Protein Acetylation Signature of ES Cell Differentiation

描述(申请人提供)：胚胎干细胞的治疗应用将取决于我们获得大量多能干细胞及其预分化后代的能力。许多努力已经引导到了解干细胞自我更新的分子机制。尽管最近取得了进展，但胚胎干细胞自我更新和分化的机制尚不清楚。我们的初步数据和文献分析促使我们提出了一个假设，认为(I)细胞内氧化还原状态，(Ii)Sirtuin蛋白家族成员，(Iii)个别调节蛋白的乙酰化状态，以及(Iv)干/祖细胞的自我更新与分化决定之间的联系。在这里，我们提出了实验，开始检验我们的一般假设，即我们将建立ES细胞与其分化的后代、类胚体之间的蛋白质乙酰化图谱的差异，以及单个sirtuins在ES细胞中的蛋白质乙酰化中的作用。我们认为，特定蛋白质靶标的差异乙酰化/去乙酰化是干细胞分化的关键调控因素之一。特别是，我们推测，Sir2家族成员介导的蛋白质去乙酰化(由细胞内氧化还原状态介导)调节干细胞的自我更新与分化决定。在目标1中，我们将建立未分化的小鼠(ES)细胞及其分化的子代胚状体的蛋白质乙酰化特征(乙酰化特征)。我们提出了一种新的质谱学方法来比较多能ES细胞及其分化的后代--类胚体中整个细胞蛋白质组的乙酰化。在目标2中，我们将把小鼠Sir2家族的单个成员的活性与小鼠ES细胞的乙酰化特征联系起来。我们将利用RNA干扰技术，在ES细胞中系统地敲除小鼠Sir2家族成员(单独和组合)，并分析ES细胞乙酰化谱的变化及其分化潜能。调节胚胎干细胞和潜在的体细胞的细胞内氧化还原状态以控制其自我更新状态或诱导分化具有重要的现实意义，并构成了未来的研究方向。自然，对小鼠ES细胞的实验将为调节人类ES细胞自我更新或诱导分化的工作奠定基础。

DESCRIPTION (provided by applicant): Therapeutic applications of embryonic stem cells will depend on our ability to obtain large amounts of pluripotent stem cells and their predifferentiated progeny. Numerous efforts have been channeled into understanding of the molecular mechanism of stem cell self-renewal. Despite recent advancements, the mechanisms.of self-renewal and differentiation of Embryonic Stem (ES) cells are poorly understood. Our preliminary data and analysis of literature prompted us to formulate a hypothesis suggesting a link between the (i) intracellular redox state, (ii) members of Sirtuin protein family, (iii) acetylation status of individual regulatory proteins, and (iv) the self-renewal vs. differentiation decision of stem/progenitor cells. Here we propose the experiments, which begin testing our general hypothesis, namely we will establish the differences in protein acetylation profile between ES cells and their differentiated progeny, embryoid bodies and the role of individual Sirtuins in protein acetylation in ES cells. We propose that differential acetylation / deacetylation of specific protein targets is one of the key modulators of stem cell differentiation. In particular, we speculate that protein deacetylation mediated by the members of Sir2 family (mediated by the intracellular redox state) regulates the stem cell self-renewal vs. differentiation decision. In Aim 1 we will establish the protein acetylation profile (acetylation signature) of undifferentiated mouse (ES) cells and their differentiated progeny embryoid bodies. We propose a novel mass spectroscopy approach to compare the acetylation of the entire cellular proteome in pluripotent ES cells and in their differentiated progeny, embryoid bodies. In Aim 2 we will link the activity of individual members of mouse Sir2 family with the acetylation signature of mouse ES cells. We will use RNA interference technique, to systematically knock-down the members of mouse Sir2 family (individual and in combination) in ES cells and analyze the changes in acetylation profile of ES cells and their differentiation potential. The modulation of the intracellular redox state of embryonic and potentially somatic stem cells in order to manipulate their self-renewal status or to induce differentiation could be of great practical importance and constitute the future directions of research. Naturally, experiments with mouse ES cells will lay the foundation for the work to modulate self-renewal or induce differentiation of human ES cells.