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1.
FEBS J ; 286(6): 1214-1229, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30633437

RESUMO

The ammonium-dependent posttranslational regulation of nitrogenase activity in Azospirillum brasilense requires dinitrogenase reductase ADP-ribosyl transferase (DraT) and dinitrogenase reductase ADP-glycohydrolase (DraG). These enzymes are reciprocally regulated by interaction with the PII proteins, GlnB and GlnZ. In this study, purified ADP-ribosylated Fe-protein was used as substrate to study the mechanism involved in the regulation of A. brasilense DraG in vitro. The data show that DraG is partially inhibited by GlnZ and that DraG inhibition is further enhanced by the simultaneous presence of GlnZ and AmtB. These results are the first to demonstrate experimentally that DraG inactivation requires the formation of a ternary DraG-GlnZ-AmtB complex in vitro. Previous structural data have revealed that when the DraG-GlnZ complex associates with AmtB, the flexible T-loops of the trimeric GlnZ bind to AmtB and become rigid; these molecular events stabilize the DraG-GlnZ complex, resulting in DraG inactivation. To determine whether restraining the flexibility of the GlnZ T-loops is a limiting factor in DraG inhibition, we used a GlnZ variant that carries a partial deletion of the T-loop (GlnZΔ42-54). However, although the GlnZΔ42-54 variant was more effective in inhibiting DraG in vitro, it bound to DraG with a slightly lower affinity than does wild-type GlnZ and was not competent to completely inhibit DraG activity either in vitro or in vivo. We, therefore, conclude that the formation of a ternary complex between DraG-GlnZ-AmtB is necessary for the inactivation of DraG.

2.
EXCLI J ; 16: 949-958, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28900375

RESUMO

The PII protein family constitutes one of the most conserved and well distributed family of signal transduction proteins in nature. These proteins play key roles in nitrogen and carbon metabolism. PII function has been well documented in Gram-negative bacteria. However, there are very few reports describing the in vitro properties and function of PII derived from Gram-positive bacteria. Here we present the heterologous expression and efficient purification protocols for untagged PII from three Actinobacteria of medical and biotechnological interest namely: Mycobacterium tuberculosis, Rhodococcus jostii and Streptomyces coelicolor. Circular dichroism and gel filtration analysis supported that the purified proteins are correctly folded. The purification protocol described here will facilitate biochemical studies and help to uncover the biochemical functions of PII proteins in Actinobacteria.

3.
Microbiology ; 162(1): 156-63, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26555736

RESUMO

Iron is an essential micronutrient for living organisms as it is involved in a broad variety of important biological processes. However, free iron inside the cell could be potentially toxic, generating hydroxyl radicals through the Fenton reaction. Dps (DNA-binding protein from starved cells) belongs to a subfamily of ferritins and can store iron atoms inside the dodecamer. The presence of a ferroxidase centre, composed of highly conserved residues, is a signature of this protein family. In this study, we analysed the role of two conserved histidine residues (H25 and H37) located at the ferroxidase centre of the Campylobacter jejuni Dps protein by replacing them with glycine residues. The C. jejuni H25G/H37G substituted variant showed reduced iron binding and ferroxidase activities in comparison with wt Dps, while DNA-binding activity remained unaffected. We also found that both CjDps wt and CjDps H25G/H37G were able to bind manganese atoms. These results indicate that the H25 and H37 residues at the ferroxidase centre of C. jejuni Dps are not strictly required for metal binding and oxidation.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Campylobacter jejuni/enzimologia , Ceruloplasmina/química , Ceruloplasmina/metabolismo , Histidina/metabolismo , Ferro/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sítios de Ligação , Campylobacter jejuni/química , Campylobacter jejuni/genética , Ceruloplasmina/genética , Sequência Conservada , Histidina/química , Histidina/genética , Cinética , Dados de Sequência Molecular , Oxirredução
4.
Protein Expr Purif ; 111: 105-10, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25707373

RESUMO

Dps proteins (DNA binding protein from starved cell) form a distinct group within the ferritin superfamily. All Dps members are composed of 12 identical subunits that assemble into a conserved spherical protein shell. Dps oxidize Fe(2+) in a conserved ferroxidase center located at the interface between monomers, the product of the reaction Fe(3+), is then stored inside the protein shell in the form of non-reactive insoluble Fe2O3. The Campylobacter jejuni Dps (CjDps) has been reported to play a plethora of functions, such as DNA binding and protection, iron storage, survival in response to hydrogen peroxide and sulfatide binding. CjDps is also important during biofilm formation and caecal colonization in poultry. In order to facilitate in vitro characterisation of CjDps, it is important to have a simple and reproducible protocol for protein purification. Here we report an observation that CjDps has an unusual high melting temperature. We exploited this property for protein purification by introducing a thermal treatment step which allowed achieving homogeneity by using only two chromatographic steps. Gel filtration chromatography, circular dichroism, mass spectrometry, DNA-binding and iron oxidation analysis confirmed that the CjDps structure and function were unaffected.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Campylobacter jejuni/química , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/isolamento & purificação , Temperatura Alta
5.
Curr Top Microbiol Immunol ; 384: 89-106, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-24934999

RESUMO

Posttranslational modification of proteins plays a key role in the regulation of a plethora of metabolic functions. Protein modification by mono-ADP-ribosylation was first described as a mechanism of action of bacterial toxins. Since these pioneering studies, the number of pathways regulated by ADP-ribosylation in organisms from all domains of life expanded significantly. However, in only a few cases the full regulatory ADP-ribosylation circuit is known. Here, we review the system where mono-ADP-ribosylation regulates the activity of an enzyme: the regulation of nitrogenase in bacteria. When the nitrogenase product, ammonium, becomes available, the ADP-ribosyltransferase (DraT) covalently links an ADP-ribose moiety to a specific arginine residue on nitrogenase switching-off nitrogenase activity. After ammonium exhaustion, the ADP-ribosylhydrolase (DraG) removes the modifying group, restoring nitrogenase activity. DraT and DraG activities are reversibly regulated through interaction with PII signaling proteins . Bioinformatics analysis showed that DraT homologs are restricted to a few nitrogen-fixing bacteria while DraG homologs are widespread in Nature. Structural comparisons indicated that bacterial DraG is closely related to Archaea and mammalian ADP-ribosylhydrolases (ARH). In all available structures, the ARH active site consists of a hydrophilic cleft carrying a binuclear Mg(2+) or Mn(2+) cluster, which is critical for catalysis.


Assuntos
Adenosina Difosfato Ribose/metabolismo , Bactérias/enzimologia , Proteínas de Bactérias/metabolismo , Eucariotos/enzimologia , Nitrogenase/metabolismo , ADP Ribose Transferases/genética , ADP Ribose Transferases/metabolismo , Animais , Bactérias/química , Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Eucariotos/química , Eucariotos/genética , Regulação Enzimológica da Expressão Gênica , Humanos , Nitrogenase/química , Nitrogenase/genética , Processamento de Proteína Pós-Traducional
6.
Nat Prod Commun ; 9(10): 1457-60, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25522535

RESUMO

Flavones have received considerable attention because of their antiproliferative properties and selective effects on cancer cells, making them good candidates for use in cancer therapy. In contrast to other flavones, little is known about the effects of the flavone core structure (2-phenyl-4H-1-benzopyran-4one) on cancer cells. Here, we report that flavone induces cell death in human hepatoma HepG2 cells. Furthermore, annexin-V+/PI- and SubG1 populations of HepG2 cells increased after flavone treatment. Exposure of HepG2 to flavone did not result in either cytochrome c release into the cytosol or changes in the mitochondrial membrane potential. Treatment of HepG2 cells with flavone for 24 h reduced the accumulation of intracellular ROS, which correlated with upregulation of Gred, CuZnSOD and MnSOD mRNA levels. Taken together, our results provided useful insights into the mechanism of cell death caused by flavones, in order to evaluate their future application in hepatocarcinoma therapy.


Assuntos
Morte Celular/efeitos dos fármacos , Flavonas/farmacologia , Citocromos c/metabolismo , Células Hep G2 , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo
7.
J Bacteriol ; 195(2): 279-86, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23144248

RESUMO

Fe protein (dinitrogenase reductase) activity is reversibly inactivated by dinitrogenase reductase ADP-ribosyltransferase (DraT) in response to an increase in the ammonium concentration or a decrease in cellular energy in Azospirillum brasilense, Rhodospirillum rubrum, and Rhodobacter capsulatus. The ADP-ribosyl is removed by the dinitrogenase reductase-activating glycohydrolase (DraG), promoting Fe protein reactivation. The signaling pathway leading to DraT activation by ammonium is still not completely understood, but the available evidence shows the involvement of direct interaction between the enzyme and the nitrogen-signaling P(II) proteins. In A. brasilense, two P(II) proteins, GlnB and GlnZ, were identified. We used Fe protein from Azotobacter vinelandii as the substrate to assess the activity of A. brasilense DraT in vitro complexed or not with P(II) proteins. Under our conditions, GlnB was necessary for DraT activity in the presence of Mg-ADP. The P(II) effector 2-oxoglutarate, in the presence of Mg-ATP, inhibited DraT-GlnB activity, possibly by inducing complex dissociation. DraT was also activated by GlnZ and by both uridylylated P(II) proteins, but not by a GlnB variant carrying a partial deletion of the T loop. Kinetics studies revealed that the A. brasilense DraT-GlnB complex was at least 18-fold more efficient than DraT purified from R. rubrum, but with a similar K(m) value for NAD(+). Our results showed that ADP-ribosylation of the Fe protein does not affect the electronic state of its metal cluster and prevents association between the Fe and MoFe proteins, thus inhibiting electron transfer.


Assuntos
ADP Ribose Transferases/metabolismo , Azospirillum brasilense/enzimologia , Proteínas de Bactérias/metabolismo , Proteínas PII Reguladoras de Nitrogênio/metabolismo , ADP Ribose Transferases/isolamento & purificação , Difosfato de Adenosina/metabolismo , Azotobacter vinelandii/enzimologia , Coenzimas , Inibidores Enzimáticos/metabolismo , Ácidos Cetoglutáricos/metabolismo , Cinética , Magnésio/metabolismo , NAD/metabolismo , Oxirredutases/isolamento & purificação , Oxirredutases/metabolismo , Ligação Proteica
8.
Proc Natl Acad Sci U S A ; 109(48): 19644-8, 2012 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-23150564

RESUMO

A doubly substituted form of the nitrogenase MoFe protein (α-70(Val)(→Ala), α-195(His→Gln)) has the capacity to catalyze the reduction of carbon dioxide (CO(2)) to yield methane (CH(4)). Under optimized conditions, 1 nmol of the substituted MoFe protein catalyzes the formation of 21 nmol of CH(4) within 20 min. The catalytic rate depends on the partial pressure of CO(2) (or concentration of HCO(3)(-)) and the electron flux through nitrogenase. The doubly substituted MoFe protein also has the capacity to catalyze the unprecedented formation of propylene (H(2)C = CH-CH(3)) through the reductive coupling of CO(2) and acetylene (HC≡CH). In light of these observations, we suggest that an emerging understanding of the mechanistic features of nitrogenase could be relevant to the design of synthetic catalysts for CO(2) sequestration and formation of olefins.


Assuntos
Acetileno/química , Alcenos/química , Dióxido de Carbono/química , Metano/química , Nitrogenase/química , Catálise , Modelos Moleculares , Oxirredução
9.
Protein Expr Purif ; 81(1): 83-88, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21963770

RESUMO

The P(II) proteins comprise a family of widely distributed signal transduction proteins that integrate the signals of cellular nitrogen, carbon and energy status, and then regulate, by protein-protein interaction, the activity of a variety of target proteins including enzymes, transcriptional regulators and membrane transporters. We have previously shown that the P(II) proteins from Azospirillum brasilense, GlnB and GlnZ, do not alter their migration behavior under native gel electrophoresis following incubated for a few minutes at 95°C. This data suggested that P(II) proteins were either resistant to high temperatures and/or that they could return to their native state after having been unfolded by heat. Here we used (1)H NMR to show that the A. brasilense GlnB is stable up to 70°C. The melting temperature (Tm) of GlnB was determined to be 84°C using the fluorescent dye Sypro-Orange. P(II) proteins from other Proteobacteria also showed a high Tm. We exploited the thermo stability of P(II) by introducing a thermal treatment step in the P(II) purification protocol, this step significantly improved the homogeneity of A. brasilense GlnB and GlnZ, Herbaspirillum seropedicae GlnB and GlnK, and of Escherichia coli GlnK. Only a single chromatography step was necessary to obtain homogeneities higher than 95%. NMR(1) and in vitro uridylylation analysis showed that A. brasilense GlnB purified using the thermal treatment maintained its folding and activity. The purification protocol described here can facilitate the study of P(II) protein family members.


Assuntos
Proteínas de Bactérias/química , Cromatografia de Afinidade/métodos , Proteínas PII Reguladoras de Nitrogênio/química , Azospirillum brasilense/enzimologia , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Ressonância Magnética Nuclear Biomolecular , Proteínas PII Reguladoras de Nitrogênio/isolamento & purificação , Proteínas PII Reguladoras de Nitrogênio/metabolismo , Conformação Proteica , Multimerização Proteica , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Temperatura de Transição
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