2007-03-012024-05-18https://scholars.lib.ntu.edu.tw/handle/123456789/699301摘要：幹細胞具有可自我複製與分化成各種細胞，進而發育成不同組織與器官的特性。 幹細胞生物學與這種細胞多能性在再生醫學基礎研究乃至於人類疾病治療的應用已成為重要的課題。 從分子層次而言，幹細胞的多能性被發現與一些轉錄因子的表現與活性有關。 Oct4 與 Nanog 透過對於數種基因的活化或抑制而被視為維持胚胎幹細胞多能性不可或缺的轉錄因子。 然而對於Oct4 與 Nanog如何調節基因表現的機制卻尚未明朗。 令一個重要的課題在於相關的共轉錄因子與調節因子如何透過與其交互作用而達成所需功能。 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Oct4 與 Nanog在幹細胞生物學上的調節角色。 運用數種生化與生物物理方法來了解其結構與功能的關聯性，並將採用蛋白質體技術來篩選可能的共轉錄因子與調節因子。 最後應用多維核磁共振技術與X光晶體學來進行結構艦定，並探討這些生物巨分子間的交互作用。 從已被證實與Oct4有關的蛋白中，FoxD3和Wwp2將是首要的研究目標。 Oct4 是FoxD3在維持胚胎分化能力的共抑制因子；而Wwp2則是透過直接的交互作用，催化與Oct4降解有關的ubiquitination。 了解其交互作用機制對於轉錄因子如何在幹細胞的分化與複製造成影響有重大的意義。 如果根據結構上的訊息設計抑制劑來破壞蛋白間的交互作用，將具有醫療與生技應用的價值。 <br> Abstract: Stem cells are defined as cells able to both extensively self-renew and differentiate into progenitors. Biology of the stem cell and its pluripotency is a topic of importance to a broad spectrum of biological investigation and treatment of human diseases. At the molecular level, pluripotency has been linked to the transcription factors, and their expression and activity appears to define whether a cell should be pluripotent. Oct4 and Nanog are two transcription factors that are indispensable in maintaining the self-renewal and pluripotency of embryonic stem cells through activating or repressing the expression of various genes. Curiously, very little is known about Oct4 and Nanog beyond their ability to regulate gene expression. Another important issue lies in the characterization of their association with potential co-factors and regulators and the functional outcome of such interactions. In this proposal, we aim to address the regulatory role of Oct4 and Nanog in stem cell biology through the structure determination of these proteins. Various biochemical and biophysical methods will be applied to acquire a better understanding of the structure-function relationship. Furthermore, we will perform proteomic screen for the potential co-factors and regulators of the transcription factors, as well as search for their in vivo binding cis-elements. Finally, the structural basis of the interaction between Oct4/Nanog and their co-factors or targets will be examined using multi-dimensional NMR and X-ray crystallography. Among the possible identified candidates, FoxD3 and Wwp2 are chosen as the preliminary targets. Oct4 functions as a corepressor of FoxD3 to provide embryonic lineage-specific transcriptional regulatory activity to maintain appropriate developmental timing. Wwp2, an E3 ubiquitin ligase, promotes the ubiquitination of Oct4 through direct interaction, which relates to the degradation of Oct4. Thus, 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 stem cells, and thus will be of great research and therapeutic value.幹細胞蛋白質交互作用結構生物學Oct4Nanogstem cellprotein interactionstructural biologyOct4Nanog