细胞色素P450与农药相互作用及其机理研究

综述了细胞色素P450的种类和功能多样性,介绍了细胞色素P450参与除草剂代谢及其作用机理．同时,简述了细胞色素P450 14DM与杀菌剂特异性作用的机制．报道了我们从抗除草剂的瑞士黑麦草中克隆CYP8181同源基因及其功能的研究,以及柑橘绿霉菌细胞色素P450 14DM基因的克隆表达．为深入研究杀菌剂作用细胞色素P450 14DM的机理从而设计以P450 14DM为特异性靶标的新型农药、以及进一步研究细胞色素P450酶系与除草剂代谢的关系打下了良好的基础．     

Crystal structure of human cytochrome P450 2C9 with bound warfarin

Cytochrome P450 proteins (CYP450s) are membrane-associated haem proteins that metabolize physiologically important compounds in many species of microorganisms, plants and animals. Mammalian CYP450s recognize and metabolize diverse xenobiotics such as drug molecules, environmental compounds and pollutants. Human CYP450 proteins CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 are the major drug-metabolizing isoforms, and contribute to the oxidative metabolism of more than 90% of the drugs in current clinical use. Polymorphic variants have also been reported for some CYP450 isoforms, which has implications for the efficacy of drugs in individuals, and for the co-administration of drugs. The molecular basis of drug recognition by human CYP450s, however, has remained elusive. Here we describe the crystal structure of a human CYP450, CYP2C9, both unliganded and in complex with the anti-coagulant drug warfarin. The structure defines unanticipated interactions between CYP2C9 and warfarin, and reveals a new binding pocket. The binding mode of warfarin suggests that CYP2C9 may undergo an allosteric mechanism during its function. The newly discovered binding pocket also suggests that CYP2C9 may simultaneously accommodate multiple ligands during its biological function, and provides a possible molecular basis for understanding complex drug-drug interactions.     

凹土玉米芯垫料对大鼠肝微粒体细胞色素P450 酶系的影响

摘要: 目的观察凹土玉米芯垫料对大鼠肝脏细胞色素P450( CytP450) 、细胞色素b5( Cytb5) 含量及大鼠肝微粒 体CYP1A2 和CYP2E1 活性的影响。方法将SD 大鼠随机分为普通刨花组、玉米秸组、凹土玉米芯组和对照组,饲 养30 d、60 d、90 d 时测定大鼠肝脏微粒体CytP450、Cytb5 的含量和CYP1A2,CYP2E1 活性。结果60-90 d 时普 通刨花组、玉米秸组与空白对照组之间CytP450 含量有显著性差异( P ＜ 0. 05) ,凹土玉米芯组与对照组之间没有显 著性差异( P ＞ 0. 05) ; 肝微粒体Cytb5 含量、CYP1A2、CYP2E1 活性各组均没有明显差异( P ＞ 0. 05) 。结论凹土玉 米芯对大鼠肝脏细胞微粒体CytP450、Cytb5、CYP1A2 和CYP2E1 没有诱导或抑制作用,普通刨花组对大鼠肝 CytP450 有较强的诱导作用,玉米秸也有不同程度的诱导作用,但低于普通刨花的诱导作用。     

川丁特罗对大鼠细胞色素P450酶的影响

研究新型抗哮喘药川丁特罗（trantinterol）对大鼠细胞色素P450酶的影响. 大鼠连续7 d灌胃给予川丁特罗后, 测定肝微粒体中CYP450的质量摩
尔浓度和主要3种亚型CYP1A2,CYP2D6和CYP3A4活性的变化. 实验结果表明, 与对照组相比,  川丁特罗给药组的大鼠肝微粒体中CYP450的总质量摩尔浓度未受影响(P>0.05), 对主要亚型CYP1A2,CYP2D6和CYP3A4的活性也无影响(P>0.05), 表明该药物对肝微粒体中的主要代谢酶无抑制或诱导作用.     

检测细胞色素P-450基因族中CYP1A1基因表达的一个简捷方法

NMR1系雄性小鼠被用来研究肺微粒体细胞色素P450家族中一个同功基因CYPlAl在香烟烟雾的诱导下表达．双轮回‘聚合酶链式反应’(PCR)和‘反向转录酶-聚合酶链式反应’(RT-PCR)被用来检测基因CYP1Al的转录水平．代表CYPlAl蛋白活性的7-乙氧基试卤灵-0-去乙基酶(EROD)的活性被用来检测基因CYPlAl的表达．双轮回‘聚合酶链式反应’(PCR)和‘反向转录酶-聚合酶链式反应’(RT-PCR)被用来检测基因CYPlAl的转录水平．测试结果表明在吸入香烟烟雾的情况下。小鼠肺微粒体EROD的活性和基因CYPlAl的转录水平是同步增加的．这个结果不同于以前在同样实验条件的一个自相矛盾的结果，即在CYPlAl蛋白增加时，基因CYPlAl的转录水平却是下降或不变．这说明本文所使用的方法可能要优于以前的实验方法.     

Up-regulation of endothelial nitric oxide synthase by cytochrome P450 arachidonic acid epoxygenase BM3F87V

Cytochrome P450 (CYP)-dependent metabolites of arachidonic acid, epoxyeicosatrienoic acids (EETs), have been suggested to be an endothelium-derived hyperpolarizing factor (EDHF). However, the interaction or relation between EDHF and endothelial nitric oxide synthase (eNOS) is still to be elucidated. In the present study, the regulation of eNOS by endogenous EDHF is examined. The cytochrome P450 epoxygenase BM3F87V is cloned into the mammalian expression vector pCB6. Cultured bovine aortic endothelial cells (BAECs) less than 4 passages are used and transfected with BM3F87V. The effects of endogenous EETs result from BM3F87V transfection on eNOS are assessed in the endothelial cells by Western blot and Northern blot, and eNOS activity is also measured by the conversion of L-arginine to L-citrulline. Compared to transfection with the empty pCB6 vector, transfection of BAECs with BM3F87V significantly elevates the levels of eNOS protein expression, which is markedly inhibited by treatment with CYP inhibitor 17-ODYA. BM3F87V transfection also elevates the eNOS mRNA level and increases the eNOS activity. This study suggests that EDHF up-regulates eNOS gene expression.     

Identification of the primary gene defect at the cytochrome P450 CYP2D locus

The mammalian cytochrome P450-dependent monooxygenase system is involved in the metabolism of drugs and chemical carcinogens. The role of these enzymes in toxicological response is exemplified by an autosomal recessive polymorphism at the cytochrome P450 CYP2D6 debrisoquine hydroxylase locus which results in the severely compromised metabolism of at least 25 drugs, and which in some cases can lead to life-threatening side-effects. In addition, this polymorphism, which affects 8-10% of the caucasian population, has been associated with altered susceptibility to lung and bladder cancer. Here we report the identification of the primary mutation responsible for this metabolic defect and the development of a simple DNA-based genetic assay to allow both the identification of most individuals at risk of drug side-effects and clarification of the conflicting reports on the association of this polymorphism with cancer susceptibility.     

Blast fungus-induction and developmental and tissue-specific expression of a rice P450<Emphasis Type="Italic">CYP72A</Emphasis> gene cluster

Cytochrome P450 gene superfamily is widely involved in diverse processes of plant development and environmental responses including defense response to pathogens.We previously isolated a rice cDNA fragment in a DD-PCR screening for blast fungus-induced genes. In the current study, we isolated a CYP72A gene cluster consisting of 7 P450 CYP72A genes (CYP72A17-23) with the conserved cDNA sequence through the public rice genome data. There are total 14 putative CYP72A members in the rice genome, with high diversity at N-terminal sequences while high homology at C-terminal sequences of those 14 putative proteins. We analyzed expression profiles of the cloned 7 CYP72A genes during pathogen infection and development. The results showed that expression of CYP72A18, 19, 22 and 23 was differentially regulated in the incompatible and compatible interactions between rice and blast fungus. Except CYP72A20, a pseudogene, other 6 CYP72A genes also exhibited temporal and spatial expression patterns, respectively.These findings provide fundamental data for rice P450 gene function analysis.     

An atypical haem in the cytochrome b(6)f complex

Photosystems I and II (PSI and II) are reaction centres that capture light energy in order to drive oxygenic photosynthesis; however, they can only do so by interacting with the multisubunit cytochrome b(6)f complex. This complex receives electrons from PSII and passes them to PSI, pumping protons across the membrane and powering the Q-cycle. Unlike the mitochondrial and bacterial homologue cytochrome bc(1), cytochrome b(6)f can switch to a cyclic mode of electron transfer around PSI using an unknown pathway. Here we present the X-ray structure at 3.1 A of cytochrome b(6)f from the alga Chlamydomonas reinhardtii. The structure bears similarities to cytochrome bc(1) but also exhibits some unique features, such as binding chlorophyll, beta-carotene and an unexpected haem sharing a quinone site. This haem is atypical as it is covalently bound by one thioether linkage and has no axial amino acid ligand. This haem may be the missing link in oxygenic photosynthesis.     

小鼠吸入香烟烟雾染毒过程的标准化和使用酶活检测在烟熏小鼠中基因CYP1A1的表达

建立了一个通过测定茄尼醇的含量来确定小鼠吸入香烟烟雾浓度的方法，因而使得来自不同实验室的相关数据的比成为可能，同时，对香烟烟雾务诱导雄性小鼠NMR1的肺微粒体中细胞色素P450异构基因CPY1A1的表达在蛋白质的水平上进行了测试，测试结果表明，代表CYP1A1蛋白活性的7-乙氧基试卤录-O-去乙基酶的活性在小鼠的肺中随着吸入香烟烟雾的增加而上升，由此可知，香烟烟雾诱导了小鼠肺中基因CYP1A1的表达。     

Cytochrome P450 2C is an EDHF synthase in coronary arteries.

In most arterial beds a significant endothelium-dependent dilation to various stimuli persists even after inhibition of nitric oxide synthase and cyclo-oxygenase. This dilator response is preceded by an endothelium-dependent hyperpolarization of vascular smooth muscle cells, which is sensitive to a combination of the calcium-dependent potassium-channel inhibitors charybdotoxin and apamin, and is assumed to be mediated by an unidentified endothelium-derived hyperpolarizing factor (EDHF). Here we show that the induction of cytochrome P450 (CYP) 2C8/34 in native porcine coronary artery endothelial cells by beta-naphthoflavone enhances the formation of 11,12-epoxyeicosatrienoic acid, as well as EDHF-mediated hyperpolarization and relaxation. Transfection of coronary arteries with CYP 2C8/34 antisense oligonucleotides results in decreased levels of CYP 2C and attenuates EDHF-mediated vascular responses. Thus, a CYP-epoxygenase product is an essential component of EDHF-mediated relaxation in the porcine coronary artery, and CYP 2C8/34 fulfils the criteria for the coronary EDHF synthase.     

褐飞虱细胞色素P450基因cDNA片段的克隆、测序及表达分析

利用反转录多聚酶链式反应(RT-PCR)技术克隆了褐飞虱细胞色素P450基因编码区的cDNA片段,并进行了序列测定.结果表明,所克隆到的cDNA片段长度为237bp,经BLAST查找比对发现,该片段所编码的氨基酸序列与来自烟草天蛾、棉铃虫、埃及伊蚊、家蝇、黑腹果蝇和线虫的CYP6家族的P450的氨基酸序列存在同源性.Northern杂交分析显示,在褐飞虱取食抗性水稻后,P450基因的表达水平明显升高.以上结果表明,P450基因的表达受抗性水稻的诱导,该基因在褐飞虱对抗性水稻的耐受性和解毒方面可能起着重要作用.     

Docking and molecular dynamics studies on CYP2D6

Drug-metabolizing enzymes, also known as cytochrome P450s, are a superfamily of hemoglobin responsible for metabolizing more than 90% clinical drugs. Cytochrome P450 2D6 (CYP2D6) is a significant member of cytochrome P450s for the reason of metabolizing about 20% clinical drugs. In this paper, molecular docking and molecular dynamic simulations are used to investi- gate the active site of CYP2D6, roles of essential amino acids within the active site and time-dependent protein energy changes. The results suggest that amino acids Glu216, Asp301, Ser304 and Ala305 in the active site are likely to form hydrogen bonding interac-tions with substrates; the benzene ring of Phe120 and aromatic ring in the substrates form ∏-∏ interactions. In addition, molecular dynamics simulations prove that the catalytic conformation of CYP2D6 without ligands can be obtained by their own atomic fluctuations. The impact of ligands on protein system energy and large conformational shift is not very large. Cytochrome P450s is known for their genetic polymorphisms, which will result in severe adverse drug reactions. Ideally, we hope to use mo- lecular modeling to investigate the differences between the substrates of wild-type and mutants while they are bonded with drugs, and predict the drug metabolizing ability of mutants. Reduce the possibility for people taking drugs that they can not metabolize, therefore reduce the rate of adverse drug reactions, and eventually establish a platform of personalized drugs to largely benefit human health.     

Alteration of mouse cytochrome P450coh substrate specificity by mutation of a single amino-acid residue

As a family of structurally-related enzymes, cytochrome P450 (P450) monooxygenases exhibit paradoxical characteristics: although collectively the enzymes display a broad range of substrate specificities, individually they are characterized by a high degree of substrate and product selectivity. Mouse P45015 alpha and P450coh, for example, which are expressed in female liver and male kidney cells, catalyse 15 alpha-hydroxylation of delta 4 3-ketone steroids, such as testosterone and 7-hydroxylation of coumarin, respectively. In spite of their divergent catalytic activities, however, these enzymes differ by only 11 amino acids within their 494 residues. To determine the structural basis of the different substrate specificities of P45015 alpha and P450coh we therefore altered each of these 11 residues by site-directed mutagenesis, expressing the mutant cytochromes in COS-1 cells. We report that the activities of both cytochromes depend critically on the identities of the amino acids at positions 117, 209 and 365 and, moreover, that a single mutation in which Phe 209 is substituted by Leu is sufficient to convert the specificity of P450coh from coumarin to steroid hydroxylation.     

诺氟沙星对水生生物细胞色素P450作用机制研究进展

环境中的抗生素主要来源于生活污水、医疗废水以及水产养殖废水等.2013年中国抗生素总使用量约为1.62×10⁵ t,其中48%为人用抗生素,其余为兽用抗生素.大量具有活性的抗生素经地表径流和城市排污管道最终积聚到河流湖泊中.这些活性物质会对水生生物产生危害,尤其是在较长的时间跨度上,这种危害会进一步扩大.文章概述了近几年基于细胞色素P450(cytochrome P450,CYP450)作为检测指标,诺氟沙星(norfloxacin,NFLX)对一些水生生物的生态毒理效应.同时介绍了NFLX对于选取的指标CYP450的影响机制,以及检测这些指标的可行性方法.     

3个白桦细胞色素P450基因生物信息学及表达分析

【目的】研究白桦细胞色素P450基因表达的组织特异性,以及在茉莉酸甲酯(MeJA)、水杨酸(SA)、赤霉素(GA₃)、脱落酸(ABA)、乙烯利和伤害处理下的基因表达模式。【方法】筛选白桦转录组,获得3个BpCYP450 基因,分别命名为BpCYP4、BpCYP5、 BpCYP14,利用生物信息学分析BpCYP450蛋白的分子结构特征及其与其他物种CYP450蛋白的亲缘关系。采用QRT-PCR技术,对白桦BpCYP450组织特异性、激素信号及伤害诱导下的表达特征进行分析。【结果】生物信息学结果表明BpCYP4、BpCYP5、BpCYP14 cDNA序列长度分别为1 569、1 584和1 530 bp,具有完整的开放阅读框(ORF),分别编码522、527和509个氨基酸,均为亲水性跨膜蛋白,主要定位于叶绿体。白桦BpCYP450与百脉根、扁豆及豌豆等豆科植物CYP450蛋白亲缘性较高。组织特异性分析结果显示,3个BpCYP450 基因在叶和根中表达较高,茎中较低。激素信号及伤害诱导结果表明,3个BpCYP450基因不同程度地响应MeJA、SA、GA₃、ABA、乙烯利激素信号及伤害诱导。【结论】3个BpCYP450基因均为CYP450基因家族的新成员,具有组织特异性及激素诱导表达特性,可能在白桦生长发育、抵御胁迫及代谢物合成中发挥重要作用。     

Odorant signal termination by olfactory UDP glucuronosyl transferase

The onset of olfactory transduction has been extensively studied, but considerably less is known about the molecular basis of olfactory signal termination. It has been suggested that the highly active cytochrome P450 monooxygenases of olfactory neuroepithelium are termination enzymes, a notion supported by the identification and molecular cloning of olfactory-specific cytochrome P450s (refs. 13-16). But as reactions catalysed by cytochrome P450 (refs 17, 18) often do not significantly alter volatility, lipophilicity or odour properties, cytochrome P450 may not be solely responsible for olfactory signal termination. In liver and other tissues, drug hydroxylation by cytochrome P450 is frequently followed by phase II biotransformation, for example by UDP glucuronosyl transferase (UGT), resulting in a major change of solubility and chemical properties. We report here the molecular cloning and expression of an olfactory-specific UGT. The olfactory enzyme, but not the one in liver microsomes, shows preference for odorants over standard UGT substrates. Furthermore, glucuronic acid conjugation abolishes the ability of odorants to stimulate olfactory adenylyl cyclase. This, together with the known broad spectrum of drug-detoxification enzymes, supports a role for olfactory UGT in terminating diverse odorant signals.     

A high-affinity conformation of Hsp90 confers tumour selectivity on Hsp90 inhibitors

Heat shock protein 90 (Hsp90) is a molecular chaperone that plays a key role in the conformational maturation of oncogenic signalling proteins, including HER-2/ErbB2, Akt, Raf-1, Bcr-Abl and mutated p53. Hsp90 inhibitors bind to Hsp90, and induce the proteasomal degradation of Hsp90 client proteins. Although Hsp90 is highly expressed in most cells, Hsp90 inhibitors selectively kill cancer cells compared to normal cells, and the Hsp90 inhibitor 17-allylaminogeldanamycin (17-AAG) is currently in phase I clinical trials. However, the molecular basis of the tumour selectivity of Hsp90 inhibitors is unknown. Here we report that Hsp90 derived from tumour cells has a 100-fold higher binding affinity for 17-AAG than does Hsp90 from normal cells. Tumour Hsp90 is present entirely in multi-chaperone complexes with high ATPase activity, whereas Hsp90 from normal tissues is in a latent, uncomplexed state. In vitro reconstitution of chaperone complexes with Hsp90 resulted in increased binding affinity to 17-AAG, and increased ATPase activity. These results suggest that tumour cells contain Hsp90 complexes in an activated, high-affinity conformation that facilitates malignant progression, and that may represent a unique target for cancer therapeutics.     

尖镰孢菌细胞色素P45055A1与NO相互作用的荧光光谱法研究

以大肠杆菌为宿主表达了尖镰孢菌细胞色素P450 55A1(CYP450 55A1),并采用荧光光谱法研究了其与NO的相互作用.结果表明:在大肠杆菌(DE3)中成功表达了具有酶活性的CYP55A1.在280 nm波长激发下,NO的加入使CYP55A1的荧光逐渐降低,但随温度的变化差异较小;在413 nm波长激发下,NO的加入使CYP55A1的荧光逐渐升高,但加入NADH后其荧光又恢复到原来的值.     

双孢蘑菇耐温差异蛋白质组学研究

利用蛋白质组学和荧光定量PCR方法,研究双孢蘑菇(Agaricus bisporus Lange (Imbach))02菌株在常温与高温胁迫下的蛋白质表达差异,发现了6个差异蛋白质点,经NCBI数据库比对分析,表明6个差异蛋白质分别为HSP70家族的HSS1热激蛋白、异柠檬酸裂解酶、细胞色素P450单氧化酶及3个未知蛋白质.这些蛋白质在双孢蘑菇受热胁迫下具有保护自身稳定的功能.