Novel 5-methylcytosine Oxidases

新型 5-甲基胞嘧啶氧化酶

• 批准号: 8822312
• 负责人: Yu Zheng
• 金额: $ 49.12万
• 依托单位: RGENE, INC.
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8822312
• 关键词: Antibodies; Biochemical; Biological; Biological Assay; Cells; Complex; DNA; Databases; Deposition; Dot Immunoblotting; Engineering; Enzymes; Escherichia coli; Family; Flagella; Generations; Genes; Genome; Genomic DNA; Genomics; Health; Human; In Vitro; Insecta; Libraries; Light; Malignant Neoplasms; Maps; Market Research; Mediating; Methods; Mus; Naegleria; Organism; Oxidases; Pathologic Processes; Pathway interactions; Phase; Process; Property; Protein Engineering; Proteins; Radioactive; Reaction; Reagent; Regulation; Role; Site-Directed Mutagenesis; Structure; System; Testing; Thin Layer Chromatography; Variant; X-Ray Crystallography; Yeasts; base; bisulfite sequencing; complex biological systems; design; enzyme substrate; epigenetic marker; epigenetic regulation; epigenome; epigenomics; expression cloning; genome-wide; improved; in vivo; insight; liquid chromatography mass spectrometry; methylome; mutant; novel; research study; screening; single molecule; three dimensional structure

DESCRIPTION: With the recent discovery of 5-hydroxymethylcytosine in mammalian DNA and the TET enzyme family responsible for its generation, it is clear that the mammalian DNA epigenome is much more complex than had been thought. A lot of questions remain to be answered about the in vivo roles and the in vitro properties of the TET enzymes. In this fast-track application, we propose studying the TET-like enzymes in the single-cell protist Naegleria gruberi. They are much smaller in size than the human TET enzymes and are much easier to produce and engineer. A panel of biochemical assays will be carried out to thoroughly characterize the in vitro properties of the active TET-like enzymes in N. gruberi. We aim at providing these enzymes as reagents to the research market. Since 5-methylcytosine (5mC) is an important epigenetic marker in mammalian genomic DNA, these enzymes should enable many creative experiments, e.g., direct sequencing of the epigenome, because they functionalize the chemically inert 5mC. To enhance their versatility, we propose using enzyme engineering to look for mutants with altered or improved properties. For this purpose, we will determine the 3D structure of at least one active TET-like enzymes. We plan to apply the wild-type enzymes or derived mutants in several epigenomic applications and determine the methylome/hydroxymethylome changes during the differentiation process of N. gruberi, which may shed lights on the similar processes in the more complex biological systems. In summary, our studies will bring a number of novel enzymatic reagents to the research market and generate scientific insights on epigenetic regulation involving 5hmC.

描述：随着哺乳动物DNA中5-羟基甲基胞嘧啶的最新发现，负责其代代相传的TET酶家族，很明显，哺乳动物DNA的表观基因组比以前认为的要复杂得多。关于体内角色和TET酶的体外特性，许多问题仍有待回答。在此快速轨道应用中，我们提出了研究单细胞小蛋白酶Naegleria Gruberi中的TET样酶。它们的尺寸比人类TET酶小得多，并且更容易生产和工程。将进行一系列生化测定，以彻底表征N. Gruberi中活性TET样酶的体外特性。我们旨在将这些酶作为研究市场的试剂。由于5-甲基胞嘧啶（5MC）是哺乳动物基因组DNA中重要的表观遗传标记，因此这些酶应该启用许多创造性的实验，例如表观基因组的直接测序，因为它们功能化化学惰性5MC。为了增强其多功能性，我们建议使用酶工程来寻找具有改变或改进特性的突变体。为此，我们将确定至少一种活性TET样酶的3D结构。我们计划在几种表观基因组应用中应用野生型酶或衍生突变体，并确定在Gruberi的分化过程中甲基甲基/羟基甲基甲基甲基的变化，这可能会在更复杂的生物学系统中的相似过程中散发出光线。总而言之，我们的研究将为研究市场带来许多新型的酶试剂，并对涉及5HMC的表观遗传调节产生科学见解。