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系統識別號 U0026-0812200913344718
論文名稱(中文) 類酯醇X 受體的變異型對細胞色素3A4 調控之研究
論文名稱(英文) Study of Pregnane X Receptor (PXR) Mutants on CYP3A4 Transactivation Regulation
校院名稱 成功大學
系所名稱(中) 基礎醫學研究所
系所名稱(英) Institute of Basic Medical Sciences
學年度 95
學期 1
出版年 96
研究生(中文) 林雲冰
研究生(英文) Yun-Ping Lim
學號 s5891101
學位類別 博士
語文別 英文
論文頁數 111頁
口試委員 口試委員-陳新
口試委員-翁祖輝
指導教授-黃金鼎
召集委員-呂增宏
口試委員-賴明德
口試委員-張明熙
中文關鍵字 細胞色素3A4  類酯醇X受體 
英文關鍵字 CYP3A4  PXR 
學科別分類
中文摘要 類酯醇X受體 (pregnane X receptor, PXR) 是許多藥物代謝基因,例如細胞色素P450 3A4 (CYP3A4) 及人類多重抗藥性基因 (P-glycoprotein, P-gp)一個主要的轉錄調控因子。而PXR的基因多型性 (single nucleotide polymorphism, SNP) 則會影響個體之間CYP3A4及P-gp的活性差異。目前已有猶太人、非裔美國人及日本人報導PXR SNP的文獻,而我們也已找到在華人族群中一個新的基因型,Q158K。我們以等位基因PCR (allele-specific PCR, AS-PCR) 的方式篩選了451個華裔個體,顯示了它的基因頻率為2.2%。利用CYP3A4啟動子報導基因的表現能力來評估此PXR變異型對CYP3A4表現的影響。結果顯示,在LS174T及HepG2細胞中,Q158K均對rifampin、paclitaxel、clotrimazole及nifedipine有降低CYP3A4 luciferase活性的作用。經由DNA親和性沉澱試驗 (DNA affinity precipitation assay, DAPA) 及電泳移動偏移分析試驗 (electrophoretic mobility shift assay, EMSA) 顯示Q158K的變異並不會改變PXR結合在CYP3A4啟動子序列上的能力。哺乳類二合法試驗 (mammalian two-hybrid assay) 顯示Q158K在有配合基的存在下與SRC-1的結合能力下降,而且進一步以免疫共同沉澱試驗(co-immunoprecipitation assay) 也證明了原生型PXR在有rifampin的存在下,與SRC-1的結合能力增強,而Q158K並不會。投予細胞SRC-1質體則可提高Q158K對CYP3A4啟動子的轉錄能力。有趣的是,此Q158氨基酸位在PXR的配合基結合區 (ligand binding domain, LBD) 上,並不參與在ligand binding cavity上,但此位置卻影響了對CYP3A4啟動子的轉錄能力。所以,單以X-ray crystallography的方式來了解PXR對於不同藥物的誘導作用似乎是不夠的。因此我們以error-prone PCR方法造成PXR隨機突變,再以CYP3A4 cell-based luciferase assay的方式,於不同配合基的誘導下,篩選出與原生型PXR差異頗大的變異株,並進一步探討這些變異株與它的coregulator之間的交互作用是否是調控了這些變異株特異性的主因。結果顯示,在Q158、W223、F257、I346及L424若有變異的話,皆會降低對所有藥物的CYP3A4 luciferase活性,而這些突變點也使得它們與coactivator (SRC-1) 的結合降低。L184及A244的突變點卻分別對rifampin具有不同的反應。CYP3A4 luciferase活性的結果與它們和SRC-1結合的結果相互符合。然而F257及I346的突變點均會使PXR不與SMRT結合,顯示PXR對於coactivator的結合是會因ligand及特殊位置的氨基酸來決定的。從我們的結果顯示,Q158K之所以在CYP3A4啟動子報導基因上有別於原生型的PXR是因為它與SRC-1的結合能力在有配合基的存在下比較弱。也由於PXR的配合基結合區是與SRC-1結合所必須的,所以我們預測在其他種族所找到的基因型 (D163G、A370T、R381W及I403V) 也影響了PXR與SRC-1結合的能力,進而改變了CYP3A4啟動子報導基因的表現。
英文摘要 The pregnane X receptor (PXR) is the main transcriptional regulator of many enzymes that metabolize xenobiotics such as P450s and drug transporters. Polymorphisms in the PXR gene contribute to population variability in CYP3A4 and P-glycoprotein levels. Single nucleotide polymorphisms (SNPs) have been reported in Caucasian, African-American and Japanese populations. In the present study, we identified the known SNP, V140M and a novel SNP, Q158K, in Chinese subjects. We developed an allele-specific polymerase chain reaction method to detect the novel allele and found its frequency in 451 Chinese subjects to be 2.2%. CYP3A4 luciferase reporter assays revealed that the Q158K variant gave rise to much lower levels of CYP3A4 promoter activity in LS174T and HepG2 cells when exposed to the PXR ligands, rifampin and paclitaxel, than did wild type PXR and with a smaller effect on induction by clotrimazole and nifedipine. By using DNA affinity precipitation assay and electrophoretic mobility shift assay, we show that Q158K does not alter the binding affinity of PXR for the CYP3A4 promoter. Instead, as shown using a mammalian two-hybrid assay, it decreased the interaction of PXR with SRC-1 in the presence of rifampin, clotrimazole, paclitaxel, or nifedipine but not in their absence. Rifampin treatment increased PXR protein in the wild type PXR-transfected cells as shown by co-immunoprecipitation but not in Q158K PXR-transfected cells. The impaired transactivation of the CYP3A4 promoter was reversed by transfecting SRC-1 expression plasmids. Interestingly, Q158 is located in helix-1 of PXR ligand binding domain, but not line in the ligand-binding pocket. Apparently, the structural basis underlying the activation of PXR under the presence of specific ligands cannot be understood by the X-ray crystallography structure alone. We tried to explore PXR variants under ligand induction by random mutagenesis based on cell-based reporter assay. Using CYP3A4 reporter assays, several PXR variants were selected within the variants library under ligand induction. These mutants were subjected to investigate the interaction between PXR and coactivator. Our results indicate that mutation on Q158, W223, F257, I346, and L424 can reduce the CYP3A4 transactivation under ligands treatment, and regulate the interaction between PXR and SRC-1 in the presence of rifampin, clotrimazole, paclitaxel, or nifedipine. Whereas, PXR-SMRT interaction was impaired in the F257L and I346T variants with or without the presence of PXR ligands. Our data indicate that the impaired induction by the Q158K variant is probably due to defective SRC-1 interaction in the presence of receptor ligand. Since the whole ligand binding domain of PXR is required for the interaction with SRC-1, we propose that, not only the Q158K variant found in Chinese, but native PXR variants in other ethnic groups (D163G, A370T, R381W, and I403V) affect CYP3A4 induction by altering SRC-1 recruitment.
論文目次 考試合格證明………………………………………………………………………II
Abstract in Chinese………………………………………………………………III
Abstract………………………………………………………………V
Acknowledgement……………………………………………………VII
Contents………………………………………………………………VIII
Table Index……………………………………………………………………XI
Figure Index…………………………………………………………………XII
Abbreviations………………………………………………………XIV

Chapter 1 Introduction…………………………………………………………………………1
I. Cytochrome P450 (CYP450) and cytochrome P450 3A4 (CYP3A4)…………………1
II. Pregnane X receptor (PXR)……………………………………………………………2
III. PXR coactivator - steroid receptor coactivator-1 (SRC-1)…………………5
IV. PXR corepressor - silencing mediator of retinoid and thyroid
hormone receptor (SMRT)……………………………………………………………7
V. Single nucleotide polymorphisms (SNPs) in pregnane X receptor……………8
VI. Purpose……………………………………………………………………9

Chapter 2 Materials and Methods……………………………………………………………11
I. Materials………………………………………………………………11
Chemicals and reagents………………………………………………………………11
Kits………………………………………………………………………14
Enzymes…………………………………………………………………14
Bacterial culture accessories and antibiotics…………………………………15
Antibodies for Western blotting …………………………………………………16
Cell culture accessories……………………………………………………………16
Instruments……………………………………………………………17
Others…………………………………………………………………17
II. Methods…………………………………………………………………19
Genomic DNA preparation………………………………………………………19
Polymerase chain reaction-single-strand conformational polymorphism
(PCR-SSCP) and allele-specific polymerase chain reaction (AS-PCR)………19
Plasmids construction…………………………………………………………19
Random mutagenesis……………………………………………………………21
Site-directed mutagenesis……………………………………………………………22
Chemicals and cell cultures…………………………………………………………22
Transfection, CYP3A4 reporter assay, and the mammalian
two-hybrid assay………………………………………………………………………22
Preparation of nuclear extracts………………………………………………… 23
Western blotting………………………………………………………………………23
DNA affinity precipitation assay (DAPA)……………………………………… 24
Electrophoretic mobility shift assay (EMSA)……………………………………25
Co-immunoprecipitation assay (Co-IP)……………………………………………25

Chapter 3 Results…………………………………………………………………………… 27
I. Detection of genetic polymorphisms in hPXR gene in Chinese subjects……27
II. Altered transactivation of the CYP3A4 proximal/enhancer reporter
by Q158K………………………………………………………………………………27
III. Identification of the PXR nuclear factor involved in the transactivation of the CYP3A4 gene………………………………………………………28
IV. Co-regulation of wild type PXR and Q158K with SRC-1………………………30
V. Binding of PXR coactivator (SRC-1) with wild type PXR and Q158K ………30
VI. Random mutagenesis created PXR variants………………………………………32
VII. Binding of PXR coregulators (SRC-1 and SMRT) with wild type PXR
and PXR variants……………………………………………………………………33

Chapter 4 Discussion………………………………………………………………35

References………………………………………………………………43
Tables…………………………………………………………………50
Figures…………………………………………………………………54
Publications…………………………………………………………90
自述………………………………………………………………………91
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