RESUMO
We studied an original radiolabeled complex of antimicrobial peptides UBI29-41 and UBI18-35, ubiquicidin derivatives, for distinguishing between bacterial and aseptic inflammation. For radiolabeling of the peptides with technetium-99m, a bifunctional chelating agent succinimide-1-yl 6-(bis(pyridin-2-ylmethyl)amino)hexanoate was used. The obtained complexes 99mТÑ-DPAH-UBI29-41 and 99mТÑ-DPAH-UBI18-35 had radiolabeling yield >80% and radiochemical purity >96%. Accumulation of the complexes in the focus of bacterial inflammation in bone structures and the absence of this complex in the site of aseptic inflammation was confirmed in a rat model of traumatic osteomyelitis by single-photon emission computed tomography.
Assuntos
Osteomielite/tratamento farmacológico , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Ribossômicas/química , Tecnécio/química , Animais , Diagnóstico Diferencial , Feminino , Ratos , Ratos Wistar , Tomografia Computadorizada de Emissão de Fóton ÚnicoRESUMO
Problems arising during treatment of tuberculosis are well known, therefore studies of Mycobacterium drug molecular targets are an area of particular importance. Members of the cytochrome P450 family (CYP) may belong to potential candidates for drug targets being involved in metabolism of biologically important molecules in the host organism. CYP124 of Mycobacterium tuberculosis (MTCYP124) catalyzes ω-hydroxylation of methyl-branched lipids. The data obtained in the present study indicate that this enzyme can also oxidize provitamin D3 (7-dehydrocholesterol) and vitamin D3. We found that the final product is different from 1α- and 25-hydroxyvitamin D3, so we propose that MTCYP124 is involved in alternative pathway for metabolism of vitamin D3.
Assuntos
Proteínas de Bactérias/metabolismo , Colecalciferol/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Desidrocolesteróis/metabolismo , Mycobacterium tuberculosis/enzimologia , Catálise , Domínio Catalítico , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Cristalografia por Raios X , Ligantes , Espectrometria de Massas , Especificidade por SubstratoRESUMO
Contaminating proteins have been identified by "shotgun" proteomic analysis in 14 recombinant preparations of human membrane heme- and flavoproteins expressed in Escherichia coli and purified by immobilized metal ion affinity chromatography. Immobilized metal ion affinity chromatography of ten proteins was performed on Ni2+-NTA-sepharose 6B, and the remaining four proteins were purified by ligand affinity chromatography on 2',5'-ADP-sepharose 4B. Proteomic analysis allowed to detect 50 protein impurities from E. coli. The most common contaminant was Elongation factor Tu2. It is characterized by a large dipole moment and a cluster arrangement of acidic amino acid residues that mediate the specific interaction with the sorbent. Peptidyl prolyl-cis-trans isomerase SlyD, glutamine-fructose-6-phosphate aminotransferase, and catalase HPII that contained repeating HxH, QxQ, and RxR fragments capable of specific interaction with the sorbent were identified among the protein contaminants as well. GroL/GroS chaperonins were probably copurified due to the formation of complexes with the target proteins. The Ni2+ cations leakage from the sorbent during lead to formation of free carboxyl groups that is the reason of cation exchanger properties of the sorbent. This was the putative reason for the copurification of basic proteins, such as the ribosomal proteins of E. coli and the widely occurring uncharacterized protein YqjD. The results of the analysis revealed variation in the contaminant composition related to the type of protein expressed. This is probably related to the reaction of E. coli cell proteome to the expression of a foreign protein. We concluded that the nature of the protein contaminants in a preparation of a recombinant protein purified by immobilized metal ion affinity chromatography on a certain sorbent could be predicted if information on the host cell proteome were available.
Assuntos
Cromatografia de Afinidade/métodos , Proteínas de Escherichia coli/isolamento & purificação , Flavoproteínas/isolamento & purificação , Hemeproteínas/isolamento & purificação , Proteômica/métodos , Sequência de Aminoácidos , Catalase/isolamento & purificação , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Flavoproteínas/genética , Flavoproteínas/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/isolamento & purificação , Proteínas de Choque Térmico/isolamento & purificação , Hemeproteínas/genética , Hemeproteínas/metabolismo , Humanos , Fator Tu de Elongação de Peptídeos/isolamento & purificação , Peptidilprolil Isomerase/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Ribossômicas/isolamento & purificação , Sefarose/análogos & derivados , Sefarose/químicaRESUMO
The exposure to anthropogenic factors has a multifaceted impact on the body of humans and animals. Due to their complex influence the detection of negative effects of the certain factors is a rather complicated task. Metabolomic methodology which permits to overcome these difficulties, has been applied in the evaluation of the nature and degree of the impact of potash fertilizers production waste on lipid profiles of experimental animals after intranasal inoculation with potassic fertilizer production waste and consumption of drinking water obtained from sources located in the zone of potential action of potassic fertilizer production. Isolation of lipids from serum was performed with the help of specially developed technique based on solid-phase extraction of samples which allows to remove cholesterin from the samples. Each sample was subjected to HPLC -MS analysis, after which the obtained chromatograms were treated with the use of the method of principal component analysis and cluster analysis. The developed technique allows to efficiently separate hydrophobic metabolites in blood serum. There was established serum lipid profile of experimental animals, in particular the content of phospholipids and oxysteroids, and there were found differences in the metabolic processes of the test and control animals. It is shown that in the serum of experimental animals, there is observed an increased concentration of hydroxysteroid as compared with the control group.
Assuntos
Exposição Ambiental/efeitos adversos , Poluentes Ambientais/toxicidade , Lipídeos/sangue , Animais , Cromatografia Líquida de Alta Pressão , Análise por Conglomerados , Misturas Complexas/química , Misturas Complexas/toxicidade , Água Potável/química , Poluentes Ambientais/química , Feminino , Fertilizantes/toxicidade , Cobaias , Masculino , Espectrometria de Massas , Metabolômica/métodos , Fosfolipídeos/sangue , Análise de Componente PrincipalRESUMO
CYP17 (steroid 17α-hydroxylase/17,20-lyase) is a key enzyme in steroid hormone biosynthesis. It catalyzes two independent reactions at the same active center and has a unique ability to differentiate Δ(4)-steroids and Δ(5)-steroids in the 17,20-lyase reaction. The present work presents a complex experimental analysis of the role of CYP17 in the metabolism of 7-dehydrosteroids. The data indicate the existence of a possible alternative pathway of steroid hormone biosynthesis using 7-dehydrosteroids. The major reaction products of CYP17 catalyzed hydroxylation of 7-dehydropregnenolone have been identified. Catalytic activity of CYP17 from different species with 7-dehydropregnenolone has been estimated. It is shown that CYP21 cannot use Δ(5)-Δ(7) steroids as a substrate.
Assuntos
Microssomos/enzimologia , Esteroide 17-alfa-Hidroxilase/metabolismo , Esteroide 21-Hidroxilase/metabolismo , Esteroides/biossíntese , Biocatálise , Humanos , Cinética , Microssomos/química , Microssomos/metabolismo , Esteroide 17-alfa-Hidroxilase/química , Esteroide 17-alfa-Hidroxilase/genética , Esteroide 21-Hidroxilase/química , Esteroide 21-Hidroxilase/genética , Esteroides/química , Especificidade por SubstratoRESUMO
In the present paper we describe studies on molecular mechanisms of protein-protein interactions between cytochrome P450 3A4 (CYP3A4) and cytochrome b(5), the latter being incorporated into the artificial recombinant protein Hmwb(5)-EGFP containing full-length cytochrome b(5) (functional module) and a mutant form of the green fluorescent protein EGFP (signal module) fused into a single polypeptide chain. It is shown that cytochrome b(5) within the fusion protein Hmwb(5)-EGFP can be reduced by NADPH-cytochrome P450 reductase in the presence of NADPH, the rate of reduction being dependent on solution ionic strength, indicating that the signal module does not prevent the interaction of the flavo- and hemeproteins. Interaction of cytochrome P450 3A4 and Hmwb(5)-EGFP was estimated based on spin equilibrium shift of cytochrome P450 3A4 to high-spin state in the presence of Hmwb(5)-EGFP, as well as based on steady-state fluorescence anisotropy of the EGFP component of the fusion protein in the presence of CYP3A4. The engineering of chimeric protein Hmwb(5)-EGFP gives an independent method to determine dissociation constant for the complex of cytochrome P450 and cytochrome b(5) that is less sensitive to environmental factors compared to spectrophotometric titration used before. Reconstitution of catalytic activity of cytochrome P450 3A4 in the reaction of testosterone 6beta-hydroxylation in the presence of Hmwb(5)-EGFP indicates that cytochrome b(5) in the fusion protein is able to stimulate the hydroxylation reaction. Using other fusion proteins containing either cytochrome b(5) or its hydrophilic domain to reconstitute catalytic activity of cytochrome P450 3A4 showed that the hydrophobic domain of cytochrome b(5) participates not only in hemeprotein interaction, but also in electron transfer from cytochrome b(5) to cytochrome P450.
Assuntos
Sistema Enzimático do Citocromo P-450/química , Citocromos b5/química , Sequência de Aminoácidos , Animais , Catálise , Citocromo P-450 CYP3A , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Citocromos b5/genética , Citocromos b5/metabolismo , Transporte de Elétrons , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Cinética , Conformação Molecular , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de SequênciaRESUMO
The conformational stabilities of chimeric protein Hmwb(5)-EGFP and its constituents (cytochrome b(5) and enhanced green fluorescent protein) in guanidine hydrochloride solutions are reported in this paper. Intensity of fluorescence of tryptophan residues, intensity of EGFP fluorescence in the visible region, absorbance of cytochrome b(5) heme and EGFP fluorophore, and fluorescence anisotropy were used to follow the unfolding process. Thermodynamic parameters of protein unfolding were obtained using different approaches. The data were analyzed using a two-stage model and a linear extrapolation method. Unfolding of protein molecules was additionally monitored by measuring Stern-Volmer constants for tryptophan fluorescence quenching by acrylamide, cesium, and iodide. The accessibility of tryptophan residues of both components in the fusion molecule is lower than in the separate molecules. The thermodynamic stability of the protein globules in the fusion protein is much lower than in the individual protein molecules in solution, the difference in free energy of unfolding being more considerable for cytochrome b(5) (29 +/- 4 and 13 +/- 2 kJ/mol) than for EGFP (26 +/- 0.9 and 20 +/- 2.7 kJ/mol). The data indicate that artificial protein fusion can greatly affect total structural stability, and in the case of cytochrome b(5) and EGFP it results in decrease in free energy of transition from native to denatured unfolded form and consequently to decrease in thermodynamic stability of protein globules compared to the separate proteins.
Assuntos
Citocromos b5/química , Proteínas de Fluorescência Verde/química , Animais , Citocromos b5/genética , Citocromos b5/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Conformação Proteica , Dobramento de Proteína , Estabilidade Proteica , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
In the present work we summarize results on construction of expression plasmid, heterologous expression in Escherichia coli, isolation and purification, as well as physicochemical characterization of chimeric protein consisting of hydrophilic domain of cytochrome b(5) and truncated from the N-terminal sequence (Delta(23)) form of NADH-cytochrome b(5) reductase. The kinetics and mechanism of electron transfer between NADH-cytochrome b(5) reductase and cytochrome b(5) in the frames of fusion protein consisting of cytochrome b(5) (94 amino acids) and truncated form of NADH-cytochrome b(5) reductase (277 amino acids) have been studied. It is shown that electron transfer takes place between redox partners belonging to two different molecules of the chimeric protein. Using computer modeling, we built the model of the tertiary structure of the fusion protein, which is in agreement with experimental data. By using Marcus theory of electron transfer in polar media, we demonstrate the inability of the hypothesis of electrostatic repulsions to explain the increase of electron transfer rate on increase of ion concentration in media due to elimination of the repulsion of similar charges. The real reason for the increase of the first order rate constant in some oxidation-reduction reactions between proteins, as shown in the present work, is a decrease of the media reorganization energy resulting in decrease of activation energy for oxidation-reduction reactions.
Assuntos
Citocromo-B(5) Redutase/genética , Citocromo-B(5) Redutase/metabolismo , Citocromos b5/genética , Citocromos b5/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Animais , Clonagem Molecular , Transporte de Elétrons/fisiologia , Escherichia coli/genética , Escherichia coli/metabolismo , Cinética , Oxirredução , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , TermodinâmicaRESUMO
In this article we report on construction of expression vector, heterologous expression in Escherichia coli, isolation, purification, and physicochemical characterization of an artificial chimeric protein HMWb(5)-EGFP consisting of full-length cytochrome b(5) (HMWb(5)) and green fluorescence protein (EGFP) from Aequorea. Optimization of expression conditions yielded an expression level up to 1500 nmol of chimeric protein per liter of culture. Recombinant chimeric protein HMWb(5)-EGFP was purified from cell membranes by using metal-affinity chromatography. It possesses physicochemical, spectral, and fluorescence properties of cytochrome b(5) and EGFP indicating independent character of protein folding in frames of the chimera. It is shown that there is a fluorescent resonance energy transfer in HMWb(5)-EGFP between the fluorophore of EGFP and heme of cytochrome b(5), and the distance between chromophores in the chimeric protein is approximately 67.3 A. The chimeric protein was shown to exist as a monomer in aqueous solution in the presence of detergents. The data indicate that the HMWb(5)-EGFP designed in the present work is a very promising model for modern biosensors and an instrument to study protein-protein interactions.