RESUMO
Mammalian cells use hydrogen peroxide (H(2)O(2)) not only to kill invading pathogens, but also as a signaling modulator. Woo et al. (2010) now show that the local inactivation of a H(2)O(2)-degrading enzyme ensures that the production of this oxidant is restricted to the signaling site.
Assuntos
Peróxido de Hidrogênio/metabolismo , Transdução de Sinais , Animais , Humanos , Camundongos , Peroxirredoxinas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismoRESUMO
Genome-scale engineering enables rational removal of dispensable genes in chassis genomes. Deviating from this approach, we applied greedy accumulation of deletions of large dispensable regions in the Bacillus subtilis genome, yielding a library of 298 strains with genomes reduced up to 1.48 Mb in size. High-throughput physiological phenotyping of these strains confirmed that genome reduction is associated with substantial loss of cell fitness and accumulation of synthetic-sick interactions. Transcriptome analysis indicated that <15% of the genes conserved in our genome-reduced strains exhibited a twofold or higher differential expression and revealed a thiol-oxidative stress response. Most transcriptional changes can be explained by loss of known functions and by aberrant transcription at deletion boundaries. Genome-reduced strains exhibited striking new phenotypes relative to wild type, including a very high resistance (increased >300-fold) to the DNA-damaging agent mitomycin C and a very low spontaneous mutagenesis (reduced 100-fold). Adaptive laboratory evolution failed to restore cell fitness, except when coupled with a synthetic increase of the mutation rate, confirming low evolvability. Although mechanisms underlying this emergent phenotype are not understood, we propose that low evolvability can be leveraged in an engineering strategy coupling reductive cycles with evolutive cycles under induced mutagenesis.
Assuntos
Bacillus subtilis , Genoma Bacteriano , Genoma Bacteriano/genética , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Fenótipo , Mutagênese , Taxa de MutaçãoRESUMO
Cell migration requires the generation of branched actin networks that power the protrusion of the plasma membrane in lamellipodia. The actin-related proteins 2 and 3 (Arp2/3) complex is the molecular machine that nucleates these branched actin networks. This machine is activated at the leading edge of migrating cells by Wiskott-Aldrich syndrome protein (WASP)-family verprolin-homologous protein (WAVE, also known as SCAR). The WAVE complex is itself directly activated by the small GTPase Rac, which induces lamellipodia. However, how cells regulate the directionality of migration is poorly understood. Here we identify a new protein, Arpin, that inhibits the Arp2/3 complex in vitro, and show that Rac signalling recruits and activates Arpin at the lamellipodial tip, like WAVE. Consistently, after depletion of the inhibitory Arpin, lamellipodia protrude faster and cells migrate faster. A major role of this inhibitory circuit, however, is to control directional persistence of migration. Indeed, Arpin depletion in both mammalian cells and Dictyostelium discoideum amoeba resulted in straighter trajectories, whereas Arpin microinjection in fish keratocytes, one of the most persistent systems of cell migration, induced these cells to turn. The coexistence of the Rac-Arpin-Arp2/3 inhibitory circuit with the Rac-WAVE-Arp2/3 activatory circuit can account for this conserved role of Arpin in steering cell migration.
Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Movimento Celular/genética , Pseudópodes/genética , Pseudópodes/metabolismo , Transdução de Sinais , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Dictyostelium/genética , Dictyostelium/metabolismo , Embrião não Mamífero , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Camundongos , Proteínas/genética , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Peixe-Zebra/genéticaRESUMO
Glutathione contributes to thiol-redox control and to extra-mitochondrial iron-sulphur cluster (ISC) maturation. To determine the physiological importance of these functions and sort out those that account for the GSH requirement for viability, we performed a comprehensive analysis of yeast cells depleted of or containing toxic levels of GSH. Both conditions triggered an intense iron starvation-like response and impaired the activity of extra-mitochondrial ISC enzymes but did not impact thiol-redox maintenance, except for high glutathione levels that altered oxidative protein folding in the endoplasmic reticulum. While iron partially rescued the ISC maturation and growth defects of GSH-depleted cells, genetic experiments indicated that unlike thioredoxin, glutathione could not support by itself the thiol-redox duties of the cell. We propose that glutathione is essential by its requirement in ISC assembly, but only serves as a thioredoxin backup in cytosolic thiol-redox maintenance. Glutathione-high physiological levels are thus meant to insulate its cytosolic function in iron metabolism from variations of its concentration during redox stresses, a model challenging the traditional view of it as prime actor in thiol-redox control.
Assuntos
Glutationa/metabolismo , Ferro/metabolismo , Saccharomyces cerevisiae/metabolismo , Compostos de Sulfidrila/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas Mitocondriais/metabolismo , Oxirredução , Dobramento de Proteína , Processamento de Proteína Pós-TraducionalRESUMO
Synthetic biology and metabolic engineering are used to develop new strategies for producing valuable compounds ranging from therapeutics to biofuels in engineered microorganisms. When developing methods for high-titer production cells, toxicity is an important element to consider. Indeed the production rate can be limited due to toxic intermediates or accumulation of byproducts of the heterologous biosynthetic pathway of interest. Conversely, highly toxic molecules are desired when designing antimicrobials. Compound toxicity in bacteria plays a major role in metabolic engineering as well as in the development of new antibacterial agents. Here, we screened a diversified chemical library of 166 compounds for toxicity in Escherichia coli. The dataset was built using a clustering algorithm maximizing the chemical diversity in the library. The resulting assay data was used to develop a toxicity predictor that we used to assess the toxicity of metabolites throughout the metabolome. This new tool for predicting toxicity can thus be used for fine-tuning heterologous expression and can be integrated in a computational-framework for metabolic pathway design. Many structure-activity relationship tools have been developed for toxicology studies in eukaryotes [Valerio (2009), Toxicol Appl Pharmacol, 241(3): 356-370], however, to the best of our knowledge we present here the first E. coli toxicity prediction web server based on QSAR models (EcoliTox server: http://www.issb.genopole.fr/â¼faulon/EcoliTox.php).
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Escherichia coli/efeitos dos fármacos , Preparações Farmacêuticas/química , Relação Estrutura-Atividade , Biotecnologia/métodos , Internet , Engenharia Metabólica/métodosRESUMO
The Escherichia coli glutaredoxins 1 and 3 (Grx1 and Grx3) are structurally similar (37% sequence identity), yet have different activities in vivo. Unlike Grx3, Grx1 efficiently reduces protein disulfides in proteins such as RR (ribonucleotide reductase), whereas it is poor at reducing S-glutathionylated proteins. An E. coli strain lacking genes encoding thioredoxins 1 and 2 and Grx1 is not viable on either rich or minimal medium; however, a M43V mutation in Grx3 restores growth under these conditions and results in a Grx1-like protein [Ortenberg, Gon, Porat and Beckwith (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 7439-7944]. To uncover the structural basis of this change in activity, we have compared wild-type and mutant Grx3 using CD and NMR spectroscopy. Ligand-induced stability measurements demonstrate that the Grx3(M43V/C65Y) mutant has acquired affinity for RR. Far-UV CD spectra reveal no significant differences, but differences are observed in the near-UV region indicative of tertiary structural changes. NMR (1)H-(15)N HSQC (heteronuclear single quantum coherence) spectra show that approximately half of the 82 residues experience significant (Deltadelta>0.03 p.p.m.) chemical shift deviations in the mutant, including nine residues experiencing extensive (Deltadelta > or =0.15 p.p.m.) deviations. To test whether the M43V mutation alters dynamic properties of Grx3, H/D (hydrogen/deuterium) exchange experiments were performed demonstrating that the rate at which backbone amides exchange protons with the solvent is dramatically enhanced in the mutant, particularly in the core of the protein. These data suggest that the Grx1-like activity of the Grx3(M43V/C65Y) mutant may be explained by enhanced intrinsic motion allowing for increased specificity towards larger substrates such as RR.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Glutarredoxinas/metabolismo , Proteínas Mutantes/metabolismo , Algoritmos , Substituição de Aminoácidos , Dicroísmo Circular , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Glutarredoxinas/química , Glutarredoxinas/genética , Dissulfeto de Glutationa/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Mutação , Oxirredução , Conformação Proteica , Estrutura Terciária de Proteína , TermodinâmicaRESUMO
Thioredoxin-1 from Escherichia coli has frequently been used as a model substrate in protein folding studies. However, for reasons of convenience, these studies have focused largely on oxidized thioredoxin and not on reduced thioredoxin, the more physiologically relevant species. Here we describe the first extensive characterization of the refolding kinetics and conformational thermodynamics of reduced thioredoxin. We have previously described a genetic screen that yielded mutant thioredoxin proteins that fold more slowly in both the oxidized and reduced forms. In this study, we apply our more detailed analysis of reduced thioredoxin folding to a larger number of folding mutants that includes those obtained from continuation of the genetic screen. We have identified mutant proteins that display folding defects specifically in the reduced state but not the oxidized state. Some of these substitutions represent unusual folding mutants in that they result in semiconservative substitutions at solvent-exposed positions in the folded conformation and do not appear to affect the conformational stability of the protein. Further, the genetic selection yields mutants at only a limited number of sites, pointing to perhaps the most critical amino acids in the folding pathway and underscoring, in particular, the role of the carboxy-terminal amino acids in the folding of thioredoxin. Our results demonstrate the importance of studying the physiologically relevant folding species.
Assuntos
Escherichia coli K12/enzimologia , Proteínas de Escherichia coli/química , Dobramento de Proteína , Tiorredoxinas/química , Substituição de Aminoácidos , Escherichia coli K12/genética , Proteínas de Escherichia coli/genética , Cinética , Mutação de Sentido Incorreto , Termodinâmica , Tiorredoxinas/genéticaRESUMO
Bacteria are sophisticated systems with high capacity and flexibility to adapt to various environmental conditions. Each prokaryote however possesses a defined metabolic network, which sets its overall metabolic capacity, and therefore the maximal growth rate that can be reached. To achieve optimal growth, bacteria adopt various molecular strategies to optimally adjust gene expression and optimize resource allocation according to the nutrient availability. The resulting physiological changes are often accompanied by changes in the growth rate, and by global regulation of gene expression. The growth-rate-dependent variation of the abundances in the cellular machineries, together with condition-specific regulatory mechanisms, affect RNA metabolism and fate and pose a challenge for rational gene expression reengineering of synthetic circuits. This article is part of a Special Issue entitled: RNA and gene control in bacteria, edited by Dr. M. Guillier and F. Repoila.
Assuntos
Bactérias/genética , Regulação Bacteriana da Expressão Gênica , RNA Bacteriano/genética , Adaptação Fisiológica , Bactérias/crescimento & desenvolvimento , Bactérias/metabolismo , Guanosina Tetrafosfato/metabolismo , RNA Bacteriano/metabolismoRESUMO
Osteoporosis is caused by excessive activity of bone-degrading osteoclasts over bone-forming osteoblast. Standard antiosteolytic treatments inhibit bone resorption by inducing osteoclast loss, with the adverse effect of hindering also bone formation. Formation of the osteoclast sealing zone requires Dock5, a guanine nucleotide exchange factor for the small GTPase Rac, and C21, a chemical inhibitor of Dock5, decreases bone resorption by cultured osteoclasts. Here we show that C21 directly inhibits the exchange activity of Dock5 and disrupts osteoclast podosome organization. Remarkably, C21 administration protects mice against bone degradation in models recapitulating major osteolytic diseases: menopause, rheumatoid arthritis and bone metastasis. Furthermore, C21 administration does not affect bone formation and is not toxic. Our results validate the pharmacological inhibition of Dock5 as a novel therapeutic route for fighting osteolytic diseases while preserving bone formation.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteogênese/efeitos dos fármacos , Osteólise/tratamento farmacológico , Sulfonamidas/uso terapêutico , Animais , Artrite/induzido quimicamente , Artrite/tratamento farmacológico , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Osteoporose/induzido quimicamente , Osteoporose/tratamento farmacológico , Sulfonamidas/química , Sulfonamidas/farmacologia , BenzenossulfonamidasRESUMO
Metabolic engineering has succeeded in biosynthesis of numerous commodity or high value compounds. However, the choice of pathways and enzymes used for production was many times made ad hoc, or required expert knowledge of the specific biochemical reactions. In order to rationalize the process of engineering producer strains, we developed the computer-aided design (CAD) tool RetroPath that explores and enumerates metabolic pathways connecting the endogenous metabolites of a chassis cell to the target compound. To experimentally validate our tool, we constructed 12 top-ranked enzyme combinations producing the flavonoid pinocembrin, four of which displayed significant yields. Namely, our tool queried the enzymes found in metabolic databases based on their annotated and predicted activities. Next, it ranked pathways based on the predicted efficiency of the available enzymes, the toxicity of the intermediate metabolites and the calculated maximum product flux. To implement the top-ranking pathway, our procedure narrowed down a list of nine million possible enzyme combinations to 12, a number easily assembled and tested. One round of metabolic network optimization based on RetroPath output further increased pinocembrin titers 17-fold. In total, 12 out of the 13 enzymes tested in this work displayed a relative performance that was in accordance with its predicted score. These results validate the ranking function of our CAD tool, and open the way to its utilization in the biosynthesis of novel compounds.
Assuntos
Desenho Assistido por Computador , Engenharia Metabólica/métodos , Redes e Vias Metabólicas/fisiologia , Software , Biotecnologia/métodos , Bases de Dados Factuais , Escherichia coli/metabolismo , Flavanonas/metabolismo , Reprodutibilidade dos Testes , Biologia SintéticaRESUMO
Malaria represents a major public health problem and an important cause of mortality and morbidity. The malaria parasites are becoming resistant to drugs used to treat the disease and still no efficient vaccine has been developed. One promising vaccine candidate is the merozoite surface protein 1 (MSP1), which has been extensively investigated as a vaccine target. The surface protein MSP1 plays an essential role in the erythrocyte invasion process and is an accessible target for the immune system. Antibodies to the carboxy-terminal region of the protein, named MSP119, can inhibit erythrocyte invasion and parasite growth. In order to develop an effective MSP119- based vaccine against malaria, production of an antigen that is recognized by protective antibodies is mandatory. To this aim, we propose a method to produce the disulfide-rich MSP119 in its native conformation based on its in vitro oxidative refolding. The native conformation of the renatured MSP119 is carefully established by immunochemical reactivity experiments, circular dichroism and NMR. MSP119 can successfully be refolded in vitro as an isolated protein or as a fusion with the maltose binding protein. The possibility to properly fold MSP119in vitro paves the way to new approaches for high titer production of native MSP119 using Escherichia coli as a host.
Assuntos
Dissulfetos/metabolismo , Proteína 1 de Superfície de Merozoito/metabolismo , Plasmodium falciparum/metabolismo , Animais , Anticorpos Antiprotozoários/imunologia , Dicroísmo Circular , Proteína 1 de Superfície de Merozoito/química , Proteína 1 de Superfície de Merozoito/imunologia , Modelos Moleculares , Plasmodium falciparum/imunologia , Dobramento de ProteínaRESUMO
Tools from metabolic engineering and synthetic biology are synergistically used in order to develop high-performance cell factories. However, the number of successful applications has been limited due to the complexity of exploring efficiently the metabolic space for the discovery of candidate heterologous pathways. To address this challenge, retrosynthetic biology provides an integrated framework to formalize and rationalize the problem of importing biosynthetic pathways into a chassis organism using methods at the interface from bottom-up and top-down strategies. Here, we describe step by step the process of implementing a retrosynthetic framework for the design of heterologous biosynthetic pathways in a chassis organism. The method consists of the following steps: choosing the chassis and the target, selection of an in silico model for the chassis, definition of the metabolic space, pathway enumeration, gene selection, estimation of yields, toxicity prediction of pathway metabolites, definition of an objective function to select the best pathway candidates, and pathway implementation and verification.
Assuntos
Engenharia Metabólica/métodos , Redes e Vias Metabólicas/genética , Modelos Biológicos , Bactérias/genética , Bactérias/metabolismo , Reatores Biológicos , Fungos/genética , Fungos/metabolismo , Expressão Gênica , Genoma Bacteriano , Genoma Fúngico , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Biologia SintéticaRESUMO
De novo biosynthetic pathways are designed, assembled and optimized to produce high-value compounds such as drugs and chemical building blocks from renewable resources. Microorganisms are used as synthetic platforms of systems biology where biochemical pathways are engineered into the host metabolic network. Retrosynthetic biology offers a creative pathway design concept that has gained interest because of its potential to identify novel metabolic ways for therapeutic production. Retrosynthetic biology uses the backward search of retrosynthetic analysis to devise and optimize tailor-made pathways. The retrosynthetic process can be seamlessly integrated into a complete circuitry system for therapeutic applications where production, sensing and delivery act as constitutive interconnecting parts. The aim of this review is to highlight recent efforts toward synthetic design for therapeutic development.
Assuntos
Vias Biossintéticas/genética , Descoberta de Drogas/métodos , Biologia Sintética/métodos , Animais , HumanosRESUMO
Progress in DNA technology, analytical methods and computational tools is leading to new developments in synthetic biology and metabolic engineering, enabling new ways to produce molecules of industrial and therapeutic interest. Here, we review recent progress in both antibiotic production and strategies to counteract bacterial resistance to antibiotics. Advances in sequencing and cloning are increasingly enabling the characterization of antibiotic biosynthesis pathways, and new systematic methods for de novo biosynthetic pathway prediction are allowing the exploration of the metabolic chemical space beyond metabolic engineering. Moreover, we survey the computer-assisted design of modular assembly lines in polyketide synthases and non-ribosomal peptide synthases for the development of tailor-made antibiotics. Nowadays, production of novel antibiotic can be tranferred into any chosen chassis by optimizing a host factory through specific strain modifications. These advances in metabolic engineering and synthetic biology are leading to novel strategies for engineering antimicrobial agents with desired specificities.
Assuntos
Antibacterianos/biossíntese , Desenho de Fármacos , Farmacorresistência Bacteriana , Biologia Sintética , Antibacterianos/química , Antibacterianos/farmacologia , Biologia Computacional , Reprodutibilidade dos TestesRESUMO
BACKGROUND: Synthetic biology is used to develop cell factories for production of chemicals by constructively importing heterologous pathways into industrial microorganisms. In this work we present a retrosynthetic approach to the production of therapeutics with the goal of developing an in situ drug delivery device in host cells. Retrosynthesis, a concept originally proposed for synthetic chemistry, iteratively applies reversed chemical transformations (reversed enzyme-catalyzed reactions in the metabolic space) starting from a target product to reach precursors that are endogenous to the chassis. So far, a wider adoption of retrosynthesis into the manufacturing pipeline has been hindered by the complexity of enumerating all feasible biosynthetic pathways for a given compound. RESULTS: In our method, we efficiently address the complexity problem by coding substrates, products and reactions into molecular signatures. Metabolic maps are represented using hypergraphs and the complexity is controlled by varying the specificity of the molecular signature. Furthermore, our method enables candidate pathways to be ranked to determine which ones are best to engineer. The proposed ranking function can integrate data from different sources such as host compatibility for inserted genes, the estimation of steady-state fluxes from the genome-wide reconstruction of the organism's metabolism, or the estimation of metabolite toxicity from experimental assays. We use several machine-learning tools in order to estimate enzyme activity and reaction efficiency at each step of the identified pathways. Examples of production in bacteria and yeast for two antibiotics and for one antitumor agent, as well as for several essential metabolites are outlined. CONCLUSIONS: We present here a unified framework that integrates diverse techniques involved in the design of heterologous biosynthetic pathways through a retrosynthetic approach in the reaction signature space. Our engineering methodology enables the flexible design of industrial microorganisms for the efficient on-demand production of chemical compounds with therapeutic applications.
Assuntos
Reatores Biológicos , Vias Biossintéticas/fisiologia , Biotecnologia/métodos , Microbiologia Industrial/métodos , Redes e Vias Metabólicas/fisiologia , Modelos Biológicos , Biologia Sintética/métodos , Antibacterianos/biossíntese , Inteligência Artificial , Bactérias , Enzimas/metabolismo , Redes e Vias Metabólicas/genética , LevedurasRESUMO
Peroxiredoxins constitute a major family of cysteine-based peroxide-scavenging enzymes. They carry an intriguing redox switch by undergoing substrate-mediated inactivation via overoxidation of their catalytic cysteine to the sulfinic acid form that is reverted by reduction catalyzed by the sulfinic acid reductase sulfiredoxin (Srx). The biological significance of such inactivation is not understood, nor is the function of Srx1. To address this question, we generated a mouse line with a null deletion of the Srx1-encoding Srxn1 gene. We show here that Srxn1(-/-) mice are perfectly viable and do not suffer from any apparent defects under laboratory conditions, but have an abnormal response to lipopolysaccharide that manifests by increased mortality during endotoxic shock. Microarray-based mRNA profiles show that although the response of Srxn1(-/-) mice to lipopolysaccharide is typical, spanning all spectrum and all pathways of innate immunity, it is delayed by several hours and remains intense when the response of Srxn1(+/+) mice has already dissipated. These data indicate that Srx1 activity protects mice from the lethality of endotoxic shock, adding this enzyme to other host factors, as NRF2 and peroxiredoxin 2, which by regulating cellular reactive oxygen species levels act as important modifiers in the pathogenesis of sepsis.
Assuntos
Lipopolissacarídeos/farmacologia , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Choque Séptico/metabolismo , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Células Cultivadas , Feminino , Engenharia Genética , Proteínas de Homeodomínio/metabolismo , Imunidade Inata , Estimativa de Kaplan-Meier , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Espécies Reativas de Oxigênio/metabolismo , Choque Séptico/imunologia , Transdução de Sinais , Transcrição GênicaRESUMO
Peroxiredoxins (Prx's) are a family of peroxidases that maintain thiol homeostasis by catalyzing the reduction of organic hydroperoxides, H2O2, and peroxynitrite. Under conditions of oxidative stress, eukaryotic Prx's can be inactivated by the substrate-dependent oxidation of the catalytic cysteine to sulfinic acid, which may regulate the intracellular messenger function of H2O2. A small redox protein, sulfiredoxin (Srx), conserved only in eukaryotes, has been shown to reduce sulfinylated 2-Cys Prx's, adding to the complexity of the H2O2 signaling network. In this study, we addressed the regulation of Srx expression in immunostimulated primary macrophages that produce both reactive oxygen species (ROS) and nitric oxide (NO(â¢)). We present genetic evidence that NO-mediated Srx up-regulation is mediated by the transcription factor nuclear factor erythroid 2-related factor (Nrf2). We also show that the NO(â¢)/Srx pathway inhibits generation of ROS. These results reveal a link between innate immunity and H2O2 signaling. We propose that an NO(â¢)/Nrf2/Srx pathway participates in the maintenance of redox homeostasis in cytokine-activated macrophages and other inflammatory settings.
Assuntos
Antioxidantes/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Óxido Nítrico/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Animais , Células Cultivadas , Peróxido de Hidrogênio/metabolismo , Imunidade Inata , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxirredução , Estresse Oxidativo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Reação em Cadeia da Polimerase , Espécies Reativas de Oxigênio/metabolismoRESUMO
The bacterial peroxiredoxin AhpC, a cysteine-dependent peroxidase, can be converted through a single amino acid insertion to a disulfide reductase, AhpC*, active in the glutathione and glutaredoxin pathway. Here we show that, whereas AhpC* is inactive as a peroxidase, other point mutants in AhpC can confer the in vivo disulfide reductase activity without abrogating peroxidase activity. Moreover, AhpC* and several point mutants tested in vitro exhibit an enhanced reductase activity toward mixed disulfides between glutathione and glutaredoxin (Grx-S-SG), consistent with the in vivo requirements for these components. Remarkably, this Grx-S-SG reductase activity relies not on the peroxidatic cysteine but rather on the resolving cysteine that plays only a secondary role in the peroxidase mechanism. Furthermore, putative conformational changes, which impart this unusual Grx-S-SG reductase activity, are transmissible across subunits. Thus, AhpC and potentially other peroxiredoxins in this widespread family can elaborate a new reductase function that alleviates disulfide stress.
Assuntos
Dissulfetos/metabolismo , Proteínas de Escherichia coli/genética , Escherichia coli/enzimologia , Glutationa/metabolismo , Mutagênese Insercional , Oxirredutases/genética , Peroxirredoxinas/genética , Mutação Puntual , Substituição de Aminoácidos , Cisteína/metabolismo , Dimerização , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/fisiologia , Glutarredoxinas/metabolismo , Peróxido de Hidrogênio/metabolismo , Modelos Moleculares , Oxirredução , Estresse Oxidativo , Oxirredutases/química , Oxirredutases/fisiologia , Peroxirredoxinas/química , Peroxirredoxinas/fisiologia , Conformação Proteica , Subunidades Proteicas , Especificidade por Substrato , Repetições de TrinucleotídeosRESUMO
Escherichia coli thioredoxin is normally a cytoplasmic protein involved in the reduction of disulfide bonds. However, thioredoxin can be translocated to the periplasm when it is attached to a cotranslational signal sequence. When exported to the periplasm, it can partially replace the activity of DsbA in promoting the formation of disulfide bonds. In contrast, when thioredoxin is fused to a posttranslational signal sequence, very little of it appears in the periplasm. We propose that this absence of posttranslational export is due to the rapid folding of thioredoxin in the cytoplasm. We sought mutants of thioredoxin that retarded its folding in the cytoplasm, which we accomplished by fusing thioredoxin to a posttranslational signal sequence and selecting for mutants in which thioredoxin was exported to the periplasm, where it could replace DsbA. The collection of mutants obtained represents a limited number of amino acid changes in the protein. In vitro studies on purified mutant proteins show that all but one are defective in the kinetics and thermodynamics of protein folding. We propose that the slower folding of the thioredoxin mutant proteins in the cytoplasm allows their export by a posttranslational pathway. We discuss some implications of this class of mutants for aspects of the folding pathway of thioredoxin and for its mechanism of export. In particular, the finding that a folding mutant that allows protein translocation alters an amino acid at the C terminus of the protein suggests that the degree to which thioredoxin folds during its translation must be severely restricted.
Assuntos
Escherichia coli/metabolismo , Mutação , Tiorredoxinas/química , Tiorredoxinas/genética , Proteínas de Bactérias/química , Western Blotting , Varredura Diferencial de Calorimetria , Dicroísmo Circular , Citoplasma/metabolismo , Bases de Dados de Proteínas , Dissulfetos , Proteínas de Escherichia coli/química , Técnicas Genéticas , Cinética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Mutagênese , Mutagênese Sítio-Dirigida , Oxigênio/química , Plasmídeos/metabolismo , Ligação Proteica , Biossíntese de Proteínas , Isomerases de Dissulfetos de Proteínas/metabolismo , Dobramento de Proteína , Processamento de Proteína Pós-Traducional , Sinais Direcionadores de Proteínas , Estrutura Terciária de Proteína , Frações Subcelulares , Tiorredoxinas/metabolismo , Fatores de TempoRESUMO
The C-terminal fragment of Plasmodium falciparum merozoite surface protein 1 (F19) is a leading candidate for the development of a malaria vaccine. Successful vaccination trials on primates, immunochemistry, and structural studies have shown the importance of its native conformation for its protective role against infection. F19 is a disulfide-rich protein, and the correct pairing of its 12 half-cystines is required for the native state of the protein. F19 has been produced in the Escherichia coli periplasm, which has an oxidative environment favorable for the formation of disulfide bonds. F19 was either expressed as a fusion with the maltose binding protein (MBP) or directly addressed to the periplasm by fusing it with the MBP signal peptide. Direct expression of F19 in the periplasm led to a misfolded protein with a heterogeneous distribution of disulfide bridges. On the contrary, when produced as a fusion protein with E. coli MBP, the F19 moiety was natively folded. Indeed, after proteolysis of the fusion protein, the resulting F19 possesses the structural characteristics and the immunochemical reactivity of the analogous fragment produced either in baculovirus-infected insect cells or in yeast. These results demonstrate that the positive effect of MBP in assisting the folding of passenger proteins extends to the correct formation of disulfide bridges in vivo. Although proteins or protein fragments fused to MBP have been frequently expressed with success, our comparative study evidences for the first time the helping property of MBP in the oxidative folding of a disulfide-rich protein.