Nuclear function of Glycine N-methyltransferase

描述(由申请人提供)：本提案的总体目标是描述甘氨酸N-甲基转移酶(GNMT)的一种新的调节功能，GNMT是一种丰富的代谢酶。GNMT以四个相同亚基的四聚体形式存在，催化一个甲基从S-腺苷蛋氨酸转移到甘氨酸，产生肌氨酸(N-甲基甘氨酸)和S-腺苷同型半胱氨酸(SAH)。据认为，这个反应调节SAM/SAH比率，这定义了细胞的甲基化潜力。GNMT被5-甲基四氢叶酸抑制，这为单碳叶酸库和甲基化循环之间的甲基流动提供了一种调节机制。因此，酶催化和相关的细胞作用在很大程度上受叶酸和蛋氨酸的供应控制，而这又取决于饮食供应。重要的是，虽然GNMT在几个人体组织中大量存在，但在肿瘤中却没有表达。我们还表明，到目前为止，在测试的八种癌细胞株中都没有检测到这种酶。这一现象的一个可能的解释来自我们最近的实验，这些实验证明了这种酶作为细胞增殖的负调节因子的功能。总体而言，以下初步数据和我们发表的研究为目前的应用奠定了基础：(I)GNMT的瞬时表达抑制癌细胞的增殖；(Ii)这种作用不能被高叶酸补充逆转；(Iii)PC3细胞激活ERK途径作为响应GNMT表达的促生存机制(但不足以拯救细胞)；(Iv)GNMT敏感的细胞显示该蛋白存在于细胞核中；(V)具有GNMT抗性的HEK293细胞不在细胞核中积累该蛋白；(V)在体外对GNMT的赖氨酸残基进行化学修饰会导致四聚体解离为单体，然后单体移位到核中。因此，我们假设GNMT通过调节基因表达来控制细胞增殖。具体地说，我们假设这一作用与SAM向SAH转化的代谢功能无关，但在转位到细胞核时发挥作用。我们进一步提出，核GNMT通过与核蛋白/染色质相互作用来影响转录。我们还提出，酶四聚体中GNMT亚基界面上的赖氨酸残基的修饰导致单体解离，并使核进入。为检验这些假设而设计的具体目标是：(1)表征GNMT的核定位。(2)阐明促进GNMT向核转位的机制。(3)区分GNMT诱导细胞毒作用的细胞质相关代谢效应和核相关效应。该项目的完成将建立GNMT核调节的机制，并确定由该酶控制的细胞过程。 与公共健康相关：叶酸是人类饮食中重要和必要的一部分，调节着许多细胞过程，包括甲基化，而叶酸缺乏会导致许多疾病。这项应用主要集中在GNMT的一种新的核相关功能上，GNMT是一种丰富的人类酶，由叶酸调节，位于叶酸代谢和甲基化的交叉点。由于GNMT可以作为肝脏和其他组织过度增殖的限制因子，了解它在细胞代谢中的作用将有助于更好地管理肝脏相关疾病。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to characterize a novel regulatory function of glycine N- methyltransferase (GNMT), an abundant metabolic enzyme. GNMT exists as a tetramer of four identical subunits and catalyses the transfer of a methyl group from S-adenosylmethionine (SAM) to glycine producing sarcosine (N-methylglycine) and S-adenosylhomocysteine (SAH). It is believed that this reaction regulates the SAM/SAH ratio, which defines the methylation potential of the cell. GNMT is inhibited by 5-methyltetrahydrofolate, which provides a regulatory mechanism for the flow of methyl groups between the one-carbon folate pool and the methylation cycle. As such, enzyme catalysis, and an associated cellular role, is controlled to a significant extent by the availability of folate and methionine, which in turn depends on a dietary supply. Importantly, while GNMT is abundant in several human tissues, its expression is lost in tumors. We have also shown that the enzyme is not detectable in eight cancer cell lines tested so far. A possible explanation of this phenomenon came from our recent experiments, which demonstrated a function of the enzyme as a negative regulator of cellular proliferation. Overall, the following preliminary data, together with our published studies, laid the ground for the present application: (i) transient expression of GNMT suppresses proliferation of cancer cells; (ii) this effect cannot be reversed by high folate supplementation; (iii) PC3 cells activate the ERK pathway as a pro-survival mechanism in response to GNMT expression (but it is not sufficient to rescue cells); (iv) GNMT-sensitive cells revealed the presence of the protein in nuclei; (v) GNMT-resistant HEK293 cells did not accumulate the protein in nuclei; (v) in vitro chemical modification of lysine residues of GNMT causes dissociation of tetramer to monomers, followed by translocation of monomers into the nuclei. Accordingly, we hypothesize that GNMT controls cellular proliferation through regulation of gene expression. Specifically, we hypothesize that this role is not associated with the metabolic function of conversion of SAM to SAH but is exerted upon translocation into nuclei. We further propose that nuclear GNMT affects transcription through interaction with nuclear proteins/chromatin. We also propose that modifications of lysine residues on the interface of GNMT subunits within the enzyme tetramer results in dissociation to monomers and enables the nuclear entry. The specific aims designed to test these hypotheses are: (1) Characterize nuclear localization of GNMT. (2) Elucidate mechanisms promoting translocation of GNMT to the nucleus. (3) Differentiate the cytoplasm-related metabolic and nucleus-associated effects of GNMT in induction of cytotoxicity. Accomplishment of this project will establish a mechanism for GNMT nuclear regulation and define cellular processes controlled by the enzyme. PUBLIC HEALTH RELEVANCE: Folate, an important and essential part of the human diet, regulates many cellular processes including methylation, while folate deficiency promotes many diseases. This application is focused on a novel nucleus-associated function of GNMT, an abundant human enzyme regulated by folate, which lies at an intersection point between folate metabolism and methylation. Since GNMT can function as a restrictor of excessive proliferation in liver and other tissues, understanding its role in cellular metabolism will help to better manage liver related diseases.