2010-08-012024-05-18https://scholars.lib.ntu.edu.tw/handle/123456789/704787摘要：表顯遺傳學即為研究表顯遺傳的繼承，是指在不改變核酸序列的情況下，能改變生物體外觀(表現型)或基因表現的方式。DNA(去氧核醣核酸)甲基化為表顯遺傳調控的可逆化學機制之一，是將Cytosine(胞嘧啶)上第五號碳進行甲基化。這種複製後訊息，對於染色質的結構、基因組的印記與其完整性等疾病相關特徵，有著重要的影響。 人類DNA methyltransferase I(DNMT1，去氧核醣核酸轉移酵素一型) 長約1620個胺基酸，屬於催化DNA甲基化過程的重要酵素家族成員之一。DNMT1是第一個被發現並主要負責在複製過程中，維持DNA甲基化程度的酵素。在哺乳動物中，DNMT1主要針對基因體中半甲基化之CpG雙核酸進行催化，而抑制DNMT1活性是對於各種不正常發育或增生型疾病－特別是癌症，提供一種相當有希望的治療策略。但是對於DNMT1與DNA辨識與其催化機制的結構訊息，目前仍瞭解甚少。另一個重要的問題在於，對於DNMT1相關蛋白的鑑定與結合功能探討也是值得關切。 我提出這份計畫書的構想主要是希望透過結構上的觀點來探討DNMT1和其功能性區塊在表顯遺傳調控上的調節角色與催化行為。運用數種生化與生物物理方法將會被運用來了解其結構與功能的關聯性。在過去幾個月，本實驗室已廣泛選殖和生產各種目標蛋白質。因此，而本三年計畫將延續我們建立的系統與結果提出了五個目標：(1)深入探討DNMT1與其功能性區塊的蛋白質穩定性與折疊；(2)探討蛋白質的修飾影響DNMT1調節與活性的機制；(3)利用蛋白質體技術與生物物理方法來篩選可能的結合蛋白並探究其功能與交互作用模式；(4)應用多維核磁共振技術、X光晶體學與低溫電子顯微鏡來進行DNMT1與其功能性區塊，及各種重要複合體的結構艦定；(5)以化學生物的觀點尋找並開發新型結合阻斷劑。 從我們的初步結果與陸陸續續不斷被發現的基因體與細胞生物證據，DNMT1與數個生物巨分子（DNA，DMAP1，PCNA和Rb蛋白和P53）的交互作用將是首要的研究目標。除了調節網絡的功能性分析，從結構觀點了解其交互作用機制與DNMT1酵素活性的運行，對於DNMT1在表顯遺傳調控的角色將能有更清楚的輪廓。 如果根據這些複合體的結構訊息篩選或設計抑制劑來破壞蛋白間的交互作用，將具有極大的研究與醫療生技應用價值。 <br> Abstract: Epigenetics is the study of epigenetic inheritance, which refers to change in phenotype (appearance) or gene expression without alternation in the underlying DNA sequence. DNA methylation, one of the epigenetic controls, involves the addition of a methyl group to the 5 position of cytosine and is a reversible chemical mechanism. This postreplicative signal is responsible for chromatin structure, genome integrity and imprinting, which associated to various deseases. Human DNA methyltranserase 1(DNMT1) is about 1620 amino acids long and belong to the important DNMTs protein family, which catalyze the chemical process of DNA methylation. DNMT1 is the first discovered and the major enzyme responsible during replication for maintenance of the DNA methylation pattern. In mammals, DNMT1 predominantly methylate hemimethylated CpG di-nucleotides in the mammalian genome and the inhibition of DNMT1 is a promising strategy for the treatment of various developmental and proliferative diseases, particularly cancer. However, structural information for DNA recognition and detailed catalytic mechanism of DNMT1 are poorly understood. Another important issue lies on the characterization of DNMT1 associated proteins and the functional outcome of such interactions. In this proposal, we aim to address the regulatory role and catalytic action of DNMT1 in epigenetic control through the structure determination on full-length and functional domains of DNMT1 protein. Various biochemical and biophysical methods will be applied to acquire a better understanding of the structure-function relationship. In the past few months, my laboratory had extensively cloned and produced various target proteins. therefore, we proposed to gain insights of (1) Understanding on the protein stabilities and foldings of DNMT1 and its functional domains; (2) Studying on the activities of DNMT1 via protein modification; (3) identification of DNA and protein targets of DNMT1 using proteomic and biophysical methods; (4) performing structural analysis of the protein/protein and protein/DNA complex using NMR and X-ray crystallography as well as cryo-EM; (5) screening potential peptide analogs or chemical compounds to disrupt the interaction between sustaining factors and their interacting proteins by chemical biology. From our previous and other reported data of the possible identified candidates, interactions between DNMT1 and its binding partners (DNA, DMAP1, PCNA, RB protein, and P53) are chosen as the preliminary targets. In addition to the functional analysis of the regulatory network, it will be of great interest to further examine, from structural perspective, the mechanism of interaction between these proteins. Another goal of the proposal is to deduce the structure of the interaction surface between them and to design inhibitor/peptide that could disrupt such interaction. Results from these studies will shed new light on the development and manipulation of DNMT1 function, and thus will be of great research and therapeutic value.表顯遺傳學蛋白質晶體學核磁共振光譜去氧核醣核酸甲基轉移酵素蛋白交互作用Epigeneticsprotein crystallographyNMR spectroscopyDNMT1protein interaction