Folate and S-adenosyimethionine in Methyl Group and One-carbon Metabolism

描述(申请人提供)：甲基化的调节与叶酸代谢密切相关。这两个代谢过程由甘氨酸N-甲基转移酶连接，甘氨酸N-甲基转移酶在肝脏、外分泌胰腺和前列腺以及肾脏和肠道粘膜中含量较低。它通过S-腺苷蛋氨酸(ADOMet)对甘氨酸进行甲基化，生成N-甲基甘氨酸和S-腺苷同型半胱氨酸(ADOHcy)。该酶的功能被认为是调节ADOMet和ADOHcy的比例。ADOMet是主要的甲基化基团，ADOMet/ADOHcy比率是细胞甲基化能力的指标。该酶还在体内和体外与作为酶抑制剂的5-甲基-四氢叶酸五谷氨酸紧密结合。这将预先形成的甲基的可用性，如蛋氨酸，与通过叶酸介导的反应从头合成甲基联系在一起。我们建立了甘氨酸N-甲基转移酶基因敲除小鼠，与WT小鼠相比，其肝脏和血浆中S-腺苷蛋氨酸水平非常高。初步数据显示，肝脏DNA并没有在全球范围内发生超甲基化。这些-/-老鼠会自发地患上脂肪肝。我们的第一个假设是，甘氨酸N-甲基转移酶水平高的动物的肝外组织也会出现组织学异常和ADOMet升高。我们的第一个具体目标是从WT和-/-小鼠的组织中检测甲硫氨酸代谢物。我们的第二个假设是，基因的启动子区域将被高甲基化，影响关键蛋白的表观遗传表达。我们的第二个特定目标是测量DNA的全球甲基化以及脂肪肝炎和蛋氨酸代谢相关基因的启动子区域。甘氨酸N-甲基转移酶在外分泌组织中含量最高。我们的第三个假设是甘氨酸N-甲基转移酶在外分泌中很重要，我们的第三个特定目标是测量WT和-/-小鼠的外分泌分泌。肝脏中含量最丰富的叶酸辅酶是5-甲基四氢叶酸。肝脏中的大部分叶酸与甘氨酸N-甲基转移酶紧密结合。我们的第四个假设是，在缺乏这种蛋白质的情况下，不同形式的叶酸将被重新分配，并导致叶酸介导的反应发生变化。我们的第四个具体目标是测量不同形式的叶酸在WT和-/-小鼠肝脏、胰腺和前列腺中的分布，以及叶酸缺乏是否会改变。在动物体内，ADOMet的水平非常高，这表明蛋白质可能被过度甲基化。我们的第五个假设是，在动物体内，His-Tone可能发生了高甲基化。我们的第五个具体目标是检测WT和-/-小鼠中组蛋白的甲基化。我们的第六个假设是，有可能通过给予作为甲基受体的化合物来降低组织中高水平的ADOMet，并减轻小鼠肝脏疾病的严重程度。我们的第六个具体目标是确定服用烟酰胺或双乙酸胍是否能做到这一点。公共卫生意义：这项名为“叶酸和S-甲硫氨酸甲硫氨酸在甲基和一碳代谢中的作用”的建议涉及甘氨酸N-甲基转移酶丢失的影响，甘氨酸N-甲基转移酶是肝脏、胰腺和前列腺中的一种主要酶。我们已经在几个儿童中发现了这种酶的突变，导致中度肝脏损伤，并开发了一种完全缺乏甘氨酸N-甲基转移酶的小鼠模型，随着他们年龄的增长，这种酶会进展为严重的肝病。这项建议的目的之一是评估几种常见的天然化合物的使用能力，以减少在该小鼠模型中肝损伤的发展，从而提高治疗人类甘氨酸N-甲基转移酶缺陷和预防严重肝损伤的可能性。

DESCRIPTION (provided by applicant): Regulation of methylation is intimately associated with folate metabolism. These two metabolic processes are connected by the enzyme, glycine N-methyltransferase, which is abundant in liver, exocrine pancreas and prostate as well as in kidney and intestinal mucosa in lower amounts. It carries out the methylation of glycine by S-adenosylmethionine (AdoMet) to form N-methylglycine and S-adenosylhomocysteine (AdoHcy). The function of the enzyme is believed to be regulation of the ratio of AdoMet to AdoHcy. AdoMet is the major do- nor of methyl groups and the AdoMet/AdoHcy ratio an index of the methylating capacity of the cell. The enzyme also tightly binds 5-methyl-tetrahydrofolate pentaglutamate in vivo and in vitro, which acts as an inhibitor of the enzyme. This links the availability of preformed methyl groups, as methionine, to the de novo synthesis of methyl groups via folate-mediated reactions. We developed a glycine N-methyltransferase knockout mouse that has very high levels of S-adenosylmethionine in the livers and plasma compared to WT mice. Preliminary data showed that liver DNA is not globally hypermethylated. These -/- mice spontaneously develop fatty liver. Our First Hypothesis is that extra-hepatic tissues from -/- animals with high levels of glycine N- methyltransferase will also have abnormal histology and elevated AdoMet. Our First Specific Aim is to ex- amine histology and measure methionine metabolites in tissues from WT, and -/- mice. Our Second Hy- pothesis is that promoter regions of genes will be hypermethylated affecting the epigenetic expression of key proteins. Our second Specific Aim is to measure DNA for global methylation and the promoter regions of genes involved in the development of steatohepatitis and methionine metabolism in tissue from WT and mice. Glycine N-methyltransferase is most abundant in exocrine tissue. Our Third Hypothesis is that glycine N-methyltransferase is important in exocrine secretion and our Third Specific Aim is to measure exocrine se- cretion in WT and -/- mice. The most abundant folate coenzyme in liver is 5-methyltetrahydrofolate. Most of the folate in the liver is tightly bound to glycine N-methyltransferase. Our Fourth Hypothesis is that in the absence of this protein the different forms of folate will be redistributed and result in alterations in folate-mediated reac- tions. Our Fourth Specific Aim is to measure the distribution of the various forms of folate in liver, pancreas and prostate of WT and -/- mice and also whether it is changed in folate deficiency. The levels of AdoMet are very high in -/- animals suggesting that proteins may be overmethylated. Our Fifth Hypothesis is that his- tones may be hypermethylated in -/- animals. Our Fifth Specific Aim is to examine the methylation of histones in WT and -/- mice. Our Sixth Hypothesis is that it is possible to reduce the high levels of AdoMet in tissues by administration of compounds that serve as acceptors of methyl groups and reduce the severity of liver dis- ease in -/- mice. Our Sixth Specific Aim is to determine whether administration of nicotinamide or guanidi- noacetate will do this. PUBLIC HEALTH RELEVANCE: This proposal "Folate and S-adenosylmethionine in Methyl Group and One-carbon Metabolism" deals with the effects of the loss of the enzyme, glycine N-methyltransferase, a major enzyme in liver, pancreas and the prostate gland. We have discovered mutations of this enzyme in several children that result in moderate liver damage and have developed a mouse model that has a complete absence of glycine N-methyltransferase that progresses to severe liver disease as they get older. One of the goals of this proposal is to evaluate the use of several common natural compounds for their ability to diminish the development of liver damage in this mouse model thus raising the possibility of treating humans with defective glycine N-methyltransferase and prevent serious liver damage.