GLYCINE N METHYL TRANSFERASE, A REGULATOR OF CYP1A1

甘氨酸 N 甲基转移酶，CYP1A1 的调节剂

• 批准号: 2733366
• 负责人: EDWARD BRESNICK
• 金额: $ 25.35万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-07-07 至 1998-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2733366
• 关键词: CHO cells; adenosine triphosphate; benzopyrenes; binding proteins; cytochrome P450; drug metabolism; enzyme activity; enzyme induction /repression; gene deletion mutation; genetically modified animals; hydroxymethyltransferases; laboratory mouse; laboratory rat; liver cells; liver metabolism; phosphoproteins; site directed mutagenesis

The overall goal of this laboratory is to understand the regulation of polycyclic hydrocarbon (PAH)- inducible expression of the cytochrome P4501A1 gene (CYP1A1). This regulation appears to be mediated by cis elements associated with the gene and trans-acting proteins. PAHs such as benzo(a)pyrene (B[a]P) can interact with high affinity and in saturable manner with a rat liver cytosolic 4S protein that has previously been shown to be identifical with glycine N-methy1transferase (GNMT). Our preliminary results indicated that B[a]P can induce the expression of CYP1A1 in the Ah receptor (AhR)-null mouse and that this PAH (as well as several others) induce CYP1A1 in CHO cells that have been stably transfected with the GNMT gene (TCDD) is ineffective in this system). The parent CHO cells have no GNMT, AhR, or Arnt expression while the transfectants elaborate only GNMT. Building on these observations, the specific aims of the current grant are to determine: a) the effects of B[a]P and 3-methylcholanathrene upon expression of CYP1A1 and upon the rate of its transcription in the livers of the Ah receptor- minus knockout mouse and the level of liver GNMT, b) the effects of introducing the GNMT coding sequence into B[a]P-nonresponsive CHO cells by measuring the steady-state CYP1A1 mRNA level and its rate of transcription before and agter B[a]P treatment and by measuring the stability of the GNMT and its mRNA, c) the site of phosphorylation of GNMT, the distribution of unphosphorylated and phosphorylated forms in cytosol and nucleus before/afterB[a]P, the effects of phosphorylation upon function of GNMT and the nature of its oligomeric form, d) the dimer-tetramer transition of GNMT by sedimentation analysis and by chemical crosslinking, and e) nature of the interaction of phosphorylated (and unphosphorylated) 4S GNMT with cis elements of CYP1A1, through the use of in vitro nuclear transcription techniques with truncated templates. We have already demonstrated that the 4S PAH-binding GNMT is a phosphorylated protein with the probable sites of phosphorylation represented by serine and/threonine. Upon definition of the specific amino acids, site-specific mutagenesis of these amino acids will be accomplished (by mutation to alanines). The hypothesis under test is that phosphorylation of the 4S protein leads to enhanced function as a PAH-binder, to increased translocation into the nucleus and to increased transcriptional activation.

该实验室的总体目标是了解 多环烃 (PAH)- 的诱导表达 细胞色素 P4501A1 基因 (CYP1A1)。 这条规定似乎是 由与基因相关的顺式元件和反式作用介导 蛋白质。 苯并(a)芘 (B[a]P) 等 PAH 可以与 与大鼠肝细胞质 4S 具有高亲和力且可饱和 先前已被证明与甘氨酸相同的蛋白质 N-甲基转移酶（GNMT）。 我们的初步结果表明 B[a]P 可诱导 Ah 受体中 CYP1A1 的表达 (AhR)-null 小鼠和该 PAH（以及其他几种） 在稳定转染的 CHO 细胞中诱导 CYP1A1 GNMT 基因（TCDD）在此系统中无效）。 家长 CHO 细胞没有 GNMT、AhR 或 Arnt 表达，而 转染子仅产生 GNMT。 基于这些观察， 当前赠款的具体目标是确定： a) 效果 CYP1A1 表达后 B[a]P 和 3-甲基胆蒽 取决于其在肝脏中 Ah 受体的转录速率- 减去敲除小鼠和肝脏 GNMT 水平，b) 的影响 将 GNMT 编码序列引入 B[a]P 无反应 通过测量稳态 CYP1A1 mRNA 水平和 B[a]P 处理前后的转录率 测量 GNMT 及其 mRNA 的稳定性，c) 的位点 GNMT 的磷酸化、未磷酸化和磷酸化的分布 B[a]P 之前/之后细胞质和细胞核中的磷酸化形式， 磷酸化对 GNMT 功能及其性质的影响 其寡聚形式，d) GNMT 的二聚体-四聚体转变 沉降分析和化学交联，以及 e) 的性质 磷酸化（和非磷酸化）4S GNMT 的相互作用 与 CYP1A1 的顺式元件，通过使用体外核 具有截断模板的转录技术。 我们已经 证明 4S PAH 结合 GNMT 是磷酸化的 具有可能的磷酸化位点的蛋白质由 丝氨酸和/苏氨酸。 根据特定氨基酸的定义， 将完成这些氨基酸的位点特异性诱变 （通过突变为丙氨酸）。 所测试的假设是 4S蛋白的磷酸化导致增强的功能 PAH 结合剂，增加进入细胞核的易位并 转录激活增加。