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The emergence of antibiotic resistance, caused by the improper use of antibiotics, is a significant challenge in combating infectious diseases, leading to millions of annual fatalities. The occurrence of antimicrobial side effects catalyzes the investigation of novel antimicrobial compounds and sources of drugs. Consequently, the research on biological activity that is conducted on plants, plant extracts, and compounds that are produced from plant components is of utmost significance. In this study, CtAC/MNPs were obtained by the reaction of activated carbon (AC) obtained from the fruits of the Celtis tournefortii (Ct) plant and magnetic nanoparticles (MNPs), and a CtAC/MNPs-Ag nanocomposite was synthesized by the reduction in silver ions added to the reaction. The synthesized CtAC/MNPs and CtAC/MNPs-Ag nanocomposites were analyzed spectroscopically (FTIR, XRD), microscopically (SEM, EDX), optically (DLS), electrochemically (zeta potential) and magnetically (VSM). The antibacterial activities of CtAC/MNPs and CtAC/MNPs-Ag nanocomposites against S. aureus and E. coli were investigated by microdilution method using minimal inhibitory concentration (MIC) and disk diffusion methods. Antioxidant activity study, including total phenolic content and DPPH and cuprac assays, revealed the remarkable effect of the CtAC/MNPs-Ag nanocomposite. This study has the advantages of obtaining CtAC/MNPs and CtAC/MNPs-Ag nanocomposites in a short time without requiring energy, and most importantly, the reaction takes place without using any toxic substances. In addition, according to the data obtained in the study, the CtAC/MNPs-Ag nanocomposite is thought to shed light on biomedical research.
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BACKGROUND: Trichobakin (TBK), a member of type I ribosome-inactivating proteins (RIPs), was first successfully cloned from Trichosanthes sp Bac Kan 8-98 in Vietnam. Previous study has shown that TBK acts as a potential protein synthesis inhibitor; however, the inhibition efficiency and specificity of TBK on cancer cells remain to be fully elucidated. METHODS AND RESULTS: In this work, we employed TBK and TBK conjugated with a part of the amino-terminal fragment (ATF) of the urokinase-type plasminogen activator (uPA), which contains the Ω-loop that primarily interacts with urokinase-type plasminogen activator receptor, and can be a powerful carrier in the drug delivery to cancer cells. Four different human tumor cell lines and BALB/c mice bearing Lewis lung carcinoma cells (LLC) were used to evaluate the role of TBK and ATF-TBK in the inhibition of tumor growth. Here we showed that the obtained ligand fused RIP (ATF-TBK) reduced the growth of four human cancer cell lines in vitro in the uPA receptor level-dependent manner, including the breast adenocarcinoma MDA-MB 231 cells and MCF7 cells, the prostate carcinoma LNCaP cells and the hepatocellular carcinoma HepG2 cells. Furthermore, the conjugate showed anti-tumor activity and prolonged the survival time of tumor-bearing mice. The ATF-TBK also did not cause the death of mice with doses up to 48 mg/kg, and they were not significantly distinct on parameters of hematology and serum biochemistry between the control and experiment groups. CONCLUSIONS: In conclusion, ATF-TBK reduced the growth of four different human tumor cell lines and inhibited lung tumor growth in a mouse model with little side effects. Hence, the ATF-TBK may be a target to consider as an anti-cancer agent for clinical trials.
Assuntos
Neoplasias Pulmonares , Neoplasias da Próstata , Humanos , Masculino , Animais , Camundongos , Ativador de Plasminogênio Tipo Uroquinase , Sistemas de Liberação de Medicamentos , Linhagem Celular TumoralRESUMO
Human InsR, IGF1R, and IRR receptor tyrosine kinases (RTK) of the insulin receptor subfamily play an important role in signaling pathways for a wide range of physiological processes and are directly associated with many pathologies, including neurodegenerative diseases. The disulfide-linked dimeric structure of these receptors is unique among RTKs. Sharing high sequence and structure homology, the receptors differ dramatically in their localization, expression, and functions. In this work, using high-resolution NMR spectroscopy supported by atomistic computer modeling, conformational variability of the transmembrane domains and their interactions with surrounding lipids were found to differ significantly between representatives of the subfamily. Therefore, we suggest that the heterogeneous and highly dynamic membrane environment should be taken into account in the observed diversity of the structural/dynamic organization and mechanisms of activation of InsR, IGF1R, and IRR receptors. This membrane-mediated control of receptor signaling offers an attractive prospect for the development of new targeted therapies for diseases associated with dysfunction of insulin subfamily receptors.
Assuntos
Desenvolvimento de Medicamentos , Receptor de Insulina , Humanos , Domínios Proteicos , Receptor de Insulina/química , Receptor de Insulina/fisiologia , Transdução de SinaisRESUMO
The search of a putative physiological electron acceptor for thiocyanate dehydrogenase (TcDH) newly discovered in the thiocyanate-oxidizing bacteria Thioalkalivibrio paradoxus revealed an unusually large, single-heme cytochrome c (CytC552), which was co-purified with TcDH from the periplasm. Recombinant CytC552, produced in Escherichia coli as a mature protein without a signal peptide, has spectral properties similar to the endogenous protein and serves as an in vitro electron acceptor in the TcDH-catalyzed reaction. The CytC552 structure determined by NMR spectroscopy reveals significant differences compared to those of the typical class I bacterial cytochromes c: a high solvent accessible surface area for the heme group and so-called "intrinsically disordered" nature of the histidine-rich N- and C-terminal regions. Comparison of the signal splitting in the heteronuclear NMR spectra of oxidized, reduced, and TcDH-bound CytC552 reveals the heme axial methionine fluxionality. The TcDH binding site on the CytC552 surface was mapped using NMR chemical shift perturbations. Putative TcDH-CytC552 complexes were reconstructed by the information-driven docking approach and used for the analysis of effective electron transfer pathways. The best pathway includes the electron hopping through His528 and Tyr164 of TcDH, and His83 of CytC552 to the heme group in accordance with pH-dependence of TcDH activity with CytC552.
Assuntos
Heme , Tiocianatos , Grupo dos Citocromos c , Ectothiorhodospiraceae , Escherichia coli/metabolismo , Heme/metabolismo , Espectroscopia de Ressonância Magnética , Oxirredução , Oxirredutases/metabolismoRESUMO
The radiothermometry (RTM) study of a cytochrome-containing system (CYP102 A1) has been conducted in order to demonstrate the applicability of RTM for monitoring changes in the functional activity of an enzyme in case of its point mutation. The study has been performed with the example of the wild-type cytochrome (WT) and its mutant type A264K. CYP102 A1 is a nanoscale protein-enzymatic system of about 10 nm in size. RTM uses a radio detector and can record the corresponding brightness temperature (Tbr) of the nanoscale enzyme solution within the 3.4-4.2 GHz frequency range during enzyme functioning. It was found that the enzymatic reaction during the lauric acid hydroxylation at the wild-type CYP102 A1 (WT) concentration of ~10-9 M is accompanied by Tbr fluctuations of ~0.5-1 °C. At the same time, no Tbr fluctuations are observed for the mutated forms of the enzyme CYP102 A1 (A264K), where one amino acid was replaced. We know that the activity of CYP102 A1 (WT) is ~4 orders of magnitude higher than that of CYP102 A1 (A264K). We therefore concluded that the disappearance of the fluctuation of Tbr CYP102 A1 (A264K) is associated with a decrease in the activity of the enzyme. This effect can be used to develop new methods for testing the activity of the enzyme that do not require additional labels and expensive equipment, in comparison with calorimetry and spectral methods. The RTM is beginning to find application in the diagnosis of oncological diseases and for the analysis of biochemical processes.
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BACKGROUND: Adenosine thiamine triphosphate (AThTP) is a nucleotide discovered in bacteria and some other living organisms more than a decade ago. No biochemical function for AThTP has been established yet, however, experimental data available indicate its possible involvement in metabolic regulation or cell signaling. Metabolism of AThTP in mammals, as well as the feasibility of its pharmacological application, is essentially unstudied. METHODS: Preparative low-pressure chromatography was employed to purify chemically synthesized AThTP with its further analysis by mass spectrometry, HPLC, UV and fluorescence spectroscopy. Enzyme activity assays along with HPLC were used to examine the effects of AThTP and thiamine on vitamin B1 metabolism in the liver of alloxan-induced diabetic rats. RESULTS: An improved procedure for AThTP synthesis and purification is elaborated. Solution stability, optical spectral properties and the molar absorption coefficient for AThTP were determined. The levels of thiamine compounds were found to be increased in the liver of diabetic rats. Neither AThTP nor thiamine treatment affected hepatic vitamin B1 metabolism. Fasting blood glucose concentration was also unchangeable after AThTP or thiamine administration. GENERAL SIGNIFICANCE: Contrast to the widespread view about thiamine deficiency in diabetes, our results clearly shows an adaptive increase in the level of B1 vitamers in the liver of alloxan diabetic rats with no further rising after AThTP or thiamine treatment at a moderate dose. Neither AThTP nor thiamine is effective in glycaemic control. These findings are to be considered in future studies dealing with thiamine or its analogues application to correct metabolic disturbances in diabetes.
Assuntos
Diabetes Mellitus Experimental , Tiamina , Trifosfato de Adenosina , Aloxano/metabolismo , Animais , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Fígado/metabolismo , Mamíferos , Ratos , Tiamina/metabolismo , Tiamina/farmacologia , Tiamina Trifosfato , VitaminasRESUMO
The current article aims to summarize all possible spectrum of protein-protein interactions for thromboxane A synthase (CYP5A1) and prostacyclin synthase (CYP8A1). These enzymes metabolize the same substrate (prostaglandin H2 ) and can participate in cardiovascular, inflammatory, immune processes, and apoptosis modulation, as well as significantly influence the risk of cancers. Binary protein-protein and multiprotein complexes are of great importance in enzyme-regulating and signal-transduction pathways. However, protein partners of CYP5A1 and CYP8A1 are not yet fully identified, although both synthases are considered as prospective drug targets. At least 36 novel protein partners of CYP5A1 and CYP8A1 were revealed from different tissue types using an approach based on affinity isolation and mass spectrometry. Enrichment analysis showed that these proteins have different molecular functions: folding (refolding), unfolded protein and chaperon binding, protein transport (export/import), posttranslational modification, protein domain-specific binding, antioxidant activity, and glutathione homeostasis. A significant part of them, belonging to molecular chaperones, were common partners for CYP5A1 and CYP8A1, while other proteins were unique with the tissue-dependent distribution. New aspects of CYP5A1 and CYP8A1 interactomics and hetero-complex formation with different protein partners, including cytochrome P450s are discussed.
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Sistema Enzimático do Citocromo P-450/metabolismo , Tromboxano-A Sintase/metabolismo , Humanos , Ligantes , Complexos Multiproteicos , Ligação ProteicaRESUMO
Potential drug-drug interactions of the antitumor drug abiraterone and the macrolide antibiotic erythromycin were studied at the stage of cytochrome P450 3A4 (CYP3A4) biotransformation. Using differential spectroscopy, we have shown that abiraterone is a type II ligand of CYP3A4. The dependence of CYP3A4 spectral changes on the concentration of abiraterone is sigmoidal, which indicates cooperative interactions of CYP3A4 with abiraterone; these interactions were confirmed by molecular docking. The dissociation constant (Kd ) and Hill coefficient (h) values for the CYP3A4-abiraterone complex were calculated as 3.8 ± 0.1 µM and 2.3 ± 0.2, respectively. An electrochemical enzymatic system based on CYP3A4 immobilized on a screen-printed electrode was used to show that abiraterone acts as a competitive inhibitor toward erythromycin N-demethylase activity of CYP3A4 (apparent Ki = 8.1 ± 1.2 µM), while erythromycin and its products of enzymatic metabolism do not affect abiraterone N-oxidation by CYP3A4. In conclusion, the inhibition properties of abiraterone toward CYP3A4-dependent N-demethylation of erythromycin and the biologically inert behavior of erythromycin toward abiraterone hydroxylation were demonstrated.
Assuntos
Androstenos/farmacologia , Antibacterianos/farmacocinética , Citocromo P-450 CYP3A/efeitos dos fármacos , Eritromicina/farmacocinética , Antineoplásicos/farmacologia , Citocromo P-450 CYP3A/metabolismo , Inibidores do Citocromo P-450 CYP3A/farmacologia , Interações Medicamentosas , Humanos , Hidroxilação , Simulação de Acoplamento MolecularRESUMO
Trichobakin (TBK) is a type-I ribosome-inactivating protein (RIP-I), acting as an extremely potent inhibitor of protein synthesis in the cell-free translation system of rabbit reticulocyte lysate (IC50: 3.5 pM). In this respect, TBK surpasses the well-studied highly homologous RIP-I trichosanthin (IC50: 20-27 pM), therefore creation of recombinant toxins based on it is of great interest. TBK needs to penetrate into cytosol through the cell membrane and specifically bind to α-sarcin/ricin loop of 28S ribosome RNA to perform the function of specific RNA depurination. At the moment, there is no detailed structural-dynamic information in solution about diverse states RIP-I can adopt at different stages on the way to protein synthesis inhibition. In this work, we report a near-complete assignment of 1H, 13C, and 15N TBK (27.3 kDa) resonances and analysis of the secondary structure based on the experimental chemical shifts data. This work will serve as a basis for further investigations of the structure, dynamics and interactions of the TBK with its molecular partners using NMR techniques.
Assuntos
N-Glicosil Hidrolases/química , Ressonância Magnética Nuclear Biomolecular , Proteínas de Plantas/química , Ribossomos/metabolismo , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Estrutura Secundária de Proteína , Espectroscopia de Prótons por Ressonância MagnéticaRESUMO
Prostacyclin synthase (PTGIS; EC 5.3.99.4) catalyzes isomerization of prostaglandin H2 to prostacyclin, a potent vasodilator and inhibitor of platelet aggregation. At present, limited data exist on functional coupling and possible ways of regulating PTGIS due to insufficient information about protein-protein interactions in which this crucial enzyme is involved. The aim of this study is to isolate protein partners for PTGIS from rat tissue lysates. Using CNBr-activated Sepharose 4B with covalently immobilized PTGIS as an affinity sorbent, we confidently identified 58 unique proteins by mass spectrometry (LC-MS/MS). The participation of these proteins in lysate complex formation was characterized by SEC lysate profiling. Several potential members of the PTGIS subinteractome have been validated by surface plasmon resonance (SPR) analysis. SPR revealed that PTGIS interacted with full-length cytochrome P450 2J2 and glutathione S-transferase (GST). In addition, PTGIS was shown to bind synthetic peptides corresponding to sequences of for GSTA1, GSTM1, aldo-keto reductase (AKR1A1), glutaredoxin 3 (GLRX3) and histidine triad nucleotide binding protein 2 (HINT2). Prostacyclin synthase could potentially be involved in functional interactions with identified novel protein partners participating in iron and heme metabolism, oxidative stress, xenobiotic and drugs metabolism, glutathione and prostaglandin metabolism. The possible biological role of the recognized interaction is discussed in the context of PTGIS functioning.
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An effective oligonucleotide preparation approach for the thermodynamically balanced, inside-out (TBIO) PCR-based assembly of long synthetic DNA molecules (synthons) is described in the current work. We replaced the necessity to purify individual oligonucleotides with just one purification procedure per approximately 500 base pairs (bp) of duplex DNA. So for an enhanced green fluorescent protein (EGFP) gene of 717 bp, we synthesized 24 oligonucleotides with a length of 50 bases and performed just two solid-phase extraction (SPE) purification procedures. It was found that the capacity of ZipTip microextractors, usually used for sample desalting in proteomics, perfectly corresponds to the gene synthesis scale (40-60 pmol). The robustness of the approach was validated with a 65-mer oligonucleotide design of the same gene. The modification of the oligonucleotide concentration gradient from the original TBIO scheme substantially increased the purity of the PCR product. We proposed a mechanism for the formation of supramolecular structures, which often occur during TBIO assembly. By using the proposed workflow, any laboratory with a standard facility for molecular biology manipulation, a 16-channel oligonucleotide synthesizer, and a conventional thermocycler has the ability to prepare one gene with a length of about 700 bp per day.
Assuntos
DNA/síntese química , Escherichia coli/genética , Oligonucleotídeos/síntese química , Reação em Cadeia da Polimerase/métodos , DNA/genética , Proteínas de Escherichia coli/genética , Genes Sintéticos , Proteínas de Fluorescência Verde/genética , Dióxido de SilícioRESUMO
The aim of the present work was to establish the thermodynamic and functional differences in the protein-protein interactions between the components of the P450-dependent mitochondrial (mit) and microsomal (mic) monooxygenase systems using 12 different isoforms of cytochromes P450 and two redox partners, NADPH-dependent cytochrome P450 reductase (CPR) and adrenodoxin (Adx). Comparative analysis of the affinity, thermodynamics, enzymatic activity and the ability for one-electron reduction has been carried out. The study of protein-protein interactions to determine the equilibrium dissociation constants (Kd) was performed using surface plasmon resonance (SPR) biosensor Biacore 3000. We demonstrated that CPR and Adx interacted with both, micCYPs and mitCYPs, with different affinities (Kd values ranged from 0.01 to 2⯵M). All complexes of microsomal (micCYP) and mitochondrial (mitCYP) cytochrome P450 with redox partners can be divided into three groups depending on the prevalent role of either enthalpy or entropy contribution. About 90% of CYP/redox partner complexes were entropy-driven, while the contribution of enthalpy and entropy differed significantly in case of mitCYP/Adx complexes. The CYP11A1/Adx complex was enthalpy-driven, while CYP11B1/Adx and CYP11B2/Adx complexes were entropy-driven. Thermodynamic discrimination of mitCYPs/Adx complexes is likely associated with the different functional impact of CYP11A1 and CYP11B. The exception was the enthalpy-entropy-driven (mixed type) CYP21A2/Adx complex. CPR and Adx were able to transfer the first electron to micCYPs while mitCYPs demonstrated high specificity to Adx. Productive catalysis for mitCYPs observed only in the presence of Adx/AdR pair, while in case of steroidogenic micCYPs (CYP17A1, CYP19A1, and CYP21A2) it was found either in the presence of a CPR or an Adx/AdR pair. From the evolutionary point of view, the type 1 electron transport system (mitCYPs, Adx and NADPH-dependent adrenodoxin reductase (AdR)) increased the specialization of protein-protein interactions (PPI) significantly, which was accompanied by an increase in the specificity of electron transfer. In contrast, the evolution of the type 2 electron transport system (micCYPs and CPR) led to an increase in versatility of PPI as demonstrated for steroidogenic microsomal cytochrome P450s. Our data enhance the current understanding of molecular recognition and summarize qualitative and thermodynamic characteristics of protein-protein interactions in the P450-dependent mitochondrial and microsomal monooxygenase systems.
Assuntos
Sistema Enzimático do Citocromo P-450/química , Domínios e Motivos de Interação entre Proteínas , Adrenodoxina/química , Animais , Transporte de Elétrons , Ferredoxina-NADP Redutase/química , Humanos , Isoenzimas/química , Modelos Moleculares , NADPH-Ferri-Hemoproteína Redutase/química , Oxirredução , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície/métodos , TermodinâmicaRESUMO
CYP2C9 plays a major role in drug metabolism. It is highly polymorphic and among the variants, CYP2C9*2 and CYP2C9*3 have been known to encode the protein with moderately to markedly reduced catalytic activity. Azole antifungals are among the most frequently used drugs in human pharmacotherapy and represent a widely used class of pesticides to which humans are inevitably exposed. Due to the similarities in CYP organization throughout species, azoles can interact not only with the target fungal CYP51 substrate-binding site but can also modulate the catalytic activity of human cytochrome P450s, including CYP2C9, causing severe adverse effects. In the present study the potency of azole-containing drugs and pesticides to inhibit recombinant wild-type CYP2C9*1 and the allelic variants CYP2C9*2 and CYP2C9*3 was evaluated. Significant differences were found in their affinity to CYP2C9*1, CYP2C9*2, and CYP2C9*3 as well as in the catalytic activity of CYP2C9 allelic variants. Moreover, addition of cytochrome b5 resulted in a decrease of CYP2C9*3 activity to diclofenac in a concentration-dependent manner. Increasing the knowledge of how azoles influence polymorphic variants of CYP2C9 could help individualize drug treatment, leading to optimization of the selection of drugs and doses for individuals based on genetic information.
Assuntos
Antifúngicos/farmacologia , Azóis/farmacologia , Inibidores do Citocromo P-450 CYP2C9/farmacologia , Citocromo P-450 CYP2C9/genética , Fungicidas Industriais/farmacologia , Citocromo P-450 CYP2C9/metabolismo , Interações Medicamentosas , Escherichia coli/genética , Humanos , Polimorfismo Genético , Proteínas Recombinantes/metabolismoRESUMO
Aptamers are short single-stranded DNA or RNA oligonucleotides that can bind to their targets with high affinity and specificity. Usually, they are experimentally selected using the SELEX method. Here, we describe an approach toward the in silico selection of aptamers for proteins. This approach involves three steps: finding a potential binding site, designing the recognition and structural parts of the aptamers and evaluating the experimental affinity. Using this approach, a set of 15-mer aptamers for cytochrome P450 51A1 was designed using docking and molecular dynamics simulation. An experimental evaluation of the synthesized aptamers using SPR biosensor showed that these aptamers interact with cytochrome P450 51A1 with Kd values in the range of 10(-6)-10(-7) M.
Assuntos
Aptâmeros de Nucleotídeos/química , Sistema Enzimático do Citocromo P-450/química , Sítios de Ligação , Modelos Moleculares , Simulação de Acoplamento Molecular/métodos , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
CYP51 (sterol 14α-demethylase) is an efficient target for clinical and agricultural antifungals and an emerging target for treatment of Chagas disease, the infection that is caused by multiple strains of a protozoan pathogen Trypanosoma cruzi. Here, we analyze CYP51A from the Y strain T. cruzi. In this protein, proline 355, a residue highly conserved across the CYP51 family, is replaced with serine. The purified enzyme retains its catalytic activity, yet has been found less susceptible to inhibition. These biochemical data are consistent with cellular experiments, both in insect and human stages of the pathogen. Comparative structural analysis of CYP51 complexes with VNI and two derivatives suggests that broad-spectrum CYP51 inhibitors are likely to be preferable as antichagasic drug candidates.
Assuntos
Inibidores de 14-alfa Desmetilase/farmacologia , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Trypanosoma cruzi/enzimologia , Inibidores de 14-alfa Desmetilase/uso terapêutico , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Biocatálise , Doença de Chagas/tratamento farmacológico , Doença de Chagas/enzimologia , Sequência Conservada , Sistema Enzimático do Citocromo P-450/genética , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Trypanosoma cruzi/efeitos dos fármacosRESUMO
Hepatic conversion to bile acids is a major elimination route for cholesterol in mammals. CYP7A1 catalyzes the first and rate-limiting step in classic bile acid biosynthesis, converting cholesterol to 7α-hydroxycholesterol. To identify the structural determinants that govern the stereospecific hydroxylation of cholesterol, we solved the crystal structure of CYP7A1 in the ligand-free state. The structure-based mutation T104L in the B' helix, corresponding to the nonpolar residue of CYP7B1, was used to obtain crystals of complexes with cholest-4-en-3-one and with cholesterol oxidation product 7-ketocholesterol (7KCh). The structures reveal a motif of residues that promote cholest-4-en-3-one binding parallel to the heme, thus positioning the C7 atom for hydroxylation. Additional regions of the binding cavity (most distant from the access channel) are involved to accommodate the elongated conformation of the aliphatic side chain. Structural complex with 7KCh shows an active site rigidity and provides an explanation for its inhibitory effect. Based on our previously published data, we proposed a model of cholesterol abstraction from the membrane by CYP7A1 for metabolism. CYP7A1 structural data provide a molecular basis for understanding of the diversity of 7α-hydroxylases, on the one hand, and cholesterol-metabolizing enzymes adapted for their specific activity, on the other hand.
Assuntos
Colesterol 7-alfa-Hidroxilase/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Domínio Catalítico , Colesterol 7-alfa-Hidroxilase/genética , Cristalografia por Raios X , Inibidores Enzimáticos/química , Humanos , Ligação de Hidrogênio , Hidroxilação , Cetocolesteróis/química , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Secundária de ProteínaRESUMO
A nanosecond laser near-infrared spectrometer was used to study singlet oxygen ((1)O2) emission in a protein matrix. Myoglobin in which the intact heme is substituted by Zn-protoporphyrin IX (ZnPP) was employed. Every collision of ground state molecular oxygen with ZnPP in the excited triplet state results in (1)O2 generation within the protein matrix. The quantum yield of (1)O2 generation was found to be equal to 0.9 ± 0.1. On the average, six from every 10 (1)O2 molecules succeed in escaping from the protein matrix into the solvent. A kinetic model for (1)O2 generation within the protein matrix and for a subsequent (1)O2 deactivation was introduced and discussed. Rate constants for radiative and nonradiative (1)O2 deactivation within the protein were determined. The first-order radiative rate constant for (1)O2 deactivation within the protein was found to be 8.1 ± 1.3 times larger than the one in aqueous solutions, indicating the strong influence of the protein matrix on the radiative (1)O2 deactivation. Collisions of singlet oxygen with each protein amino acid and ZnPP were assumed to contribute independently to the observed radiative as well as nonradiative rate constants.
Assuntos
Luminescência , Mioglobina/química , Processos Fotoquímicos , Protoporfirinas/química , Oxigênio Singlete/química , Algoritmos , Animais , Cavalos , Cinética , Lasers , Modelos Moleculares , Oxigênio/química , Teoria Quântica , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Água/químicaRESUMO
Oxysterols and neurosteroids are important signaling molecules produced by monooxygenases of the cytochrome P450 family that realize their effect through nuclear receptors. CYP7B1 catalyzes the 6- or 7-hydroxylation of both steroids and oxysterols and thus is involved in the metabolism of neurosteroids and bile acid synthesis, respectively. The dual physiological role of CYP7B1 is evidenced from different diseases, liver failure and progressive neuropathy, caused by enzyme malfunction. Here we present biochemical characterization of CYP7B1 at the molecular level to understand substrate specificity and susceptibility to azole drugs. Based on our experiments with purified enzyme, the requirements for CYP7B1 hydroxylation of steroid molecules are as follows: C5 hydrogen in the α-configuration (or double bond at C5), a polar group at C17, a hydroxyl group at C3, and the absence of the hydroxyl group at C20-C24 in the C27-sterol side chain. 21-hydroxy-pregnenolone was identified as a new substrate, and overall low activity toward pregnanes could be related to the increased potency of 7-hydroxy derivatives produced by CYP7B1. Metabolic conversion (deactivation) of oxysterols by CYP7B1 in a reconstituted system proceeds via two sequential hydroxylations. Two mutations that are found in patients with diseases, Gly57Arg and Phe216Ser, result in apo-P450 (devoid of heme) protein formation. Our CYP7B1 homology model provides a rationale for understanding clinical mutations and relatively broad substrate specificity for steroid hydroxylase.
Assuntos
Esteroide Hidroxilases/química , Esteroide Hidroxilases/metabolismo , 17-alfa-Hidroxipregnenolona/metabolismo , Substituição de Aminoácidos , Azóis/metabolismo , Domínio Catalítico , Família 7 do Citocromo P450 , Humanos , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Dobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrofotometria , Esteroide Hidroxilases/genética , Homologia Estrutural de Proteína , Especificidade por SubstratoRESUMO
Most prostate and breast cancers are hormone dependent. The inhibition of steroid 17α-hydroxylase/17,20- lyase (CYP17), which is a crucial enzyme for steroid hormone biosynthesis, is widely used to treat androgen-dependent prostate cancer (PC). CYP17 has dual enzymatic activity: 17alpha-hydroxylase activity (utilizing delta4- C21 steroids as substrates) and the 17,20-lyase activity (using delta5- C21 steroids as substrates). The steroid biosynthetic pathway is directed to either the production of corticosteroids or sex hormones depending on the activity of CYP17. In this review, the current information on the genetics, molecular structure, substrate specificity and inhibitors of CYP17 is analyzed and discussed.
Assuntos
Esteroide 17-alfa-Hidroxilase/genética , Esteroide 17-alfa-Hidroxilase/metabolismo , Corticosteroides/biossíntese , Androgênios/biossíntese , Neoplasias da Mama/metabolismo , Carcinogênese/efeitos dos fármacos , Carcinogênese/metabolismo , Carcinógenos Ambientais/metabolismo , Carcinógenos Ambientais/toxicidade , Citocromos b5/metabolismo , Feminino , Humanos , Masculino , Neoplasias Hormônio-Dependentes/metabolismo , Neoplasias da Próstata/metabolismo , Conformação Proteica , Esteroide 17-alfa-Hidroxilase/antagonistas & inibidores , Especificidade por Substrato , Xenobióticos/metabolismo , Xenobióticos/toxicidadeRESUMO
Aldosterone is a major mineralocorticoid hormone that plays a key role in the regulation of electrolyte balance and blood pressure. Excess aldosterone levels can arise from dysregulation of the renin-angiotensin-aldosterone system and are implicated in the pathogenesis of hypertension and heart failure. Aldosterone synthase (cytochrome P450 11B2, CYP11B2) is the sole enzyme responsible for the production of aldosterone in humans. Blocking of aldosterone synthesis by mediating aldosterone synthase activity is thus a recently emerging pharmacological therapy for hypertension, yet a lack of structural information has limited this approach. Here, we present the crystal structures of human aldosterone synthase in complex with a substrate deoxycorticosterone and an inhibitor fadrozole. The structures reveal a hydrophobic cavity with specific features associated with corticosteroid recognition. The substrate binding mode, along with biochemical data, explains the high 11ß-hydroxylase activity of aldosterone synthase toward both gluco- and mineralocorticoid formation. The low processivity of aldosterone synthase with a high extent of intermediates release might be one of the mechanisms of controlled aldosterone production from deoxycorticosterone. Although the active site pocket is lined by identical residues between CYP11B isoforms, most of the divergent residues that confer additional 18-oxidase activity of aldosterone synthase are located in the I-helix (vicinity of the O(2) activation path) and loops around the H-helix (affecting an egress channel closure required for retaining intermediates in the active site). This intrinsic flexibility is also reflected in isoform-selective inhibitor binding. Fadrozole binds to aldosterone synthase in the R-configuration, using part of the active site cavity pointing toward the egress channel. The structural organization of aldosterone synthase provides critical insights into the molecular mechanism of catalysis and enables rational design of more specific antihypertensive agents.