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1.
Molecules ; 24(24)2019 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-31842508

RESUMO

Biofilm-associated infections are difficult to manage or treat as biofilms or biofilm-embedded bacteria are difficult to eradicate. Antimicrobial peptides have gained increasing attention as a possible alternative to conventional drugs to combat drug-resistant microorganisms because they inhibit the growth of planktonic bacteria by disrupting the cytoplasmic membrane. The current study investigated the effects of synthetic peptides (PS1-2, PS1-5, and PS1-6) and conventional antibiotics on the growth, biofilm formation, and biofilm reduction of drug-resistant Pseudomonas aeruginosa and Staphylococcus aureus. The effects of PS1-2, PS1-5, and PS1-6 were also tested in vivo using a mouse model. All peptides inhibited planktonic cell growth and biofilm formation in a dose-dependent manner. They also reduced preformed biofilm masses by removing the carbohydrates, extracellular DNA, and lipids that comprised extracellular polymeric substances (EPSs) but did not affect proteins. In vivo, PS1-2 showed the greatest efficacy against preformed biofilms with no cytotoxicity. Our findings indicate that the PS1-2 peptide has potential as a next-generation therapeutic drug to overcome multidrug resistance and to regulate inflammatory response in biofilm-associated infections.


Assuntos
Anti-Infecciosos , Peptídeos Catiônicos Antimicrobianos , Biofilmes/efeitos dos fármacos , Farmacorresistência Bacteriana/efeitos dos fármacos , Plâncton/fisiologia , Pseudomonas aeruginosa/fisiologia , Staphylococcus aureus/fisiologia , Anti-Infecciosos/síntese química , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/síntese química , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Biofilmes/crescimento & desenvolvimento
2.
Biochem Biophys Res Commun ; 505(2): 478-484, 2018 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-30268502

RESUMO

The increasing emergence of drug-resistant bacteria creates a requirement for new antibiotics and various types of antibiotic materials such as proteins, peptides, polymers, and chemical compounds. Among these, antimicrobial peptides (AMPs) are considered to be promising antibiotic candidates for clinical treatments. In this study, we have designed a novel series of peptides with repeated sequences of minimum membrane-active motif, 'XWZX' basic sequence (X: lysine or arginine, Z: leucine, tyrosine, valine, or glycine), and an α-helical secondary structure. Some peptides displayed a potent antibacterial activity via membranolytic action and high therapeutic index (toxic dose/minimum inhibitory concentration) in vitro. Furthermore, in vivo experiments using bacterial ear-skin infection models verified that these peptides have the potential to be powerful and safe antibiotics. The present study provides a lead sequence for designing peptide antibiotics against bacterial membranes and information for cell-selectivity of hydrophobic amino acids with aromatic side chains such as Trp and Tyr.


Assuntos
Antibacterianos/química , Peptídeos/química , Peptídeos/farmacologia , Triptofano/química , Tirosina/química , Animais , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Bactérias/efeitos dos fármacos , Bactérias/metabolismo , Linhagem Celular , Permeabilidade da Membrana Celular , Farmacorresistência Bacteriana , Humanos , Lipossomos , Camundongos Endogâmicos BALB C , Camundongos Nus , Peptídeos/metabolismo , Peptídeos/uso terapêutico , Conformação Proteica em alfa-Hélice , Estrutura Secundária de Proteína , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/patologia , Staphylococcus aureus
3.
Int J Mol Sci ; 19(12)2018 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-30486512

RESUMO

Increases in the numbers of immunocompromised patients and the emergence of drug-resistance fungal pathogens have led to the need for new, safe, efficacious antifungal agents. In this study, we designed a histidine-lysine-lysine (HKK) motif and synthesized six HKK peptides with repetitions of the motif. These peptides showed length-dependent antifungal activity against drug-susceptible and drug-resistant fungal pathogens via membranolytic or non-membranolytic action. None of the peptides were cytotoxic to rat erythrocytes or NIH3T3 mouse embryonic fibroblasts. Short-length peptides were directly translocated in fungal cytosol and reacted with mitochondria, resulting in apoptosis. Membrane-permeabilizing activity occurred in the presence of long peptides, and peptides were able to transfer to the cytosol and induce reactive oxygen species. Our results suggest that peptides composed only of cationic amino acids may be good candidates as antifungal agents.


Assuntos
Antifúngicos/química , Histidina/química , Lisina/química , Peptídeos/química , Peptídeos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Células NIH 3T3 , Ratos , Espécies Reativas de Oxigênio/metabolismo , Relação Estrutura-Atividade
4.
Extremophiles ; 18(3): 537-44, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24638259

RESUMO

AAA(+) ATPases are ubiquitous enzymes that can function as molecular chaperones, employing the energy obtained from ATP hydrolysis to remodel macromolecules. In this report, the MoxR enzyme from Thermococcus kodakarensis KOD1 (TkMoxR) was shown to have two native forms: a two-stack hexameric ring and a hexameric structure, under physiological conditions and cold stress, respectively. TkMoxR was altered to a microtubule-like form in the presence of ATP and tightly interacted with dsDNA molecules of various lengths. In addition, the two-stack hexameric protein catalyzed dsDNA decomposition to form and then release ssDNA, whereas the hexamer TkMoxR structure interacted with but did not release dsDNA. These results suggest that TkMoxR has DNA helicase activity involved in gene expression control.


Assuntos
Proteínas Arqueais/metabolismo , DNA Helicases/metabolismo , Thermococcus/enzimologia , Sequência de Aminoácidos , Proteínas Arqueais/química , Proteínas Arqueais/genética , DNA Helicases/química , DNA Helicases/genética , Estabilidade Enzimática , Temperatura Alta , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Thermococcus/genética
5.
Extremophiles ; 17(2): 193-203, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23283522

RESUMO

Enzymes from many archaea colonizing extreme environments are of great interest because of their potential for various biotechnological processes and scientific value of evolution. Many enzymes from archaea have been reported to catalyze promiscuous reactions or moonlight in different functions. Here, we summarize known archaeal enzymes of both groups that include different kinds of proteins. Knowledge of their biochemical properties and three-dimensional structures has proved invaluable in understanding mechanism, application, and evolutionary implications of this manifestation. In addition, the review also summarizes the methods to unravel the extra function which almost was discovered serendipitously. The study of these amazing enzymes will provide clues to optimize protein engineering applications and how enzymes might have evolved on Earth.


Assuntos
Archaea/enzimologia , Archaea/genética , Evolução Molecular , Glicólise , Hidrolases/química , Hidrolases/genética , Hidrolases/metabolismo , Via de Pentose Fosfato , Estrutura Terciária de Proteína , Transferases/química , Transferases/genética , Transferases/metabolismo
6.
Extremophiles ; 16(1): 87-93, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22083128

RESUMO

Sarcosine oxidase (SOX) catalyzes the oxidation of the methyl group in sarcosine and transfer of the oxidized methyl group into the one-carbon metabolic pool. Here, we separately cloned and expressed α and ß subunit of SOX from Thermococcus kodakarensis KOD1 (TkSOX) in Escherichia coli and the recombinant proteins were purified to homogeneity. Gel filtration chromatography and transmission electron microscopy analysis showed that the α subunit formed a dimeric structure and behaved as an NADH dehydrogenase; ß subunit was a tetramer that had sarcosine oxidase and L: -proline dehydrogenase activity. The TkSOX complex assembled into the hetero-octameric (αß)(4) form and had NADH dehydrogenase activity. Gold-label analysis indicated that α and ß subunits were oriented in the alternative form. Based on these results, we suggested that TkSOX was a multifunctional enzyme and that each subunit and (αß)(4) complex may separately exist as a function enzyme in different conditions.


Assuntos
Sarcosina Oxidase/metabolismo , Thermococcus/enzimologia , Sequência de Bases , Biocatálise , Cromatografia em Gel , Primers do DNA , Microscopia Eletrônica , Reação em Cadeia da Polimerase , Sarcosina Oxidase/química
7.
Extremophiles ; 16(4): 619-25, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22622648

RESUMO

Phospholipases can catalyze the hydrolysis of one or more ester and phosphodiester bonds and have a considerable interest in the food, oil leather and pharmaceutical industries. In this report, a lysophospholipase gene from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (LysoPL-tk) was cloned. The gene of 783 bp encodes a 260-amino acid protein with a molecular mass of 29 kDa. LysoPL-tk has a consensus motif (GxSxG) and a catalytic triad (S, D, H) of esterases in the deduced amino acid sequence. LysoPL-tk was expressed in Escherichia coli and purified to homogeneity. The enzyme can degrade substrates with both short and long acyl chain lengths. The apparent K (m) value for p-nitrophenyl butyrate was 607.1 µM with V (max) values of 95.5 U/mg. The enzyme was active at a broad range of pH (5-8) and temperatures (70-95 °C) with the optimum pH and temperature being 8.0 and 85 °C, respectively. The high yield, broad substrate range along with its thermo-stability indicates that LysoPL-tk is a potential enzyme in industrial application.


Assuntos
Proteínas Arqueais/biossíntese , Proteínas Arqueais/química , Lisofosfolipase/biossíntese , Lisofosfolipase/química , Thermococcus/enzimologia , Motivos de Aminoácidos , Proteínas Arqueais/genética , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Temperatura Alta , Concentração de Íons de Hidrogênio , Lisofosfolipase/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Especificidade por Substrato/fisiologia , Thermococcus/genética
8.
Proc Natl Acad Sci U S A ; 106(14): 5978-83, 2009 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-19293385

RESUMO

We found that Arabidopsis AtTDX, a heat-stable and plant-specific thioredoxin (Trx)-like protein, exhibits multiple functions, acting as a disulfide reductase, foldase chaperone, and holdase chaperone. The activity of AtTDX, which contains 3 tetratricopeptide repeat (TPR) domains and a Trx motif, depends on its oligomeric status. The disulfide reductase and foldase chaperone functions predominate when AtTDX occurs in the low molecular weight (LMW) form, whereas the holdase chaperone function predominates in the high molecular weight (HMW) complexes. Because deletion of the TPR domains results in a significant enhancement of AtTDX disulfide reductase activity and complete loss of the holdase chaperone function, our data suggest that the TPR domains of AtTDX block the active site of Trx and play a critical role in promoting the holdase chaperone function. The oligomerization status of AtTDX is reversibly regulated by heat shock, which causes a transition from LMW to HMW complexes with concomitant functional switching from a disulfide reductase and foldase chaperone to a holdase chaperone. Overexpression of AtTDX in Arabidopsis conferred enhanced heat shock resistance to plants, primarily via its holdase chaperone activity.


Assuntos
Proteínas de Arabidopsis/fisiologia , Resposta ao Choque Térmico , Tiorredoxinas/fisiologia , Dimerização , Resposta ao Choque Térmico/genética , Chaperonas Moleculares , Peso Molecular , NADH NADPH Oxirredutases
9.
Extremophiles ; 15(3): 337-46, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21409597

RESUMO

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays an essential role in glycolysis by catalyzing the conversion of D-glyceraldehyde 3-phosphate (D-G3P) to 1,3-diphosphoglycerate using NAD(+) as a cofactor. In this report, the GAPDH gene from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (GAPDH-tk) was cloned and the protein was purified to homogeneity. GAPDH-tk exists as a homotetramer with a native molecular mass of 145 kDa; the subunit molecular mass was 37 kDa. GAPDH-tk is a thermostable protein with a half-life of 5 h at 80-90°C. The apparent K (m) values for NAD(+) and D-G3P were 77.8 ± 7.5 µM and 49.3 ± 3.0 µM, respectively, with V (max) values of 45.1 ± 0.8 U/mg and 59.6 ± 1.3 U/mg, respectively. Transmission electron microscopy (TEM) and image processing confirmed that GAPDH-tk has a tetrameric structure. Interestingly, GAPDH-tk migrates as high molecular mass forms (~232 kDa and ~669 kDa) in response to oxidative stress.


Assuntos
Proteínas Arqueais/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Thermococcus/enzimologia , Sequência de Aminoácidos , Proteínas Arqueais/química , Proteínas Arqueais/genética , Proteínas Arqueais/isolamento & purificação , Domínio Catalítico , Clonagem Molecular , Ácidos Difosfoglicéricos/metabolismo , Estabilidade Enzimática , Gliceraldeído 3-Fosfato/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/química , Gliceraldeído-3-Fosfato Desidrogenases/genética , Gliceraldeído-3-Fosfato Desidrogenases/isolamento & purificação , Meia-Vida , Temperatura Alta , Cinética , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Peso Molecular , Mutação , NAD/metabolismo , Estresse Oxidativo , Estrutura Quaternária de Proteína , Relação Estrutura-Atividade , Thermococcus/genética
10.
Biosci Biotechnol Biochem ; 75(6): 1160-6, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21670507

RESUMO

Deblocking aminopeptidase (DAP) is an exoprotease that can release N-terminal amino acids from blocked peptides. Three DAP homologous (TkDAP1, TkDAP2, and TkDAP3) are annotated in the genome data base of Thermococcus kodakarensis KOD1. TkDAP2 and TkDAP3 were identified as proteins that are overexpressed in response to heat and oxidative stress by two-dimensional electrophoresis. In this study, the TkDAP1 and TkDAP2 genes were cloned and expressed in Escherichia coli. The two proteins were purified homogeneity and analyzed by gel filtration chromatography and electron microscopy. TkDAP1 showed two oligomers, which were identified as an octodecimer and a dodecamer. TkDAP2 produced three native forms: octodecimer, dodecamer, and trimer. Dodecamer assembly was the main form in the two proteins. Finally, TkDAP1 was found to have higher deblocking aminopeptidase activity on the substrates of Ac-Leu-pNA and Ac-Ala-Ala-Ala, while TkDAP2 had higher aminopeptidase activity on the substrates of Leu-pNA and Ala-Ala-Ala-pNA.


Assuntos
Aminopeptidases/metabolismo , Proteínas Arqueais/metabolismo , Isoenzimas/metabolismo , Proteínas Recombinantes/metabolismo , Thermococcus/enzimologia , Sequência de Aminoácidos , Aminopeptidases/química , Aminopeptidases/genética , Aminopeptidases/ultraestrutura , Proteínas Arqueais/química , Proteínas Arqueais/genética , Proteínas Arqueais/ultraestrutura , Cromatografia em Gel , Clonagem Molecular , Eletroforese em Gel Bidimensional , Escherichia coli , Expressão Gênica , Temperatura Alta , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/ultraestrutura , Microscopia Eletrônica , Dados de Sequência Molecular , Oligopeptídeos/metabolismo , Plasmídeos , Polimerização , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/ultraestrutura , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Especificidade por Substrato , Thermococcus/genética , Transformação Bacteriana
11.
Plant Mol Biol ; 72(6): 569-83, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20047069

RESUMO

Sulfite reductase (SiR) performs dual functions, acting as a sulfur assimilation enzyme and as a chloroplast (cp-) nucleoid binding protein. In this study, we examined the in vivo effects of SiR deficiency on chloroplast development in Nicotiana benthamiana. Virus-induced gene silencing of NbSiR resulted in leaf yellowing and growth retardation phenotypes, which were not rescued by cysteine supplementation. NbSiR:GFP fusion protein was targeted to chloroplasts and colocalized with cp-nucleoids. Recombinant full-length NbSiR protein and the C-terminal half of NbSiR possessed cp-DNA compaction activities in vitro, and expression of full-length NbSiR in E. coli caused condensation of genomic DNA. NbSiR silencing differentially affected expression of plastid-encoded genes, inhibiting expression of several genes more severely than others. In the later stages, depletion of NbSiR resulted in chloroplast ablation. In NbSiR-silenced plants, enlarged cp-nucleoids containing an increased amount of cp-DNA were observed in the middle of the abnormal chloroplasts, and the cp-DNAs were predominantly of subgenomic sizes based on pulse field gel electrophoresis. The abnormal chloroplasts developed prolamellar body-like cubic lipid structures in the light without accumulating NADPH:protochlorophyllide oxidoreductase proteins. Our results suggest that NbSiR plays a role in cp-nucleoid metabolism, plastid gene expression, and thylakoid membrane development.


Assuntos
Cloroplastos/genética , Nicotiana/genética , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Proteínas de Plantas/genética , Cloroplastos/metabolismo , Cloroplastos/ultraestrutura , Cisteína/farmacologia , DNA de Plantas/metabolismo , Inativação Gênica , Proteínas de Fluorescência Verde/análise , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestrutura , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/fisiologia , Fenótipo , Proteínas de Plantas/fisiologia , Proteínas Recombinantes de Fusão/análise , Glycine max/genética , Nicotiana/efeitos dos fármacos , Nicotiana/ultraestrutura
12.
Antibiotics (Basel) ; 9(12)2020 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-33352972

RESUMO

Several antimicrobial peptides (AMPs) have been discovered, developed, and purified from natural sources and peptide engineering; however, the clinical applications of these AMPs are limited owing to their lack of abundance and side effects related to cytotoxicity, immunogenicity, and hemolytic activity. Accordingly, to improve cell selectivity for pseudin-2, an AMP from Pseudis paradoxa skin, in mammalian cells and pathogenic fungi, the sequence of pseudin-2 was modified by alanine or lysine at each position of two amino acids within the leucine-zipper motif. Alanine-substituted variants were highly selective toward fungi over HaCaT and erythrocytes and maintained their antifungal activities and mode of action (membranolysis). However, the antifungal activities of lysine-substituted peptides were reduced, and the compound could penetrate into fungal cells, followed by induction of mitochondrial reactive oxygen species and cell death. In vivo antifungal assays of analogous peptide showed excellent antifungal efficiency in a Candida tropicalis skin infection mouse model. Our results demonstrated the usefulness of selective amino acid substitution in the repeated sequence of the leucine-zipper motif for the design of AMPs with potent antimicrobial activities and low toxicity.

13.
Biochim Biophys Acta ; 1784(5): 783-8, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18319068

RESUMO

Osmotically inducible protein C (OsmC) is involved in the cellular defense mechanism against oxidative stress caused by exposure to hyperoxides or elevated osmolarity. OsmC was identified by two-dimensional electrophoresis (2DE) analysis as a protein that is overexpressed in response to osmotic stress, but not under heat and oxidative stress. Here, an OsmC gene from T. kodakaraensis KOD1 was cloned and expressed in Escherichia coli. TkOsmC showed a homotetrameric structure based on gel filtration and electron microscopic analyses. TkOsmC has a significant peroxidase activity toward both organic and inorganic peroxides in high, but not in low temperature.


Assuntos
Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Thermococcus/metabolismo , Sequência de Aminoácidos , Proteínas Arqueais/ultraestrutura , Cromatografia em Gel , Reagentes de Ligações Cruzadas/farmacologia , Temperatura Alta , Peróxido de Hidrogênio/metabolismo , Dados de Sequência Molecular , Pressão Osmótica/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Peroxidase/metabolismo , Estrutura Quaternária de Proteína , Homologia de Sequência de Aminoácidos , Thermococcus/efeitos dos fármacos , Thermococcus/enzimologia , terc-Butil Hidroperóxido/metabolismo
14.
Antioxidants (Basel) ; 8(12)2019 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-31795318

RESUMO

Although there are many antimicrobial proteins in plants, they are not well-explored. Understanding the mechanism of action of plant antifungal proteins (AFPs) may help combat fungal infections that impact crop yields. In this study, we aimed to address this gap by screening Oryza sativa leaves to isolate novel AFPs. We identified a thioredoxin protein with antioxidant properties. Being ubiquitous, thioredoxins (Trxs) function in the redox balance of all living organisms. Sequencing by Edman degradation method revealed the AFP to be O. sativa Thioredoxin m-type isoform (OsTrxm). We purified the recombinant OsTrxm and its cysteine mutant proteins (OsTrxm C/S) in Escherichia coli. The recombinant OsTrxm proteins inhibited the growth of various pathogenic fungal cells. Interestingly, OsTrxm C/S mutant showed higher antifungal activity than OsTrxm. A growth inhibitory assay against various fungal pathogens and yeasts confirmed the pertinent role of cysteine residues. The OsTrxm protein variants penetrated the fungal cell wall and membrane, accumulated in the cells and generated reactive oxygen species. Although the role of OsTrxm in chloroplast development is known, its biochemical and molecular functions have not been elucidated. These findings suggest that in addition to redox regulation, OsTrxm also functions as an antimicrobial agent.

15.
Mol Cells ; 23(2): 252-7, 2007 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-17464204

RESUMO

Escherichia coli HslVU is an ATP-dependent protease consisting of two heat shock proteins, the HslU ATPase and HslV peptidase. In the reconstituted enzyme, HslU stimulates the proteolytic activity of HslV by one to two orders of magnitude, while HslV increases the rate of ATP hydrolysis by HslU several-fold. Here we show that HslV alone can efficiently degrade certain unfolded proteins, such as unfolded lactalbumin and lysozyme prepared by complete reduction of disulfide bonds, but not their native forms. Furthermore, HslV alone cleaved a lactalbumin fragment sandwiched by two thioredoxin molecules, indicating that it can hydrolyze the internal peptide bonds of lactalbumin. Surprisingly, ATP inhibited the degradation of unfolded proteins by HslV. This inhibitory effect of ATP was markedly diminished by substitution of the Arg86 residue located in the apical pore of HslV with Gly, suggesting that interaction of ATP with the Arg residue blocks access of unfolded proteins to the proteolytic chamber of HslV. These results suggest that uncomplexed HslV is inactive under normal conditions, but may can degrade unfolded proteins when the ATP level is low, as it is during carbon starvation.


Assuntos
Trifosfato de Adenosina/farmacologia , Endopeptidases/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Dobramento de Proteína , Proteases Dependentes de ATP/genética , Proteases Dependentes de ATP/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Carbono/metabolismo , Endopeptidases/genética , Ativação Enzimática , Proteínas de Escherichia coli/genética , Proteínas de Choque Térmico/metabolismo , Hidrólise , Lactalbumina/metabolismo , Dados de Sequência Molecular , Muramidase/metabolismo
17.
Mol Cells ; 21(1): 129-34, 2006 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-16511355

RESUMO

Lon, also known as protease La, belongs to a class of ATP-dependent serine protease. It plays an essential role in degradation of abnormal proteins and of certain short-lived regulatory proteins, and is thought to possess a Ser-Lys catalytic dyad. To examine the structural organization of Lon, we performed an electron microscope analysis. The averaged images of Lon with end-on orientation revealed a six-membered, ring-shaped structure with a central cavity. The side-on view showed a two-layered structure with an equal distribution of mass across the equatorial plane of the complex. Since a Lon subunit possesses two large regions containing nucleotide binding and proteolytic domains, each layer of the Lon hexamer appears to consist of the side projections of one of the major domains arranged in a ring. Lon showed a strong tendency to form hexamers in the presence of Mg(2+), but dissociated into monomers and/or dimers in its absence. Moreover, Mg(2+)-dependent hexamer formation was independent of ATP. These results indicate that Lon has a hexameric ring-shaped structure with a central cavity, and that the establishment of this configuration requires Mg(2+), but not ATP.


Assuntos
Proteases Dependentes de ATP/química , Escherichia coli/enzimologia , Polímeros , Protease La/química , Proteases Dependentes de ATP/ultraestrutura , Trifosfato de Adenosina/farmacologia , Escherichia coli/efeitos dos fármacos , Magnésio/farmacologia , Modelos Biológicos , Protease La/ultraestrutura , Estrutura Quaternária de Proteína
18.
Front Microbiol ; 6: 605, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26150806

RESUMO

The thermophilic species, Thermococcus kodakarensis KOD1, a model microorganism for studying hyperthermophiles, has adapted to optimal growth under conditions of high temperature and salinity. However, the environmental conditions for the strain are not always stable, and this strain might face different stresses. In the present study, we compared the proteome response of T. kodakarensis to heat, oxidative, and salt stresses using two-dimensional electrophoresis, and protein spots were identified through MALDI-TOF/MS. Fifty-nine, forty-two, and twenty-nine spots were induced under heat, oxidative, and salt stresses, respectively. Among the up-regulated proteins, four proteins (a hypothetical protein, pyridoxal biosynthesis lyase, peroxiredoxin, and protein disulphide oxidoreductase) were associated with all three stresses. Gene ontology analysis showed that these proteins were primarily involved metabolic and cellular processes. The KEGG pathway analysis suggested that the main metabolic pathways involving these enzymes were related to carbohydrate metabolism, secondary metabolite synthesis, and amino acid biosynthesis. These data might enhance our understanding of the functions and molecular mechanisms of thermophilic Archaea for survival and adaptation in extreme environments.

19.
Protein Pept Lett ; 22(5): 443-8, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25808014

RESUMO

Peroxiredoxins (Prxs) are ubiquitous and conserved proteins that can catalyze the reduction of inorganic and organic hydroperoxides to protect against damage by reactive oxygen species. In this study, a Prx subfamily member, and specifically a bacterioferritin comigratory protein from hyperthermophilic Thermococcus kodakaraensis KOD1 (TkBcp), was overexpressed, purified and characterized. Based on the conserved cysteine (Cys) residues in its amino acids sequence, TkBcp can be grouped into 1-Cys Prx family. Size exclusion chromatography analysis showed that TkBcp exists in three oligomeric forms: 700 kDa, 70 kDa, and 20 kDa. The peroxidase function was found to predominate in the lowmolecular- weight (MW) form, whereas the high-MW complex has the chaperone function. Oxidative reagents caused the protein structure of TkBcp to shift from low-MW form to high-MW complexes, whereas reducing reagents caused a shift in the reverse direction. Furthermore, the high-MW form of TkBcp preferred to tightly bind DNA. The relationship of TkBcp with other homologs was also examined.


Assuntos
Proteínas Arqueais/metabolismo , Proteínas de Bactérias/metabolismo , Grupo dos Citocromos b/metabolismo , Ferritinas/metabolismo , Chaperonas Moleculares/metabolismo , Peroxirredoxinas/metabolismo , Thermococcus/metabolismo , Proteínas Arqueais/química , Proteínas de Bactérias/química , Grupo dos Citocromos b/química , Ferritinas/química , Chaperonas Moleculares/química , Peroxirredoxinas/química , Agregados Proteicos , Thermococcus/química
20.
PLoS One ; 10(5): e0125325, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25933432

RESUMO

Peroxiredoxins (Prxs) act against hydrogen peroxide (H2O2), organic peroxides, and peroxynitrite. Thermococcus kodakaraensis KOD1, an anaerobic archaeon, contains many antioxidant proteins, including three Prxs (Tk0537, Tk0815, and Tk1055). Only Tk0537 has been found to be induced in response to heat, osmotic, and oxidative stress. Tk0537 was found to belong to a 1-Cys Prx6 subfamily based on sequence analysis and was named 1-Cys TkPrx. Using gel filtration chromatography, electron microscopy, and blue-native polyacrylamide gel electrophoresis, we observed that 1-Cys TkPrx exhibits oligomeric forms with reduced peroxide reductase activity as well as decameric and dodecameric forms that can act as molecular chaperones by protecting both proteins and DNA from oxidative stress. Mutational analysis showed that a cysteine residue at the N-terminus (Cys46) was responsible for the peroxide reductase activity, and cysteine residues at the C-terminus (Cys205 and Cys211) were important for oligomerization. Based on our results, we propose that interconversion between different oligomers is important for regulating the different functions of 1-Cys TkPrx.


Assuntos
Proteínas Arqueais/metabolismo , Dano ao DNA , DNA Bacteriano/metabolismo , Chaperonas Moleculares/metabolismo , Peroxirredoxinas/metabolismo , Estresse Fisiológico , Thermococcus/metabolismo , Proteínas Arqueais/química , Clonagem Molecular , Radical Hidroxila/metabolismo , Chaperonas Moleculares/química , Proteínas Mutantes/química , Oxirredução , Peroxidase/metabolismo , Peroxirredoxinas/química , Mutação Puntual , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Análise de Sequência de Proteína , Temperatura , Regulação para Cima
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