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: 2377219
• 负责人: EDWARD BRESNICK
• 金额: $ 24.61万
• 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 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2377219
• 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.

本实验室的总体目标是了解 多环烃（PAH）诱导表达的 细胞色素P4501 A1基因（CYP 1A 1）。 这项规定似乎是 由与基因相关的顺式元件介导， proteins. 苯并（a）芘（B[a]P）等多环芳烃可与 与大鼠肝细胞溶质4S具有高亲和力且可饱和的方式 一种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受体表达CYP 1A 1 （AhR）-null小鼠，这种PAH（以及其他几种） 在已经稳定转染CYP 1A 1 CHO细胞中诱导CYP 1A 1 GNMT基因（TCDD）在该系统中无效）。 母 CHO细胞没有GNMT、AhR或Arnt表达，而 转染子仅阐述GNMT。 在这些观察的基础上， 目前赠款的具体目标是确定：a）影响 B[a]P和3-甲基胆蒽烯对CYP 1A 1表达的影响， 根据其在Ah受体肝脏中的转录速率， 减基因敲除小鼠和肝脏GNMT水平，B）影响 将GNMT编码序列引入B[a] P-无反应的 通过测定稳态CYP 1A 1 mRNA水平测定CHO细胞， 在B[a]P处理前后， 测量GNMT及其mRNA的稳定性， GNMT的磷酸化，未磷酸化和 B [a]P前/后胞浆和核中磷酸化形式， 磷酸化对GNMT功能的影响以及 d）GNMT的二聚体-四聚体转变， 沉降分析和化学交联，和e）性质 磷酸化（和非磷酸化）4S GNMT的相互作用 与CYP 1A 1的顺式元件，通过使用体外核 用截短的模板转录技术。 我们已经 证明了4S PAH结合GNMT是磷酸化的 可能具有磷酸化位点的蛋白质， 丝氨酸和/或苏氨酸。 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 (by突变为丙氨酸）。 正在测试的假设是， 4S蛋白的磷酸化导致增强的功能， PAH结合剂，增加易位到细胞核中， 增加转录激活。