RESUMO
CYP2A7 is one of the most understudied human cytochrome P450 enzymes and its contributions to either drug metabolism or endogenous biosynthesis pathways are not understood, as its only known enzymatic activities are the conversions of two proluciferin probe substrates. In addition, the CYP2A7 gene contains four single-nucleotide polymorphisms (SNPs) that cause missense mutations and have minor allele frequencies (MAFs) above 0.5. This means that the resulting amino acid changes occur in the majority of humans. In a previous study, we employed the reference standard sequence (called CYP2A7*1 in P450 nomenclature). For the present study, we created another CYP2A7 sequence that contains all four amino acid changes (Cys311, Glu169, Gly479, and Arg274) and labeled it CYP2A7-WT. Thus, it was the aim of this study to identify new substrates and inhibitors of CYP2A7 and to compare the properties of CYP2A7-WT with CYP2A7*1. We found several new proluciferin probe substrates for both enzyme variants (we also performed in silico studies to understand the activity difference between CYP2A7-WT and CYP2A7*1 on specific substrates), and we show that while they do not act on the standard CYP2A6 substrates nicotine, coumarin, or 7-ethoxycoumarin, both can hydroxylate diclofenac (as can CYP2A6). Moreover, we found ketoconazole, 1-benzylimidazole, and letrozole to be CYP2A7 inhibitors.
Assuntos
Hidrocarboneto de Aril Hidroxilases , Inibidores Enzimáticos , Humanos , Hidrocarboneto de Aril Hidroxilases/antagonistas & inibidores , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Polimorfismo de Nucleotídeo Único , Especificidade por SubstratoRESUMO
Cytochromes P450 (CYP) and UDP-glucuronosyltransferases (UGT) are two enzyme families that play an important role in drug metabolism, catalyzing either the functionalization or glucuronidation of xenobiotics. However, their mutual interactions are poorly understood. In this study, the functional interactions of human CYP2D6 with four human UGTs (UGT1A7, UGT1A8, UGT1A9, and UGT2A1) were investigated using our previously established co-expression model system in the fission yeast Schizosaccharomyces pombe. The substrate employed was propranolol because it is well metabolized by CYP2D6. Moreover, the CYP2D6 metabolite 4-hydroxypropranolol is a known substrate for the four UGTs included in this study. Co-expression of either UGT1A7, UGT1A8, or UGT1A9 was found to increase the activity of CYP2D6 by a factor of 3.3, 2.1 or 2.8, respectively, for the conversion of propranolol to 4-hydroxypropranolol. In contrast, UGT2A1 co-expression did not change CYP2D6 activity. On the other hand, the activities of all four UGTs were completely suppressed by co-expression of CYP2D6. This data corroborates our previous report that CYP2D6 is involved in functional CYP-UGT interactions and suggest that such interactions can contribute to both adverse drug reactions and changes in drug efficacy.
RESUMO
2-Alkylquinolones are a class of microbial natural products primarily produced in the Pseudomonas and Burkholderia genera that play a key role in modulating quorum sensing. Bacterial alkylquinolones were synthesized and then subjected to oxidative biotransformation using human cytochrome P450 enzyme CYP4F11, heterologously expressed in the fission yeast Schizosaccharomyces pombe. This yielded a range of hydroxylated and carboxylic acid derivatives which had undergone ω-oxidation of the 2-alkyl chain, the structures of which were determined by analysis of NMR and MS data. Oxidation efficiency depended on chain length, with a chain length of eight or nine carbon atoms proving optimal for high yields. Homology modeling suggested that Glu233 was relevant for binding, due to the formation of a hydrogen bond from the quinolone nitrogen to Glu233, and in this position only the longer alkyl chains could come close enough to the heme moiety for effective oxidation. In addition to the direct oxidation products, a number of esters were also isolated, which was attributed to the action of endogenous yeast enzymes on the newly formed ω-hydroxy-alkylquinolones. ω-Oxidation of the alkyl chain significantly reduced the antimicrobial and antibiofilm activity of the quinolones.
Assuntos
Bactérias , Sistema Enzimático do Citocromo P-450 , Humanos , Oxirredução , Sistema Enzimático do Citocromo P-450/metabolismo , Família 4 do Citocromo P450/metabolismoRESUMO
Cytochrome P450 enzymes (CYPs) are one of the most important classes of oxidative enzymes in the human body, carrying out metabolism of various exogenous and endogenous substrates. In order to expand the knowledge of these enzymes' specificity and to obtain new natural product derivatives, CYP4F11, a cytochrome P450 monooxygenase, was used in the biotransformation of dialkylresorcinols 1 and 2, a pair of antibiotic microbial natural products. This investigation resulted in four biotransformation products including two oxidative products: a hydroxylated derivative (3) and a carboxylic acid derivative (4). In addition, acetylated (5) and esterified products (6) were isolated, formed by further metabolism by endogenous yeast enzymes. Oxidative transformations were highly regioselective, and took place exclusively at the ω-position of the C-5 alkyl chain. Homology modeling studies revealed that optimal hydrogen bonding between 2 and the enzyme can only be established with the C-5 alkyl chain pointing towards the heme. The closely-related CYP4F12 was not capable of oxidizing the dialkylresorcinol 2. Modeling experiments rationalize these differences by the different shapes of the binding pockets with respect to the non-oxidized alkyl chain. Antimicrobial testing indicated that the presence of polar groups on the side-chains reduces the antibiotic activity of the dialkylresorcinols.
Assuntos
Antibacterianos , Sistema Enzimático do Citocromo P-450 , Resorcinóis , Humanos , Antibacterianos/metabolismo , Biotransformação , Sistema Enzimático do Citocromo P-450/metabolismo , Oxirredução , Resorcinóis/metabolismoRESUMO
Propranolol, a non-selective beta-blocker medication, has been utilized in the treatment of cardiovascular diseases for several decades. Its hydroxynaphthyl metabolites have been recognized to possess varying degrees of beta-blocker activity due to the unaltered side-chain. This study achieved the successful separation and identification of diastereomeric glucuronic metabolites derived from 4-, 5-, and 7-hydroxypropranolol (4-OHP, 5-OHP, and 7-OHP) in human urine. Subsequently, reaction phenotyping of 5- and 7-hydroxypropranolol by different uridine 5'-diphospho-glucuronosyltransferases (UGTs) was carried out, with a comparison to the glucuronidation of 4-hydroxypropranolol (4-OHP). Among the 19 UGT enzymes examined, UGT1A1, UGT1A3, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2A1, and UGT2A2 were found to be involved in the glucuronidation of 5-OHP. Furthermore, UGT1A6 exhibited glucuronidation activity towards 7-OHP, along with the aforementioned eight UGTs. Results obtained by glucuronidation of corresponding methoxypropranolols and MS/MS analysis of 1,2-dimethylimidazole-4-sulfonyl (DMIS) derivatives of hydroxypropranolol glucuronides suggest that both the aromatic and aliphatic hydroxy groups of the hydroxypropranolols may be glucuronidated in vitro. However, the analysis of human urine samples collected after the administration of propranolol leads us to conclude that aromatic-linked glucuronidation is the preferred pathway under physiological conditions.
Assuntos
Glucuronídeos , Microssomos Hepáticos , Humanos , Glucuronídeos/metabolismo , Microssomos Hepáticos/metabolismo , Propranolol/metabolismo , Espectrometria de Massas em Tandem , Glucuronosiltransferase/metabolismo , Antagonistas Adrenérgicos beta , CinéticaRESUMO
PURPOSE: CYP4Z1 is a human cytochrome P450 enzyme involved in breast cancer progression and prognosis, but its functional role in these processes is not understood. In order to gain more insight into CYP4Z1's properties it was recombinantly expressed in a host animal that does not have an endogenous homologue. METHODS: We generated a transgenic mouse model that specifically expresses human CYP4Z1 in breast tissue under the control of the whey acidic protein promoter. Complementary experiments were done using cell lines derived from human breast cell. RESULTS: Induction of CYP4Z1 expression led to reduction of body weight, activity, and birth rates. Histological analysis revealed no evidence for tumor formation. However, a strong increase in estrogen receptor alpha was observed by immunohistochemistry; weaker but significantly increased immunoreactivity was also detected for collagen I and fibronectin. Overexpression of CYP4Z1 in the human breast cancer cell line MCF7 also led to increased ERα expression. Moreover, increased expression of both CYP4Z1 and ERα was observed in MCF-10A normal breast cells upon cocultivation with MCF-7 cells (with or without overexpression of CYP4Z1). CONCLUSION: These data suggest that CYP4Z1 facilitates breast cancer development by induction of ERα expression via an as yet undefined mechanism.
Assuntos
Neoplasias da Mama , Receptor alfa de Estrogênio , Animais , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Família 4 do Citocromo P450/genética , Família 4 do Citocromo P450/metabolismo , Receptor alfa de Estrogênio/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Transgênicos , Regulação para CimaRESUMO
The secondary metabolite pseudopyronine B, isolated from Pseudomonas mosselii P33, was biotransformed by human P450 enzymes, heterologously expressed in the fission yeast Schizosaccharomyces pombe. Small-scale studies confirmed that both CYP4F2 and CYP4F3A were capable of oxidizing the substrate, with the former achieving a higher yield. In larger-scale studies using CYP4F2, three new oxidation products were obtained, the structures of which were elucidated by UV-vis, 1D and 2D NMR, and HR-MS spectroscopy. These corresponded to hydroxylated, carboxylated, and ester derivatives (1-3) of pseudopyronine B, all of which had been oxidized exclusively at the ω-position of the C-6 alkyl chain. In silico homology modeling experiments highlighted key interactions between oxygen atoms of the pyrone ring and two serine residues and a histidine residue of CYP4F2, which hold the substrate in a suitable orientation for oxidation at the terminus of the C-6 alkyl chain. Additional modeling studies with all three pseudopyronines revealed that the seven-carbon alkyl chain of pseudopyronine B was the perfect length for oxidation, with the terminal carbon lying close to the heme iron. The antibacterial activity of the substrates and three oxidation products was also assessed, revealing that oxidation at the ω-position removes all antimicrobial activity. This study both increases the range of known substrates for human CYF4F2 and CYP4F3A enzymes and demonstrates their utility in producing additional natural product derivatives.
Assuntos
Antibacterianos , Sistema Enzimático do Citocromo P-450 , Pironas , Humanos , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Biotransformação , Sistema Enzimático do Citocromo P-450/metabolismo , Família 4 do Citocromo P450/metabolismo , Hidroxilação , Oxirredução , Pironas/química , Pironas/metabolismo , Pironas/farmacologia , Schizosaccharomyces/enzimologiaRESUMO
1. Olanzapine is an atypical antipsychotic primarily used to treat schizophrenia and bipolar disorder. An intronic single nucleotide polymorphism (SNP) that highly significantly predicts increased olanzapine clearance (rs472660) was previously identified in the CYP3A43 gene, which encodes a cytochrome P450 enzyme. But until now there was no experimental evidence for the metabolism of olanzapine by the CYP3A43 enzyme.2. In the present study we provide this evidence, together with a thorough analysis of olanzapine metabolism by all human CYP3A enzymes. We also rationalise our findings by molecular docking experiments. Moreover, we describe the activities of several CYP3A43 mutants and present the first enzymatic activity data for the CYP3A43.3 variant; with respect to prostate cancer, this polymorphic variant is associated with both increased risk and increased mortality. The catalytic properties of the wild type enzyme and the tumour mutant were analysed by molecular dynamics simulations, which fit very well with the observed experimental results.3. Our findings suggest that the SNP rs472660 likely causes an increased CYP3A43 expression level and demonstrate that, depending on the substrate under study, the tumour mutant CYP3A43.3 can have increased activity in comparison to the wild type enzyme CYP3A43.1.
Assuntos
Antipsicóticos , Antipsicóticos/uso terapêutico , Benzodiazepinas , Citocromo P-450 CYP3A/genética , Sistema Enzimático do Citocromo P-450/genética , Humanos , Masculino , Simulação de Acoplamento Molecular , OlanzapinaRESUMO
Propranolol is a competitive non-selective beta-receptor antagonist that is available on the market as a racemic mixture. In the present study, glucuronidation of propranolol and its equipotent phase I metabolite 4-hydroxypropranolol by all 19 members of the human UGT1 and UGT2 families was monitored. UGT1A7, UGT1A9, UGT1A10 and UGT2A1 were found to glucuronidate propranolol, with UGT1A7, UGT1A9 and UGT2A1 mainly acting on (S)-propranolol, while UGT1A10 displays the opposite stereoselectivity. UGT1A7, UGT1A9 and UGT2A1 were also found to glucuronidate 4-hydroxypropranolol. In contrast to propranolol, 4-hydroxypropranolol was found to be glucuronidated by UGT1A8 but not by UGT1A10. Additional biotransformations with 4-methoxypropanolol demonstrated different regioselectivities of these UGTs with respect to the aliphatic and aromatic hydroxy groups of the substrate. Modeling and molecular docking studies were performed to explain the stereoselective glucuronidation of the substrates under study.
Assuntos
Glucuronosiltransferase , Microssomos Hepáticos , Propranolol , Glucuronídeos/metabolismo , Glucuronosiltransferase/metabolismo , Humanos , Microssomos Hepáticos/metabolismo , Simulação de Acoplamento Molecular , Propranolol/análogos & derivados , Propranolol/farmacologia , UDP-Glucuronosiltransferase 1ARESUMO
Purpose: Polymorphisms in the gene that codes for the human cytochrome P450 enzyme CYP4V2 are a cause of Bietti crystalline dystrophy (BCD). Therefore, inhibition of CYP4V2 activity may well be a cause of visual disability. However, monitoring the fatty acid hydroxylation reactions catalyzed by this enzyme is tedious and not well suited for inhibitor screening. Methods: We investigated the use of proluciferin compounds as probe substrates for efficient and convenient determination of CYP4V2 activity. Results: Ten proluciferins were tested for conversion by CYP4V2, and eight were found to be substrates of this enzyme. One point inhibitor assays were performed using luciferin 6' 3-furfuryl ether methyl ester (luciferin-3FEME) as the probe substrate and 12 test compounds. As expected, HET0016 had by far the strongest effect, while two other compounds (including osilodrostat) also displayed statistically significant inhibitory potency. The half maximal inhibitory concentration (IC50) for HET0016 was determined to be 179 nM. A recently identified potent inhibitor of human CYP4Z1 was found not to inhibit CYP4V2. To explore the selectivity of this compound between CYP4Z1 and CYP4V2, we developed a homology model of CYP4V2 and conducted docking experiments. Conclusions: We provide the first protocol for a robust and convenient CYP4V2 inhibitor assay that does not depend on fatty acid analysis but can be simply monitored with luminescence. Moreover, we demonstrate additional evidence for the concern that compounds with CYP-inhibitory properties may inhibit CYP4V2 activity and thus, possibly cause visual disability.
Assuntos
Distrofias Hereditárias da Córnea , Família 4 do Citocromo P450/antagonistas & inibidores , Doenças Retinianas , Família 4 do Citocromo P450/genética , Humanos , Luciferinas , MutaçãoRESUMO
Human cytochrome P450 enzymes (CYPs or P450s) are known to be reduced by their electron transfer partners in the absence of substrate and in turn to reduce other acceptor molecules such as molecular oxygen, thereby creating superoxide anions (O2-â¢). This process is known as futile cycling. Using our previously established fission yeast expression system we have monitored cells expressing each one of the 50 human microsomal CYPs in the absence of substrate for oxidation of dihydroethidium in living cells by flow cytometry. It was found that 38 of these display a statistically significant increase in O2-⢠production. More specifically, cells expressing some CYPs were found to be intermediate strength O2-⢠producers, which means that their effect was comparable to that of treatment with 3â¯mMâ¯H2O2. Cells expressing other CYPs had an even stronger effect, with those expressing CYP2B6, CYP5A1, CYP2A13, CYP51A1, or CYP1A2, respectively, being the strongest producers of O2-â¢.
Assuntos
Sistema Enzimático do Citocromo P-450 , Peróxido de Hidrogênio/metabolismo , Microssomos/enzimologia , Schizosaccharomyces , Superóxidos/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Humanos , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genéticaRESUMO
Metandienone and methyltestosterone are orally active anabolic-androgenic steroids with a 17α-methyl structure that are prohibited in sports but are frequently detected in anti-doping analysis. Following the previously reported detection of long-term metabolites with a 17ξ-hydroxymethyl-17ξ-methyl-18-nor-5ξ-androst-13-en-3ξ-ol structure in the chlorinated metandienone analog dehydrochloromethyltestosterone ("oral turinabol"), in this study we investigated the formation of similar metabolites of metandienone and 17α-methyltestosterone with a rearranged D-ring and a fully reduced A-ring. Using a semi-targeted approach including the synthesis of reference compounds, two diastereomeric substances, viz. 17α-hydroxymethyl-17ß-methyl-18-nor-5ß-androst-13-en-3α-ol and its 5α-analog, were identified following an administration of methyltestosterone. In post-administration urines of metandienone, only the 5ß-metabolite was detected. Additionally, 3α,5ß-tetrahydro-epi-methyltestosterone was identified in the urines of both administrations besides the classical metabolites included in the screening procedures. Besides their applicability for anti-doping analysis, the results provide new insights into the metabolism of 17α-methyl steroids with respect to the order of reductions in the A-ring, the participation of different enzymes, and alterations to the D-ring.
Assuntos
Anabolizantes/metabolismo , Anabolizantes/urina , Metandrostenolona/metabolismo , Metandrostenolona/urina , Metiltestosterona/metabolismo , Metiltestosterona/urina , Anabolizantes/química , Cromatografia Gasosa-Espectrometria de Massas , Voluntários Saudáveis , Humanos , Metandrostenolona/química , Metiltestosterona/química , Pessoa de Meia-Idade , Padrões de Referência , Espectrometria de Massas em TandemRESUMO
CYP20A1 is a well-conserved member of the human cytochrome P450 enzyme family for which no endogenous or xenobiotic substrate is known. We have recently shown that this enzyme has moderate activity towards two proluciferin probe substrates. In order to facilitate the search for physiological substrates we have tested nine additional proluciferins in this study and identified three such probe substrates that give much higher product yields. Using one of these probes, we demonstrate inhibition of CYP20A1 activity by 1-benzylimidazole, ketoconazole and letrozole. Finally, we show that the combination of two common single nucleotide polymorphisms (SNPs) of CYP20A1 leads to an enzyme (CYP20A1Leu97Phe346) with reduced activity.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Imidazóis/farmacologia , Cetoconazol/farmacologia , Letrozol/farmacologia , Sistema Enzimático do Citocromo P-450/genética , Humanos , Estrutura Molecular , Especificidade por SubstratoRESUMO
The development of convenient assays for the in vitro study of drug metabolizing enzymes (DMEs) such as cytochromes P450 (CYPs) and UDP-glucuronosyltransferases (UGTs) greatly facilitates metabolism studies of candidate drug compounds and other xenobiotics. We have developed and optimized an experimental approach that combines the advantages of recombinant expression in yeast with a microsomal-like biotransformation and thus allows for rapid and convenient enzymatic assays. Recombinant strains of the fission yeast Schizosaccharomyces pombe have previously been demonstrated to functionally express human CYPs and UGTs. Permeabilization of such cells with Triton X-100 results in the formation of enzyme bags, which can be used as biocatalysts. This protocol describes the preparation of such enzyme bags (3 h) and their application in enzyme activity assays (4 h) utilizing either pro-luminescent substrates and luminescence measurements or non-luminescent substrates and liquid chromatography coupled to mass spectrometry (LC-MS). Both applications provide practical tools for investigating CYP and UGT reactions in vitro without the need for additional sophisticated instrumentation or expertise.
Assuntos
Bioensaio , Sistema Enzimático do Citocromo P-450/metabolismo , Glucuronosiltransferase/metabolismo , Proteínas Recombinantes/genética , Biotransformação , Sistema Enzimático do Citocromo P-450/genética , Ativação Enzimática , Enzimas , Glucuronosiltransferase/genética , Humanos , Octoxinol , Permeabilidade , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genética , Espectrometria de Fluorescência , Especificidade por SubstratoRESUMO
OBJECTIVES: Development of an open-loop fed-batch protocol for highly reproducible fermentation of fission yeast that starts from batch cultures instead of glucose-limited aerobic chemostat cultures. RESULTS: A new strategy was employed that consists of an exponential feeding phase followed by a starvation period and then a linear feeding phase. A comparison of several independent fed-batch fermentations of a recombinant fission yeast strain showed that while during the initial phase process parameters such as glucose consumption and CO2 evolution varied considerably as expected, they were much more uniform during the third phase. For instance, the normalized standard deviation of glucose consumption was thirty times higher during the exponential feeding phase of the fermentation that during the linear feeding phase. CONCLUSION: These data demonstrate the usefulness of the proposed strategy. It is expected that by variation of only two parameters (the total amount of glucose fed in the initial phase and the time frame of the starvation phase) the protocol can easily be adapted to other microbes.
Assuntos
Técnicas de Cultura Celular por Lotes/métodos , Fermentação/fisiologia , Schizosaccharomyces , Biomassa , Dióxido de Carbono/análise , Dióxido de Carbono/metabolismo , Glucose/análise , Glucose/metabolismo , Schizosaccharomyces/citologia , Schizosaccharomyces/metabolismoRESUMO
BACKGROUND: Recently the expression patterns of several cytochrome P450 (CYP) genes in different human osteoblast models were reported. However, the various expression patterns of CYPs in human osteoblasts during different stages of osteogenic differentiation have not been investigated and the effect of inflammatory cytokines on CYPs expression in osteoblasts is unknown. METHODS: The expression levels of nine different CYP genes in the human osteoblast cell line MG63 and in primary human osteoblasts (HOB) during osteogenic differentiation with or without treatment with tumor necrosis factor-α (TNFα) were analyzed by quantitative real-time PCR. RESULTS: The expression levels of most CYPs under study show a significant time dependence during osteogenic differentiation. Overall, more highly significant CYP expression level changes occur in HOB than in MG63 cells. Treatment with TNFα causes a variety of CYP expression level changes in both HOB and MG63 cells. CONCLUSIONS: Our findings indicate that TNFα treatment reduces steroid hormone production in MG63 cells (but not in HOB) at the level of lanosterol-demethylation during cholesterol biosynthesis. By contrast, TNFα treatment of HOB cells (but not in MG63) leads to the upregulation of several key enzymes involved in the biosynthesis of sex steroids, which is proposed to lead to higher levels of estrogen production. These data also suggest that at least with respect to the topic of this study the cell line MG63 is not a good representative for osteoblasts and that it is preferential to use primary osteoblasts instead.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Osteoblastos/metabolismo , Osteogênese/genética , Adulto , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Feminino , Humanos , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
The human cytochrome P450 enzyme CYP8B1 is a crucial regulator of the balance of cholic acid (CA) and chenodeoxycholic acid (CDCA) in the liver. It was previously shown to catalyze the conversion of 7α-hydroxycholest-4-en-3-one, a CDCA precursor, to 7α,12α-dihydroxycholest-4-en-3-one, which is an intermediate of CA biosynthesis. In this study we demonstrate that CYP8B1 can also convert CDCA itself to CA. We also show that five derivatives of luciferin are metabolized by CYP8B1 and established a rapid and convenient inhibitor test system. In this way we were able to identify four new CYP8B1 inhibitors, which are aminobenzotriazole, exemestane, ketoconazole and letrozole.
Assuntos
Ácido Quenodesoxicólico/metabolismo , Ácido Cólico/biossíntese , Esteroide 12-alfa-Hidroxilase/metabolismo , Biocatálise , Ácido Quenodesoxicólico/química , Ácido Cólico/química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Corantes Fluorescentes/química , Humanos , Estrutura Molecular , Esteroide 12-alfa-Hidroxilase/antagonistas & inibidoresRESUMO
Protein secretion in yeast is a complex process and its efficiency depends on a variety of parameters. We performed a comparative proteome analysis of a set of Schizosaccharomyces pombe strains producing the α-glucosidase maltase in increasing amounts to investigate the overall proteomic response of the cell to the burden of protein production along the various steps of protein production and secretion. Proteome analysis of these strains, utilizing an isobaric labeling/two dimensional LC-MALDI MS approach, revealed complex changes, from chaperones and secretory transport machinery to proteins controlling transcription and translation. We also found an unexpectedly high amount of changes in enzyme levels of the central carbon metabolism and a significant up-regulation of several amino acid biosyntheses. These amino acids were partially underrepresented in the cellular protein compared with the composition of the model protein. Additional feeding of these amino acids resulted in a 1.5-fold increase in protein secretion. Membrane fluidity was identified as a second bottleneck for high-level protein secretion and addition of fluconazole to the culture caused a significant decrease in ergosterol levels, whereas protein secretion could be further increased by a factor of 2.1. In summary, we show that high level protein secretion causes global changes of protein expression levels in the cell and that precursor availability and membrane composition limit protein secretion in this yeast. In this respect, comparative proteome analysis is a powerful tool to identify targets for an efficient increase of protein production and secretion in S. pombe Data are available via ProteomeXchange with identifiers PXD002693 and PXD003016.
Assuntos
Proteômica/métodos , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Cromatografia Líquida , Regulação Fúngica da Expressão Gênica , Schizosaccharomyces/classificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , alfa-Glucosidases/metabolismoRESUMO
Cytochromes P450 (CYPs) are important for bone homeostasis, but only limited information is available on their expression in human bone cells. We analyzed the expression levels of eight CYPs in osteoblasts cultured in human bone pieces, in osteoblasts differentiated from human periosteum mesenchymal stem cells, in primary human osteoblasts and in the human osteoblast cell line MG63, respectively. Our results confirm previous reports about the presence of CYP11A1, CYP17A1, CYP24A1 and CYP27B1, while demonstrating expression of CYP2E1, CYP26A1, CYP39A1 and CYP51A1 for the first time. However, expression patterns in the four models were remarkably different from each other.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Modelos Biológicos , Osteoblastos/metabolismo , Células Cultivadas , Sistema Enzimático do Citocromo P-450/metabolismo , Humanos , Osteoblastos/citologia , Reação em Cadeia da PolimeraseRESUMO
OBJECTIVES: To use permeabilized cells of the fission yeast, Schizosaccharomyces pombe, that expresses human UDP-glucose 6-dehydrogenase (UGDH, EC 1.1.1.22), for the production of UDP-glucuronic acid from UDP-glucose. RESULTS: In cell extracts no activity was detected. Therefore, cells were permeabilized with 0.3 % (v/v) Triton X-100. After washing away all low molecular weight metabolites, the permeabilized cells were directly used as whole cell biocatalyst. Substrates were 5 mM UDP-glucose and 10 mM NAD(+). Divalent cations were not added to the reaction medium as they promoted UDP-glucose hydrolysis. With this reaction system 5 mM UDP-glucose were converted into 5 mM UDP-glucuronic acid within 3 h. CONCLUSIONS: Recombinant permeabilized cells of S. pombe can be used to synthesize UDP-glucuronic acid with 100 % yield and selectivity.