Nuclear function of Glycine N-methyltransferase

描述（由申请人提供）：本提案的总体目标是表征甘氨酸N-甲基转移酶（GNMT）（一种丰富的代谢酶）的新型调节功能。GNMT作为四个相同亚基的四聚体存在，并催化甲基从S-腺苷甲硫氨酸（SAM）转移到甘氨酸，产生肌氨酸（N-甲基甘氨酸）和S-腺苷高半胱氨酸（SAH）。据信，该反应调节SAM/SAH比率，其定义了细胞的甲基化潜力。GNMT被5-甲基四氢叶酸抑制，5-甲基四氢叶酸为甲基在一碳叶酸库和甲基化循环之间的流动提供了调节机制。因此，酶催化和相关的细胞作用在很大程度上受叶酸和甲硫氨酸的可用性控制，而叶酸和甲硫氨酸的可用性又取决于膳食供应。重要的是，虽然GNMT在几种人体组织中丰富，但其表达在肿瘤中丢失。我们还表明，这种酶在迄今为止测试的八种癌细胞系中检测不到。对这种现象的一个可能的解释来自我们最近的实验，该实验证明了这种酶作为细胞增殖的负调节剂的功能。总体而言，以下初步数据以及我们发表的研究为本申请奠定了基础：（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）PC 3细胞激活ERK途径作为响应GNMT表达的促存活机制（iv）GNMT敏感性细胞揭示蛋白质存在于细胞核中;（v）GNMT抗性HEK 293细胞不积累蛋白质于细胞核中;（V）GNMT的赖氨酸残基的体外化学修饰导致四聚体解离成单体，随后单体易位到细胞核中。因此，我们假设GNMT通过调节基因表达来控制细胞增殖。具体而言，我们假设这种作用与SAM向SAH转化的代谢功能无关，而是在易位到细胞核中时发挥作用。我们进一步提出核GNMT通过与核蛋白/染色质的相互作用影响转录。我们还提出，在酶四聚体内GNMT亚基的界面上的赖氨酸残基的修饰导致解离成单体，并使核进入。具体的研究目的是：（1）研究GNMT的核定位。(2)阐明促进GNMT易位到细胞核的机制。(3)区分GNMT诱导细胞毒性的细胞质相关代谢和细胞核相关效应。该项目的完成将建立GNMT核调节机制并定义由该酶控制的细胞过程。 公共卫生相关性：叶酸是人类饮食中重要和必不可少的一部分，调节许多细胞过程，包括甲基化，而叶酸缺乏会促进许多疾病。本申请集中于GNMT的新的核相关功能，GNMT是一种由叶酸调节的丰富的人类酶，其位于叶酸代谢和甲基化之间的交叉点。由于GNMT可以作为肝脏和其他组织过度增殖的限制器，因此了解其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.