RESUMO
We reported recently that the Parkinsonism-associated protein DJ-1 and its bacterial homologs Hsp31, YhbO and YajL function as deglycases that repair proteins and nucleotides from endogeneous glycation by glyoxal and methylglyoxal, two reactive by-products of glucose metabolism responsible for up to 60% of glycation damage. Here, we show that DJ-1, deglycase 1 and deglycase 2 repair glyoxal- and methylglyoxal-glycated substrates, whereas deglycase 3 principally repairs glyoxal-glycated substrates. Moreover, deglycase 1 and 2 are overexpressed in stationary phase, whereas deglycase 3 is steadily expressed throughout bacterial growth. Finally, deglycase mutants overexpress glyoxalases, aldoketoreductases, glutathione-S-transferase and efflux pumps to alleviate carbonyl stress. In the discussion, we present an overview of the multiple functions of DJ-1 proteins. Our thourough work on deglycases provides compelling evidence that their previously reported glyoxalase III activity merely reflects their deglycase activity. Moreover, for their deglycase activity the Maillard deglycases likely recruit: i) their chaperone activity to interact with glycated proteins, ii) glyoxalase 1 activity to catalyze the rearrangement of Maillard products (aminocarbinols and hemithioacetals) into amides and thioesters, respectively, iii) their protease activity to cleave amide bonds of glycated arginine, lysine and guanine, and iv) glyoxalase 2 activity to cleave thioester bonds of glycated cysteine. Finally, because glycation affects many cellular processes, the discovery of the Maillard deglycases, awaited since 1912, likely constitutes a major advance for medical research, including ageing, cancer, atherosclerosis, neurodegenerative, post-diabetic, renal and autoimmune diseases.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Proteína Desglicase DJ-1/metabolismo , Proteínas Ribossômicas/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , Glicosilação , Glioxal/metabolismo , Humanos , Aldeído Pirúvico/metabolismoRESUMO
We discovered recently that Parkinsonism-associated DJ-1 and its bacterial homologs function as protein deglycases that repair glyoxal- and methylglyoxal-glycated proteins. Protein glycation levels are 2- to 10-fold increased in deglycase-depleted cells, and deglycase mutants display up to 500-fold loss of viability in methylglyoxal or glucose-containing media, suggesting that these deglycases play important roles in protecting cells against electrophile and carbonyl stress. Although the deglycase activity of DJ-1 is well supported by extensive biochemical work, Pfaff et al. (J. Biol. Chem. in presshttp://dx.doi.org/10.1074/jbc.M116.743823) claimed in a recent study that deglycation of the hemithioacetal formed upon cysteine glycation by methylglyoxal results from a Tris buffer artefact. Here, we show that this is not the case, and that DJ-1 and its homologs are the bona fide deglycases awaited since the Maillard discovery.
Assuntos
Proteínas Oncogênicas/metabolismo , Proteína Desglicase DJ-1/metabolismo , Acetilcisteína/química , Artefatos , Meios de Cultura/química , Cisteína/química , Cisteína/metabolismo , Escherichia coli/metabolismo , Glucose/química , Produtos Finais de Glicação Avançada/metabolismo , Glicosilação , Glioxal/química , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mutação , Transtornos Parkinsonianos/metabolismo , Aldeído Pirúvico/metabolismoRESUMO
DJ-1 and its prokaryotic homologs, Hsp31, YhbO and YajL from Escherichia coli and PfpI from Pyrococcus furiosus, repair proteins from glycation by glyoxals (R-CO-CHO), which constitute their major glycating agents. Glycation is a non-enzymatic covalent reaction discovered by Louis Camille Maillard in 1912, between reactive carbonyls (reducing sugars and glyoxals) and amino acids (cysteine, arginine and lysine), which inactivates proteins. By degrading Maillard adducts formed between carbonyls and thiols or amino groups, the DJ-1 family Maillard deglycases prevent the formation of the so-called advanced glycation end products (AGEs) that arise from Maillard adducts after dehydrations, oxidations and rearrangements. Since glycation is involved in ageing, cancer, atherosclerosis and cataracts, as well as post-diabetic, neurovegetatives and renal and autoimmune diseases, the DJ-1 deglycases are likely to play an important role in preventing these diseases. These deglycases, especially those from thermophilic organisms, may also be used to prevent the formation of dietary AGEs during food processing, sterilization and storage. They also prevent acrylamide formation in food, likely by degrading the asparagine/glyoxal Maillard adducts responsible for its formation. Since Maillard adducts are the substrates of the DJ-1 family deglycases, we propose renaming them Maillard deglycases.
Assuntos
Glioxal/metabolismo , Reação de Maillard , Proteína Desglicase DJ-1/metabolismo , Processamento de Proteína Pós-Traducional , Acrilamida/química , Acrilamida/metabolismo , Animais , Arginina/química , Arginina/metabolismo , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Cisteína/química , Cisteína/metabolismo , Produtos Finais de Glicação Avançada/química , Produtos Finais de Glicação Avançada/metabolismo , Glicosilação , Glioxal/química , Humanos , Lisina/química , Lisina/metabolismoRESUMO
Glycation is an inevitable nonenzymatic covalent reaction between proteins and endogenous reducing sugars or dicarbonyls (methylglyoxal, glyoxal) that results in protein inactivation. DJ-1 was reported to be a multifunctional oxidative stress response protein with poorly defined function. Here, we show that human DJ-1 is a protein deglycase that repairs methylglyoxal- and glyoxal-glycated amino acids and proteins by acting on early glycation intermediates and releases repaired proteins and lactate or glycolate, respectively. DJ-1 deglycates cysteines, arginines, and lysines (the three major glycated amino acids) of serum albumin, glyceraldehyde-3-phosphate dehydrogenase, aldolase, and aspartate aminotransferase and thus reactivates these proteins. DJ-1 prevented protein glycation in an Escherichia coli mutant deficient in the DJ-1 homolog YajL and restored cell viability in glucose-containing media. These results suggest that DJ-1-associated Parkinsonism results from excessive protein glycation and establishes DJ-1 as a major anti-glycation and anti-aging protein.
Assuntos
Arginina/química , Cisteína/química , Glioxal/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisina/química , Proteínas Oncogênicas/metabolismo , Transtornos Parkinsonianos/metabolismo , Aldeído Pirúvico/química , Acetilcisteína/química , Albuminas/química , Apoptose , Aspartato Aminotransferases/metabolismo , Catálise , Sobrevivência Celular , Escherichia coli/metabolismo , Frutose-Bifosfato Aldolase/metabolismo , Glucose/química , Glicolatos/química , Humanos , Lactatos/química , Espectrometria de Massas , Estresse Oxidativo , Proteína Desglicase DJ-1RESUMO
Reducing sugars and dicarbonyls form covalent adducts with proteins through a nonenzymatic process known as glycation, which inactivates proteins, is increased in diabetic patients and is associated with diabetic complications, including retinopathy, cataracts, nephropathy, neuropathy, cardiomyopathy and skin defects. We recently characterized DJ-1/Park7 as a protein deglycase that repairs proteins from glycation by glyoxal and methylglyoxal, two major glycating agents which are responsible for up to 65% of glycation events. In this study, we investigated the ability of DJ-1 to prevent protein glycation in keratinocytes. Glycation of collagen and keratinocyte proteins was tested by measuring ultraviolet absorption and fluorescence emission. Protein glycation in HaCaT keratinocytes was investigated by immunodetection with anti-advanced glycation endproduct antibodies, after DJ-1 depletion or overexpression. In vitro, DJ-1 prevented glycation of collagen and keratinocyte protein extracts. In cell culture, DJ-1 depletion by small interfering RNAs resulted in a 3-fold increase in protein glycation levels. Moreover, protein glycation levels were decreased several-fold in cells overexpressing DJ-1 after addition of the Nrf2 inducer sulforaphane or after transfection with a DJ-1 plasmid. Thus, the DJ-1 deglycase plays a major role in preventing protein glycation in eukaryotic cells and might be important for preventing skin glycation.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Queratinócitos/metabolismo , Proteínas Oncogênicas/metabolismo , Aldeídos/química , Carboidratos/química , Linhagem Celular , Complicações do Diabetes/metabolismo , Inativação Gênica , Produtos Finais de Glicação Avançada/metabolismo , Glicosilação , Glioxal/química , Humanos , Isotiocianatos/química , Queratinócitos/citologia , Fator 2 Relacionado a NF-E2/metabolismo , Proteína Desglicase DJ-1 , Pele/efeitos dos fármacos , Pele/metabolismo , Envelhecimento da Pele , SulfóxidosRESUMO
YhbO and YajL belong to the PfpI/Hsp31/DJ-1 superfamily. Both proteins are involved in protection against environmental stresses. Here, we show that, like DJ-1 and Hsp31, they repair glyoxal- and methylglyoxal-glycated proteins. YhbO and YajL repair glycated serum albumin, collagen, glyceraldehyde-3-phosphate dehydrogenase, and fructose biphosphate aldolase. Bacterial extracts from deglycase mutants display increased glycation levels, whereas deglycase overexpression decreases protein glycation. Moreover, yhbO and yajL mutants display decreased viability in methylglyoxal- or glucose-containing media. Finally, the apparent glyoxalase activities of YhbO and YajL reflect their deglycase activities.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Glioxal/metabolismo , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Aldeído Pirúvico/metabolismo , Proteínas Ribossômicas/metabolismo , Citoproteção/fisiologia , Produtos Finais de Glicação Avançada/metabolismoRESUMO
The presence of acrylamide in food is a worldwide concern because it is carcinogenic, reprotoxic and neurotoxic. Acrylamide is generated in the Maillard reaction via condensation of reducing sugars and glyoxals arising from their decomposition, with asparagine, the amino acid forming the backbone of the acrylamide molecule. We reported recently the discovery of the Maillard deglycases (DJ-1/Park7 and its prokaryotic homologs) which degrade Maillard adducts formed between glyoxals and lysine or arginine amino groups, and prevent glycation damage in proteins. Here, we show that these deglycases prevent acrylamide formation, likely by degrading asparagine/glyoxal Maillard adducts. We also report the discovery of a deglycase from the hyperthermophilic archaea Pyrococcus furiosus, which prevents acrylamide formation at 100 °C. Thus, Maillard deglycases constitute a unique enzymatic method to prevent acrylamide formation in food without depleting the components (asparagine and sugars) responsible for its formation.
Assuntos
Acrilamida/metabolismo , Reação de Maillard , Família Multigênica , Proteína Desglicase DJ-1/metabolismo , Asparagina/metabolismo , Frutose/metabolismo , Glucose/metabolismo , Glioxal/metabolismo , Pyrococcus furiosus/enzimologiaRESUMO
Hsp31 belongs to the PfpI/Hsp31/DJ-1 superfamily, and has been reported to display chaperone, peptidase and glutathione-independent glyoxalase activities. Here, we show that Hsp31 repairs glyoxal- and methylglyoxal-glycated amino acids and proteins and releases repaired proteins and lactate or glycolate, respectively. Hsp31 deglycates cysteine, arginine and lysine by acting on early glycation intermediates (hemithioacetals and aminocarbinols) and prevents the formation of Schiff bases and advanced glycation endproducts. Hsp31 repairs glycated serum albumin, glyceraldehyde-3-phosphate dehydrogenase, fructose biphosphate aldolase and aspartate aminotransferase. Moreover, we show that bacterial extracts from the hchA mutant display increased glycation levels and that the apparent glyoxalase activity of Hsp31 reflects its deglycase activity. Our results suggest that other Hsp31 members, previously characterized as glutathione-independent glyoxalases, likely function as protein deglycases.
Assuntos
Proteínas de Escherichia coli/fisiologia , Glioxal/farmacologia , Chaperonas Moleculares/fisiologia , Aldeído Pirúvico/farmacologia , Arginina/metabolismo , Cisteína/metabolismo , Glucose/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Lisina/metabolismo , Bases de SchiffRESUMO
YajL is the closest prokaryotic homologue of Parkinson's disease-associated DJ-1, a protein of undefined function involved in the oxidative stress response. We reported recently that YajL and DJ-1 protect cells against oxidative stress-induced protein aggregation by acting as covalent chaperones for the thiol proteome, including the NuoG subunit of NADH dehydrogenase 1, and that NADH dehydrogenase 1 activity is negligible in the yajL mutant. We report here that this mutant compensates for low NADH dehydrogenase activity by utilizing NADH-independent alternative dehydrogenases, including pyruvate oxidase PoxB and d-amino acid dehydrogenase DadA, and mixed acid aerobic fermentations characterized by acetate, lactate, succinate and ethanol excretion. The yajL mutant has a low adenylate energy charge favouring glycolytic flux, and a high NADH/NAD ratio favouring fermentations over pyruvate dehydrogenase and the Krebs cycle. DNA array analysis showed upregulation of genes involved in glycolytic and pentose phosphate pathways and alternative respiratory pathways. Moreover, the yajL mutant preferentially catabolized pyruvate-forming amino acids over Krebs cycle-related amino acids, and thus the yajL mutant utilizes pyruvate-centred respiro-fermentative metabolism to compensate for the NADH dehydrogenase 1 defect and constitutes an interesting model for studying eukaryotic respiratory complex I deficiencies, especially those associated with Alzheimer's and Parkinson's diseases.
Assuntos
Escherichia coli/enzimologia , Escherichia coli/metabolismo , NADH Desidrogenase/deficiência , Proteínas Ribossômicas/deficiência , Aerobiose , Proteínas de Escherichia coli , Fermentação , Perfilação da Expressão Gênica , Humanos , Análise do Fluxo Metabólico , Análise em Microsséries , Modelos Biológicos , Dados de Sequência Molecular , Transtornos Parkinsonianos/patologia , Análise de Sequência de DNARESUMO
BACKGROUND: Lactococcus lactis, a lactic acid bacterium traditionally used to ferment milk and manufacture cheeses, is also, in the biotechnology field, an interesting host to produce proteins of medical interest, as it is "Generally Recognized As Safe". Furthermore, as L. lactis naturally secretes only one major endogenous protein (Usp45), the secretion of heterologous proteins in this species facilitates their purification from a protein-poor culture medium. Here, we developed and optimized protein production and secretion in L. lactis to obtain proteins of high quality, both correctly folded and pure to a high extent. As proteins to be produced, we chose the two transmembrane members of the HtrA protease family in Staphylococcus aureus, an important extra-cellular pathogen, as these putative surface-exposed antigens could constitute good targets for vaccine development. RESULTS: A recombinant ORF encoding a C-terminal, soluble, proteolytically inactive and tagged form of each staphylococcal HtrA protein was cloned into a lactococcal expression-secretion vector. After growth and induction of recombinant gene expression, L. lactis was able to produce and secrete each recombinant rHtrA protein as a stable form that accumulated in the culture medium in similar amounts as the naturally secreted endogenous protein, Usp45. L. lactis growth in fermenters, in particular in a rich optimized medium, led to higher yields for each rHtrA protein. Protein purification from the lactococcal culture medium was easily achieved in one step and allowed recovery of highly pure and stable proteins whose identity was confirmed by mass spectrometry. Although rHtrA proteins were monomeric, they displayed the same secondary structure content, thermal stability and chaperone activity as many other HtrA family members, indicating that they were correctly folded. rHtrA protein immunogenicity was established in mice. The raised polyclonal antibodies allowed studying the expression and subcellular localization of wild type proteins in S. aureus: although both proteins were expressed, only HtrA1 was found to be, as predicted, exposed at the staphylococcal cell surface suggesting that it could be a better candidate for vaccine development. CONCLUSIONS: In this study, an efficient process was developed to produce and secrete putative staphylococcal surface antigens in L. lactis and to purify them to homogeneity in one step from the culture supernatant. This allowed recovering fully folded, stable and pure proteins which constitute promising vaccine candidates to be tested for protection against staphylococcal infection. L. lactis thus proved to be an efficient and competitive cell factory to produce proteins of high quality for medical applications.
Assuntos
Antígenos de Bactérias/química , Proteínas de Bactérias/química , Vacinas Bacterianas/química , Lactococcus lactis/genética , Peptídeo Hidrolases/química , Staphylococcus aureus/enzimologia , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/isolamento & purificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/isolamento & purificação , Vacinas Bacterianas/genética , Vacinas Bacterianas/imunologia , Humanos , Lactococcus lactis/metabolismo , Camundongos , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/imunologia , Peptídeo Hidrolases/isolamento & purificação , Dobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/prevenção & controle , Staphylococcus aureus/química , Staphylococcus aureus/imunologiaRESUMO
YajL is the most closely related Escherichia coli homolog of Parkinsonism-associated protein DJ-1, a protein with a yet-undefined function in the oxidative-stress response. YajL protects cells against oxidative-stress-induced protein aggregation and functions as a covalent chaperone for the thiol proteome, including FeS proteins. To clarify the cellular responses to YajL deficiency, transcriptional profiling of the yajL mutant was performed. Compared to the parental strain, the yajL mutant overexpressed genes coding for chaperones, proteases, chemical chaperone transporters, superoxide dismutases, catalases, peroxidases, components of thioredoxin and glutaredoxin systems, iron transporters, ferritins and FeS cluster biogenesis enzymes, DNA repair proteins, RNA chaperones, and small regulatory RNAs. It also overexpressed the RNA polymerase stress sigma factors sigma S (multiple stresses) and sigma 32 (protein stress) and activated the OxyR and SoxRS oxidative-stress transcriptional regulators, which together trigger the global stress response. The yajL mutant also overexpressed genes involved in septation and adopted a shorter and rounder shape characteristic of stressed bacteria. Biochemical experiments showed that this upregulation of many stress genes resulted in increased expression of stress proteins and improved biochemical function. Thus, protein defects resulting from the yajL mutation trigger the onset of a robust and global stress response in a prokaryotic model of DJ-1-associated Parkinsonism.
Assuntos
Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Choque Térmico/biossíntese , Estresse Oxidativo/genética , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Reparo do DNA , DNA Bacteriano/metabolismo , Escherichia coli/genética , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Proteínas de Choque Térmico/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ferro/metabolismo , Chaperonas Moleculares/biossíntese , Chaperonas Moleculares/genética , Mutação , Proteínas Oncogênicas/genética , Oxirredução , Transtornos Parkinsonianos/genética , Proteína Desglicase DJ-1RESUMO
YajL is the closest Escherichia coli homolog of the Parkinsonism-associated protein DJ-1, a multifunctional oxidative stress response protein whose biochemical function remains unclear. We recently reported the aggregation of proteins in a yajL mutant in an oxidative stress-dependent manner and that YajL exhibits chaperone activity. Here, we show that YajL displays covalent chaperone and weak protein oxidoreductase activities that are dependent on its exposed cysteine 106. It catalyzes reduced RNase oxidation and scrambled RNase isomerization and insulin reduction and forms mixed disulfides with many cellular proteins upon oxidative stress. The formation of mixed disulfides was detected by immunoblotting bacterial extracts with anti-YajL antibodies under nonreducing conditions. Disulfides were purified from bacterial extracts on a YajL affinity column, separated by nonreducing-reducing SDS-PAGE, and identified by mass spectrometry. Covalent YajL substrates included ribosomal proteins, aminoacyl-tRNA synthetases, chaperones, catalases, peroxidases, and other proteins containing cysteines essential for catalysis or FeS cluster binding, such as glyceraldehyde-3-phosphate dehydrogenase, aldehyde dehydrogenase, aconitase, and FeS cluster-containing subunits of respiratory chains. In addition, we show that DJ-1 also forms mixed disulfides with cytoplasmic proteins upon oxidative stress. These results shed light on the oxidative stress-dependent chaperone function of YajL and identify YajL substrates involved in translation, stress protection, protein solubilization, and metabolism. They reveal a crucial role for cysteine 106 and suggest that DJ-1 also functions as a covalent chaperone. These findings are consistent with several defects observed in yajL or DJ-1 mutants, including translational defects, protein aggregation, oxidative stress sensitivity, and metabolic deficiencies.
Assuntos
Proteínas de Escherichia coli/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/química , Chaperonas Moleculares/metabolismo , Proteínas Oncogênicas/química , Proteoma/metabolismo , Proteínas Ribossômicas/metabolismo , Homologia de Sequência de Aminoácidos , Compostos de Sulfidrila/metabolismo , Dissulfetos/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Mutação , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Estresse Oxidativo , Oxirredutases/metabolismo , Proteína Desglicase DJ-1 , Isomerases de Dissulfetos de Proteínas/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genéticaRESUMO
YajL is the closest prokaryotic homolog of the parkinsonism-associated protein DJ-1 (40% sequence identity and similar three-dimensional structure), a protein of unknown function involved in the cellular response to oxidative stress. We report here that a yajL mutant of Escherichia coli displays an increased sensitivity to oxidative stress. It also exhibits a protein aggregation phenotype in aerobiosis, but not in anaerobiosis or in aerobic cells overexpressing superoxide dismutase, suggesting that protein aggregation depends on the presence of reactive oxygen species produced by respiratory chains. The protein aggregation phenotype of the yajL mutant, which can be rescued by the wild-type yajL gene, but not by the corresponding cysteine 106 mutant allele, is similar to that of multiple mutants deficient in superoxide dismutases and catalases, although intracellular hydrogen peroxide levels were not increased in the yajL mutant, suggesting that protein aggregation in this strain does not result from a hydrogen peroxide detoxification defect. Aggregation-prone proteins included 17 ribosomal proteins, the ATP synthase beta subunit, flagellin, and the outer membrane proteins OmpA and PAL; all of them are part of multiprotein complexes, suggesting that YajL might be involved in optimal expression of these complexes, especially during oxidative stress. YajL stimulated the renaturation of urea-unfolded citrate synthase and the solubilization of the urea-unfolded ribosomal proteins S1 and L3 and was more efficient as a chaperone in its oxidized form than in its reduced form. The mRNA levels of several aggregated proteins of the yajL mutant were severely affected, suggesting that YajL also acts at the level of gene expression. These two functions of YajL might explain the protein aggregation phenotype of the yajL mutant.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/química , Mutação/genética , Proteínas Oncogênicas/química , Estresse Oxidativo , Aerobiose , Anaerobiose , Catalase/metabolismo , Citrato (si)-Sintase/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Humanos , Peróxido de Hidrogênio/metabolismo , Conformação Proteica , Proteína Desglicase DJ-1 , Dobramento de Proteína , Espécies Reativas de Oxigênio/metabolismo , Proteína Ribossômica L3 , Proteínas Ribossômicas/metabolismo , Superóxido Dismutase/metabolismoRESUMO
Escherichia coli contains two thioredoxins, Trx1 and Trx2, and a thioredoxin-like protein, YbbN, that displays both redox and chaperone properties. Since three out of the six proteins of the YbbN interactome (Butland et al., 2005) are components of DNA polymerase 3 holoenzyme (i.e. the ß-clamp DnaN, the θ subunit HolE and the δ' subunit HolB), we investigated whether the ybbN mutant presents DNA replication defects. We found that this mutant incorporates (3)H-thymidine at higher rates than the parental strain and displays overinitiation, hypermutator and filamentation phenotypes with the occurrence of anucleated cells. Moreover, YbbN functions as a bona fide chaperone in the refolding of the urea-unfolded ß-clamp. These results suggest that the DNA replication and cell division defects of the ybbN mutant might best be explained by chaperone functions of YbbN in the biogenesis of DNA polymerase 3 holoenzyme.
Assuntos
Replicação do DNA/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Chaperonas Moleculares/genética , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Tiorredoxinas/genética , DNA Polimerase III/química , Escherichia coli/ultraestrutura , Proteínas de Escherichia coli/química , Citometria de Fluxo , Microscopia , Chaperonas Moleculares/química , Mutação , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/química , Desnaturação Proteica , Tiorredoxinas/química , Ureia/químicaRESUMO
We report here that YajL is associated with ribosomes and interacts with many ribosomal proteins and that a yajL mutant of Escherichia coli displays decreased translation accuracy, as well as increased dissociation of 70S ribosomes into 50S and 30S subunits after oxidative stress.
Assuntos
Escherichia coli/fisiologia , Biossíntese de Proteínas , Proteínas Ribossômicas/deficiência , Escherichia coli/metabolismo , Proteínas de Escherichia coli , Ribossomos/metabolismoRESUMO
We used preS2-S'-beta-galactosidase, a three domain fusion protein that aggregates extensively at 43 degrees C in the cytoplasm of Escherichia coli to search for multicopy suppressors of protein aggregation and inclusion bodies formation, and took advantage of the known differential solubility of preS2-S'-beta-galactosidase at 37 and 43 degrees C to develop a selection procedure for the gene products that would prevent its aggregation in vivo at 43 degrees C. First, we demonstrate that the differential solubility of preS2-S'-beta-galactosidase results in a lactose-positive phenotype at 37 degrees C as opposed to a lactose-negative phenotype at 43 degrees C. We searched for multicopy suppressors of preS2-S'-beta-galactosidase aggregation at 43 degrees C by selecting pink lactose-positive colonies on a background of white lactose-negative colonies after transformation of bacteria with an E. coli gene bank. We found only two multicopy suppressors of preS2-S'-beta-galactosidase aggregation at 43 degrees C, protein isoaspartate methyltransferase (PIMT) and the membrane components ChbBC of the N,N'-diacetylchitobiose phosphotransferase transporter. We have previously shown that PIMT overexpression reduces the level of isoaspartate in preS2-S'-beta-galactosidase, increases its thermal stability and consequently helps in its solubilization at 43 degrees C (Kern et al., J. Bacteriol. 187, 1377-1383). In the present work, we show that ChbBC overexpression targets a fraction of preS2-S'-beta-galactosidase to the membrane, and decreases its amount in inclusion bodies, which results in its decreased thermodenaturation and in a lactose-positive phenotype at 43 degrees C. Cross-linking experiments show that the inner membrane protein ChbC interacts with preS2-S'-beta-galactosidase. Our results suggest that membrane docking of aggregation-prone proteins might be a useful method for their solubilization.
Assuntos
Proteínas de Escherichia coli/metabolismo , Corpos de Inclusão/metabolismo , beta-Galactosidase/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Teste de Complementação Genética , Corpos de Inclusão/genética , Lactose/metabolismo , Modelos Biológicos , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Solubilidade , Temperatura , beta-Galactosidase/química , beta-Galactosidase/genéticaRESUMO
Escherichia coli contains two thioredoxins, Trx1 and Trx2, and a thioredoxin-like protein, YbbN, which presents a strong homology in its N-terminal part with thioredoxins, and possesses a 20kDa C-terminal part of unknown function. We reported previously that YbbN displays both protein oxido-reductase and chaperone properties in vitro. In this study, we show that an ybbN-deficient strain displays an increased sensitivity to thermal stress but not to oxidative stress, a normal redox state of its cellular proteins but a decreased expression of several cytoplasmic proteins, including EF-Tu, DnaK, GroEL, trigger factor and several Krebs cycle enzymes, suggesting that the chaperone properties of YbbN are more important in vivo than its redox properties. YbbN specifically interacts with DnaK and GroEL, as shown by reverse purification. It increases 4-fold the rate of protein renaturation in vitro by the DnaK chaperone machine, suggesting that it cooperates with DnaK for the optimal expression of several cytoplasmic proteins.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Temperatura Alta , Chaperonas Moleculares/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Tiorredoxinas/metabolismo , Membrana Celular/enzimologia , Citoplasma/enzimologia , Dissulfetos/metabolismo , Escherichia coli/enzimologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Choque Térmico HSP70/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/isolamento & purificação , Mutação , Estresse Oxidativo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/isolamento & purificação , Periplasma/enzimologia , Proteômica , Tiorredoxinas/genética , Tiorredoxinas/isolamento & purificaçãoRESUMO
We cloned, overexpressed and purified the Escherichia coli yeaG gene product, whose amino acid sequence displays homology to prokaryotic serine protein kinases. The gene coding for YeaG was generated by amplifying the yeaG gene from E. coli by polymerase chain reaction. It was inserted into the expression plasmid pET-21a, under the transcriptional control of the bacteriophage T7 promoter and lac operator. A BL21(DE3) E. coli strain transformed with the YeaG-expression vector pET-21a-yeaG accumulates large amounts of a soluble protein with a molecular mass of 76kDa in SDS-PAGE, which matches the expected YeaG molecular weight of 74.5kDa. YeaG, although soluble, has a marked tendency to aggregate in the absence of detergents, so that it was purified in the presence of 0.1% Triton X-100, by ion exchange chromatography and hydroxyapatite chromatography. The purified protein is monomeric and displays autokinase and casein kinase activities which are optimal in the presence of 10mM Mn(2+). The purification of the active protein kinase YeaG described in this study should allow us to characterize its biochemical target(s) in E. coli extracts.
Assuntos
Proteínas de Escherichia coli/biossíntese , Proteínas Serina-Treonina Quinases/biossíntese , Caseínas/metabolismo , Cromatografia por Troca Iônica , Clonagem Molecular , Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/metabolismo , Peso Molecular , Fosforilação , Proteínas Serina-Treonina Quinases/isolamento & purificação , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
The "Bioénergétique et Ingénierie des Protéines (BIP)" laboratory, CNRS (France), organized its first French workshop on molecular chaperone proteins and protein folding in November 2017. The goal of this workshop was to gather scientists working in France on chaperone proteins and protein folding. This initiative was a great success with excellent talks and fruitful discussions. The highlights were on the description of unexpected functions and post-translational regulation of known molecular chaperones (such as Hsp90, Hsp33, SecB, GroEL) and on state-of-the-art methods to tackle questions related to this theme, including Cryo-electron microscopy, Nuclear Magnetic Resonance (NMR), Electron Paramagnetic Resonance (EPR), simulation and modeling. We expect to organize a second workshop in two years that will include more scientists working in France in the chaperone field.