Nuclear function of Glycine N-methyltransferase

甘氨酸 N-甲基转移酶的核功能

• 批准号: 7990236
• 负责人: Natalia Ivanovna Krupenko
• 金额: $ 22.13万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7990236
• 关键词: Affect; Benzo(a)pyrene; Cancer cell line; Catalysis; Cell Nucleus; Cell Proliferation; Cell physiology; Cells; Chemicals; Chromatin; Cytochrome P-450 CYP1A1; Cytoplasm; Cytosol; Data; Diet; Diffusion; Disease; Dissociation; Enzymes; Fluorescein; Fluoresceins; Folate; Folic Acid Deficiency; Gene Expression; Gene Expression Regulation; Genetic Transcription; Genome; Glyceraldehyde-3-Phosphate Dehydrogenases; Glycine; Goals; Green Fluorescent Proteins; Human; In Vitro; Isothiocyanates; Liver; Lysine; Malignant Neoplasms; Mammalian Cell; Metabolic; Metabolism; Methionine; Methylation; Modification; Nuclear; Nuclear Protein; Nuclear Proteins; Nuclear Translocation; One Carbon Pool by Folate Pathway; Pathway interactions; Proteins; Publishing; Rattus; Reaction; Regulation; Regulator Genes; Reporting; Resistance; Role; Safety; Sarcosine; Signal Transduction; Supplementation; Testing; Tetrahydrofolates; Tissues; Transcriptional Regulation; Ubiquitin; cancer cell; cell growth regulation; cytosolic receptor; cytotoxicity; design; dimer; folic acid metabolism; glycine N-methyltransferase; human tissue; in vivo; methyl group; methylcobalamin-coenzyme M methyltransferase; monomer; mutant; novel; prevent; public health relevance; research study; response; tumor

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.

描述（由申请人提供）：该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 以四个相同亚基的四聚体形式存在，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，该比率定义了细胞的甲基化潜力。 GNMT 被 5-甲基四氢叶酸抑制，它为单碳叶酸库和甲基化循环之间的甲基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蛋氨酸的可用性控制，而叶酸和蛋氨酸又取决于饮食供应。重要的是，虽然 GNMT 在多种人体组织中丰富，但其表达在肿瘤中丢失。我们还表明，迄今为止在测试的八种癌细胞系中无法检测到该酶。对这种现象的一个可能的解释来自我们最近的实验，该实验证明了该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) 这种效应不能通过补充大量叶酸来逆转； (iii) PC3细胞响应GNMT表达而激活ERK通路作为促生存机制（但这不足以拯救细胞）； (iv) GNMT 敏感细胞显示细胞核中存在该蛋白质； (v) GNMT 抗性 HEK293 细胞不会在细胞核中积累蛋白质； (v) GNMT 赖氨酸残基的体外化学修饰导致四聚体解离为单体，然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。具体来说，我们假设这种作用与 SAM 转化为 SAH 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/染色质相互作用影响转录。我们还提出，酶四聚体内 GNMT 亚基界面上的赖氨酸残基的修饰会导致解离为单体并能够进入核。测试这些假设的具体目标是：（1）表征 GNMT 的核定位。 (2)阐明促进GNMT转位至细胞核的机制。 (3) 区分 GNMT 在细胞毒性诱导中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。 公众健康相关性：叶酸是人类饮食的重要组成部分，调节包括甲基化在内的许多细胞过程，而叶酸缺乏会促进许多疾病。该应用的重点是 GNMT 的一种新的细胞核相关功能，GNMT 是一种受叶酸调节的丰富的人类酶，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，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。