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。 幹細胞生物學與其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是相當重要與被關注的課題。 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如何與其相關的共轉錄因子和調節因子透過交互作用,而達成維持細胞多能性的機制卻仍未明朗。 另一個有趣的觀察是在分化後的細胞便找不到Oct4與Nanog蛋白的蹤跡,而在2007年11月27號於science期刊有研究指出將Oct4, Nanog, Sox2和LIN28轉殖回體細胞都可以讓體細胞轉成具多能性的幹細胞。 這種誘發性幹細胞在新的疾病模型研究、移植醫學與藥物發展將會有相當的助益。 因此瞭解Oct4與Nanog的蛋白質穩定性及被修飾與降解的途徑將可提供此新興領域重要的分子觀點。
在過去半年來執行前期計畫的結果,我們已能獲得Oct4與Nanog的DNA結合區塊的重組蛋白,而全長基因和其他部分亦已獲得或質體建構完成,實驗發現此兩蛋白的HD區塊具有不同穩定性的特徵,同時我們也開始著手進行結構鑑定,此發現與未來深入探討將有助於釐清為何蛋白只出現細胞具多能性的時期。 此外,DNA甲基化酵素一型(DNMT1)被證實會調控Oct4表現進而與發育或其他癌症相關疾病有關。 我們也著手進行DNMT1與其抑制劑的結構探討,並已有部分結果發表期刊。 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Oct4與Nanog的蛋白質穩定性與泛素修飾(ubiquitination)之蛋白質降解機制;(2)探討核酸或蛋白質的修飾影響Oct4與Nanog表現與活性的機制;(3)利用蛋白質體技術來篩選可能的共轉錄因子與調節因子;(4)利用生物物理方法探討這些生物巨分子間的交互作用,並應用多維核磁共振技術與X光晶體學來進行結構艦定;(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,Oct4與FoxD3、 Oct4與-catenin以及Nanog與Smad1的交互作用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;-catenin與Oct4結合可負調節Nanog表現;而Nanog與Smad1結合阻礙Smad複合體形成的可抑制細胞分化。 此外從酵素的觀點,Wwp2則是透過直接的交互作用,催化與Oct4降解有關的ubiquitination;而DNMTs可調節Oct4與Nanog的表現。了解其交互作用機制與酵素活性對於這些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,將具有醫療與生技應用的價值。
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. However, the network of interaction among extrinsic and intrinsic determinants of ES cell pluripotency is currently poorly understood. Interestingly, Oct4 and Nanog are found only in undifferentiated ES cells. In addition, the phenotypes of both Oct4- and Nanog-deficient embryos confirm that they play crucial roles for the ES cell potency and self-renewal. In a paper published online in Science on 27 November, described that four factors (OCT4, SOX2, NANOG, and LIN28) are sufficient to reprogram human somatic cells to pluripotent stem cells that exhibit the essential characteristics of embryonic stem cells. Such human induced pluripotent cell lines should be useful in the production of new disease models and in application in transplantation medicine as well as drug development. An associated arising task is to study the protein stabilities and the degradation pathways of Oct4 and Nanog.
In the past eight months, my laboratory had extensively cloned the various target genes, and also the different constructions and protein productions of these genes had been performed. Biophysical studies indicate that the homeodomains of Oct4 and Nanog present different structural characteristics of protein stability. The structural determinations of the proteins are in progress. In addition, the related protein DNMT1 has been demonstrated to control the Oct4 gene expression by DNA methylation in cell differentiation. We also study on DNMT1 and its inhibitor. Part of the result has been published in the journal. In this renewal project, we proposed to gain insights of (1) Understanding on the protein stabilities and degradation mechanism by ubiquitination of Oct4 and Nanog; (2) Studying on the activities of Oct4 and Nanog via DNA or protein modification; (3) identification of DNA and protein targets of Oct4 and Nanog using proteomic and biophysical methods; (4) performing structural analysis of the protein/protein and protein/DNA complex using NMR and X-ray crystallography; (5) screening potential peptide analogs or chemical compounds to disrupt the interaction between sustaining factors and their interacting proteins by chemical biology.
We aim to address the regulatory role of Oct4 and Nanog in stem cell biology through the structure determination of these proteins. Another important issue lies in the characterization of their association with potential co-factors and regulators and the functional outcome of such interactions. 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. From our previous and other reported data of the possible identified candidates, interactions between Oct4 and FoxD3, Nanog and Smad1 as well as Oct4 and -catenin 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; Nanog binds to Smad1 for interfering with the recruitment of coactivators to the active Smad transcriptional complexes and then block BMP-induced mesoderm differentiation of ES cells; -catenin up-regulates Nanog through interaction with Oct4 physically. For the molecular enzymology studies, Wwp2, an E3 ubiquitin ligase, promotes the ubiquitination of Oct4 through direct interaction, which relates to the degradation of Oct4; DNMTs can regulate the gene expressions of Oct4 and Nanog via DNA methylation. 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.