细胞色素P450酶系的异源表达研究

传统的药物代谢主要以实验动物为对象进行药物早期及临床研究,近几年来,结合生物化学与分子生物学的发展,药物早期代谢研究进入到药物体外代谢的研究阶段,主要围绕人肝内参与代谢的细胞色素P450家族展开.本文就近年来对此家族酶的异源表达及其在药物代谢中的应用进行了综述.     

Jasmonate and salicylate induce expression of herbivore cytochrome P450 genes

Jasmonate and salicylate are plant-produced signals that activate plant defence genes after herbivory or pathogen attack. Amplification of these signals, evoked by either enemy attack or experimental manipulation, leads to an increase in the synthesis of toxic compounds (allelochemicals) and defence proteins in the plants. Although the jasmonate and salicylate signal cascades activate different sets of plant defence genes, or even act antagonistically, there is substantial communication between the pathways. Jasmonate and salicylate also contribute to protecting plants against herbivores by causing plants that experience insect damage to increase their production of volatile molecules that attract natural enemies of herbivorous insects. In response to plant defences, herbivores increase their production of enzymes that detoxify allelochemicals, including cytochrome P450s (refs 15, 16). But herbivores are potentially vulnerable to toxic allelochemicals in the duration between ingesting toxins and induction of detoxification systems. Here we show that the corn earworm Helicoverpa zea uses jasmonate and salicylate to activate four of its cytochrome P450 genes that are associated with detoxification either before or concomitantly with the biosynthesis of allelochemicals. This ability to 'eavesdrop' on plant defence signals protects H. zea against toxins produced by host plants.     

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.     

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.     

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.     

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．     

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。     

人细胞色素P450氧化还原酶的克隆表达和抗体的制备

细胞色素P450还原酶(CPR)是人细胞色素P450酶系的电子供给者,对后者活性的发挥起到重要作用.本研究将从人胚胎cDNA文库中得到的人CPR的编码基因,连接入pT7450表达载体中,在大肠杆菌BL21(DE3)中高效表达并纯化.每升发酵液可得到纯化蛋白49 mg,占总蛋白含量的10%,以细胞色素C为底物检测酶活性,比活为65 u/mg.利用纯化的CPR作为抗原,常规免疫纯系大耳家兔,获得高效价、高特异性的抗血清,抗体滴度为1∶200 000(ELISA法),纯化后抗体应用于不同组织中CPR含量的评价,证明重组CPR及其抗体可用于细胞色素P450的体外药物代谢及细胞色素P450与CPR作用机理的研究.     

Structures of cytochrome P450 17A1 with prostate cancer drugs abiraterone and TOK-001

Cytochrome P450 17A1 (also known as CYP17A1 and cytochrome P450c17) catalyses the biosynthesis of androgens in humans. As prostate cancer cells proliferate in response to androgen steroids, CYP17A1 inhibition is a new strategy to prevent androgen synthesis and treat lethal metastatic castration-resistant prostate cancer, but drug development has been hampered by lack of information regarding the structure of CYP17A1. Here we report X-ray crystal structures of CYP17A1, which were obtained in the presence of either abiraterone, a first-in-class steroidal inhibitor recently approved by the US Food and Drug Administration for late-stage prostate cancer, or TOK-001, an inhibitor that is currently undergoing clinical trials. Both of these inhibitors bind the haem iron, forming a 60° angle above the haem plane and packing against the central I helix with the 3β-OH interacting with aspargine 202 in the F helix. Notably, this binding mode differs substantially from those that are predicted by homology models and from steroids in other cytochrome P450 enzymes with known structures, and some features of this binding mode are more similar to steroid receptors. Whereas the overall structure of CYP17A1 provides a rationale for understanding many mutations that are found in patients with steroidogenic diseases, the active site reveals multiple steric and hydrogen bonding features that will facilitate a better understanding of the enzyme's dual hydroxylase and lyase catalytic capabilities and assist in rational drug design. Specifically, structure-based design is expected to aid development of inhibitors that bind only CYP17A1 and solely inhibit its androgen-generating lyase activity to improve treatment of prostate and other hormone-responsive cancers.     

细胞色素P450酶生物转化及新催化反应的最新进展

细胞色素P450酶(P450s或CYPs)是一类广泛存在于生命体中,依赖于亚铁血红素催化多种底物的单加氧酶。一方面,它涉及许多高活性天然产物生物合成的关键步骤,对其生物转化研究有助于提高活性分子的产率和活性;另一方面,其催化涉及金属元素以及辅助因子,其蛋白质改造和新催化反应的研发也不断取得新突破。本文就细胞色素P450酶这两方面的最近研究进展进行了总结与论述。     

Characterization of the common genetic defect in humans deficient in debrisoquine metabolism

In population studies of individuals given the antihypertensive drug debrisoquine, two distinct phenotypes have been described: extensive metabolizers excrete 10-200 times more of the urinary metabolite 4-hydroxydebrisoquine than poor metabolizers. In family studies the poor-metabolizer phenotype behaves as an autosomal recessive trait with an incidence between 5% and 10% in the white population of Europe and North America, and extends to the deficient metabolism of more than 20 commonly prescribed drugs. Clinical studies have shown that such individuals are at high risk for the development of adverse side effects from these and probably many other drugs. Here we show that poor metabolizers have negligible amounts of the cytochrome P450 enzyme P450db1. We have cloned the human P450db1 complementary DNA and expressed it in mammalian cell culture. Furthermore, by directly cloning and sequencing cDNAs from several poor-metabolizer livers, we have identified three variant messenger RNAs that are products of mutant genes producing incorrectly spliced db1 pre-mRNA, providing a molecular explanation for one of man's most commonly defective genes (frequency of mutant alleles 35-43%).     

Cytochrome P450 Monooxygenase Immobilization as a Model of Herbicide Metabolism in vitro

To investigate herbicide metabolism in vitro by cytochrome P450 with stable enzymatic activity, cy-tochrome P450 is immobilized in silk fibroin. The enzymatic activity of immobilized cytochrome P450 is maintained above 80% after repeated batch experiments for 10 times. Moreover, the enzymatic activity of immobilized cytochrome P450 is kept as relatively high as 73.8% after storage for two months at 4℃. In addition, immobilization can improve the temperature and pH stability of cytochrome P450. Immobilized cytochrome P450 has the similar affinity Km values for herbicide chlorsulfuron and triasulfuron as the free cytochrome P450. In the case of chlorosulfuron, affinity Km value is 53μmol/L for free cytochrome P450,and 63μmol/L for immobilized cytochrome P450, respectively. In the case of triasulfuron affinity, Km value is 36μmol/L for free cytochrome P450, and 44μmol/L for immobilized cytochrome P450, respec-tivily. Immobilized cytochrome P450 will be convenient, rapid, stable and continuous for herbicide metabolism in micro-bioreactor in vitro.     

RGS2 regulates signal transduction in olfactory neurons by attenuating activation of adenylyl cyclase III

The heterotrimeric G-protein Gs couples cell-surface receptors to the activation of adenylyl cyclases and cyclic AMP production (reviewed in refs 1, 2). RGS proteins, which act as GTPase-activating proteins (GAPs) for the G-protein alpha-subunits alpha(i) and alpha(q), lack such activity for alpha(s) (refs 3-6). But several RGS proteins inhibit cAMP production by Gs-linked receptors. Here we report that RGS2 reduces cAMP production by odorant-stimulated olfactory epithelium membranes, in which the alpha(s) family member alpha(olf) links odorant receptors to adenylyl cyclase activation. Unexpectedly, RGS2 reduces odorant-elicited cAMP production, not by acting on alpha(olf) but by inhibiting the activity of adenylyl cyclase type III, the predominant adenylyl cyclase isoform in olfactory neurons. Furthermore, whole-cell voltage clamp recordings of odorant-stimulated olfactory neurons indicate that endogenous RGS2 negatively regulates odorant-evoked intracellular signalling. These results reveal a mechanism for controlling the activities of adenylyl cyclases, which probably contributes to the ability of olfactory neurons to discriminate odours.     

The S. cerevisiae SEC65 gene encodes a component of yeast signal recognition particle with homology to human SRP19.

Translocation of proteins across the endoplasmic reticulum (ER) membrane represents the first step in the eukaryotic secretory pathway. In mammalian cells, the targeting of secretory and membrane protein precursors to the ER is mediated by signal recognition particle (SRP), a cytosolic ribonucleoprotein complex comprising a molecule of 7SL RNA and six polypeptide subunits (relative molecular masses 9, 14, 19, 54, 68 and 72K). In Saccharomyces cerevisiae, a homologue of the 54K subunit (SRP54) co-purifies with a small cytoplasmic RNA, scR1 (refs 4, 5). Genetic data indicate that SRP54 and scR1 are involved in translocation in vivo, suggesting the existence of an SRP-like activity in yeast. Whether this activity requires additional components similar to those found in mammalian SRP is not known. We have recently reported a genetic selection that led to the isolation of a yeast mutant, sec65-1, which is conditionally defective in the insertion of integral membrane proteins into the ER. Here we report the cloning and sequencing of the SEC65 gene, which encodes a 31.2K protein with significant sequence similarity to the 19K subunit of human SRP (SRP19). We also report the cloning of a multicopy suppressor of sec65-1, and its identification as the previously defined SRP54 gene, providing genetic evidence for an interaction between these gene products in vivo.     

Primary structure and functional expression of a cyclic nucleotide-activated channel from olfactory neurons.

Odorant signal transduction occurs in the specialized cilia of the olfactory sensory neurons. Considerable biochemical evidence now indicates that this process could be mediated by a G protein-coupled cascade using cyclic AMP as an intracellular second messenger. A stimulatory G protein alpha subunit is expressed at high levels in olfactory neurons and is specifically enriched in the cilia, as is a novel form of adenylyl cyclase. This implies that the olfactory transduction cascade might involve unique molecular components. Electrophysiological studies have identified a cyclic nucleotide-activated ion channel in olfactory cilia. These observations provide evidence for a model in which odorants increase intracellular cAMP concentration, which in turn activates this channel and depolarizes the sensory neuron. An analogous cascade regulating a cGMP-gated channel mediates visual transduction in photoreceptor cells. The formal similarities between olfactory and visual transduction suggest that the two systems might use homologous channels. Here we report the molecular cloning, functional expression and characterization of a channel that is likely to mediate olfactory transduction.     

A flagellin-induced complex of the receptor FLS2 and BAK1 initiates plant defence

Plants sense potential microbial invaders by using pattern-recognition receptors to recognize pathogen-associated molecular patterns (PAMPs). In Arabidopsis thaliana, the leucine-rich repeat receptor kinases flagellin-sensitive 2 (FLS2) (ref. 2) and elongation factor Tu receptor (EFR) (ref. 3) act as pattern-recognition receptors for the bacterial PAMPs flagellin and elongation factor Tu (EF-Tu) (ref. 5) and contribute to resistance against bacterial pathogens. Little is known about the molecular mechanisms that link receptor activation to intracellular signal transduction. Here we show that BAK1 (BRI1-associated receptor kinase 1), a leucine-rich repeat receptor-like kinase that has been reported to regulate the brassinosteroid receptor BRI1 (refs 6,7), is involved in signalling by FLS2 and EFR. Plants carrying bak1 mutations show normal flagellin binding but abnormal early and late flagellin-triggered responses, indicating that BAK1 acts as a positive regulator in signalling. The bak1-mutant plants also show a reduction in early, but not late, EF-Tu-triggered responses. The decrease in responses to PAMPs is not due to reduced sensitivity to brassinosteroids. We provide evidence that FLS2 and BAK1 form a complex in vivo, in a specific ligand-dependent manner, within the first minutes of stimulation with flagellin. Thus, BAK1 is not only associated with developmental regulation through the plant hormone receptor BRI1 (refs 6,7), but also has a functional role in PRR-dependent signalling, which initiates innate immunity.     

The nuclear receptor CAR mediates specific xenobiotic induction of drug metabolism

Organisms encounter a wide range of foreign compounds--or 'xenobiotics'--with potentially harmful consequences. The cytochrome P450 (CYP) enzymes metabolize xenobiotics and thus are a primary defence against these compounds. Increased expression of specific CYP genes in response to particular xenobiotics is a central component of this defence, although such induction can also increase production of toxic metabolites. Here we show that the nuclear receptor CAR mediates the response evoked by a class of xenobiotics known as the 'phenobarbital-like inducers'. The strong activation of Cyp2b10 gene expression by phenobarbital, or by the more potent TCPOBOP, is absent in mice lacking the CAR gene. These animals also show decreased metabolism of the classic CYP substrate zoxazolamine and a complete loss of the liver hypertrophic and hyperplastic responses to these inducers. Cocaine causes acute hepatotoxicity in wild-type mice previously exposed to phenobarbital-like inducers and this toxicity is also absent in the CAR-deficient animals. Thus, loss of CAR function alters sensitivity to toxins, increasing or decreasing it depending on the compound. Modulation of CAR activity in humans may significantly affect metabolism of drugs and other xenobiotics.     

永生化胚胎肝细胞生物学特性及功能研究

研究永生化胚胎肝细胞的生物学特性及功能,研究并观察永生化胚胎肝细胞的形态及结构特征,测定其生长曲线,观察克隆形成能力并作软琼脂试验,对细胞染色体进行核型分析,同时对细胞合成白蛋白、尿素及细胞色素P450能力进行检测。结果显示永生化胚胎肝细胞克隆形成率为31.2%,染色体核型分析表明细胞核型无明显异常,软琼脂集落形成试验表明细胞在软琼脂中不能生长。细胞已在体外传56代仍保持正常肝细胞的形态特征,而且具有合成白蛋白、尿素及细胞色素P450的功能。说明该永生化胚胎肝细胞可以成为生物人工肝及肝细胞移植研究中的理想细胞材料。     

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.