GLYCINE N METHYL TRANSFERASE, A REGULATOR OF CYP1A1

本实验室的总体目标是了解 多环烃(PAH)诱导表达 细胞色素P4501A1基因(CYP1A1)。这项规定似乎是 由与基因和反式作用相关的顺式元件介导 蛋白质。多环芳烃，如苯并(A)芘(B[a]P)可与 与大鼠肝细胞浆4S的高亲和力和可饱和性 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型受体细胞色素P1A1的表达 (AHR)-空鼠标，此PAH(以及其他几个) 稳定表达细胞色素P4501A1的CHO细胞的诱导 GNMT基因(TCDD)在该系统中无效)。父辈 CHO细胞不表达GNMT、AhR或Arnt，而 转染者只阐述GNMT。以这些观察为基础， 目前赠款的具体目的是确定：a)效果 苯并[a]磷和3-甲基胆碱对细胞色素P1A1和 根据其在肝脏中的转录速度，阿氏受体- 减去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-无响应 通过检测CHO细胞稳态细胞色素P4501A1的mRNA水平和 它在B[a]P处理前后和通过 测量GNMT及其mRNA的稳定性，c) GNMT的磷酸化、未磷酸化的分布和 B[a]P前后胞质和胞核中的磷酸化形式， 磷酸化对GNMT功能的影响及其性质 其寡聚体形式，d)通过 沉淀物分析和化学交联，以及e)性质 磷酸化(和非磷酸化)4S GNMT的相互作用 用细胞色素P1A1的顺式元件，通过使用体外核 使用截断模板的转录技术。我们已经这么做了 证明了4S PAH结合GNMT是一种磷酸化的 具有可能的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， 这些氨基酸的定点突变将被完成 (通过突变为丙氨酸)。正在测试的假设是 4S蛋白的磷酸化导致作为一种 多环芳烃结合剂，以增加转位到细胞核和 转录活性增强。

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.