lymnaea , Camundongos , Camundongos Endogâmicos ICR , Dados de Sequência Molecular , Peso Molecular , Peroxirredoxinas/química , Peroxirredoxinas/classificação , Filogenia , RNA de Helmintos/isolamento & purificação , Coelhos , Alinhamento de SequênciaRESUMO
Peroxiredoxins are ubiquitous proteins that catalyze the reduction of hydroperoxides, thus conferring resistance to oxidative stress. Using high-resolution mass spectrometry, we recently reclassified one such peroxiredoxin, bacterioferritin comigratory protein (BCP) of Escherichia coli, as an atypical 2-Cys peroxiredoxin that functions through the formation of an intramolecular disulfide bond between the active and resolving cysteine. An engineered E. coli BCP, which lacked the resolving cysteine, retained enzyme activity through a novel catalytic pathway. Unlike the active cysteine, the resolving cysteine of BCP peroxiredoxins is not conserved across all members of the family. To clarify the catalytic mechanism of native BCP enzymes that lack the resolving cysteine, we have investigated the BCP homologue of Burkholderia cenocepacia. We demonstrate that the B. cenocepacia BCP (BcBCP) homologue functions through a 1-Cys catalytic pathway. During catalysis, BcBCP can utilize thioredoxin as a reductant for the sulfenic acid intermediate. However, significantly higher peroxidase activity is observed utilizing glutathione as a resolving cysteine and glutaredoxin as a redox partner. Introduction of a resolving cysteine into BcBCP changes the activity from a 1-Cys pathway to an atypical 2-Cys pathway, analogous to the E. coli enzyme. In contrast to the native B. cenocepacia enzyme, thioredoxin is the preferred redox partner for this atypical 2-Cys variant. BCP-deficient B. cenocepacia exhibit a growth-phase-dependent hypersensitivity to oxidative killing. On the basis of sequence alignments, we believe that BcBCP described herein is representative of the major class of bacterial BCP peroxiredoxins. To our knowledge, this is the first detailed characterization of their catalytic activity. These studies support the subdivision of the BCP family of peroxiredoxins into two classes based on their catalytic activity.