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 多环芳烃（PAH） - 可诱导表达的表达 细胞色素P4501A1基因（CYP1A1）。 该法规似乎是 由与基因和反式作用相关的顺式元素介导 蛋白质。 诸如苯并（a）pyrene（b [a] p）之类的PAH可以与 高亲和力，以大鼠肝脏胞质4s的饱和方式饱和 以前已显示与甘氨酸鉴定的蛋白质 N-METHY1转移酶（GNMT）。 我们的初步结果表明 B [A] P可以诱导CYP1A1在AH受体中的表达 （ahr） - 无鼠标和该pah（以及其他几个） 诱导与已稳定转染的CHO细胞中的CYP1A1 GNMT基因（TCDD）在该系统中无效）。 父母 CHO细胞没有GNMT，AHR或ARNT表达 转染剂仅详细详细介绍GNMT。 以这些观察为基础 当前赠款的具体目的是确定：a）效果 CYP1A1和 根据其在AH受体肝脏中转录的速率 负小鼠和肝脏GNMT的水平，b）效果 将GNMT编码序列引入B [A] p-nonresponsive 通过测量稳态CYP1A1 mRNA水平和 它之前的转录速率和agter b [a] p处理以及 测量GNMT及其mRNA的稳定性，c） GNMT的磷酸化，未磷酸化和 磷酸化形式在胞质和核之前/源前核[a] p之前，磷酸化形式， 磷酸化对GNMT功能的影响和 它的寡聚形式，d）GNMT的二聚体四聚体过渡 沉积分析和化学交联，E）的性质 磷酸化（和未磷酸化）4S GNMT的相互作用 使用CYP1A1的顺式元素，通过使用体外核 带有截短模板的转录技术。 我们已经 证明4S PAH结合GNMT是磷酸化的 蛋白质具有磷酸化的可能位点 丝氨酸和/苏氨酸。 根据特定氨基酸的定义 这些氨基酸的位点特异性诱变将得到完成 （通过突变到丙氨酸）。 正在测试的假设是 4S蛋白的磷酸化导致功能增强 pah-binder，增加易位到细胞核和到 增加转录激活。