Protein Acetylation Signature of ES Cell Differentiation

ES 细胞分化的蛋白质乙酰化特征

• 批准号: 7031897
• 负责人: ALEXEY V TERSKIKH
• 金额: $ 25.79万
• 依托单位: SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-06-06 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7031897
• 关键词: RNA interference; acetylation; amidohydrolases; cell differentiation; embryonic stem cell; laboratory mouse; mass spectrometry; oxidation reduction reaction; protein structure function; transcription factor

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.

描述（由申请人提供）：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。为了理解干细胞自我更新的分子机制，人们付出了许多努力。尽管最近取得了进展，但胚胎干细胞的自我更新和分化机制仍知之甚少。我们的初步数据和文献分析促使我们提出一个假设，表明 (i) 细胞内氧化还原状态、(ii) Sirtuin 蛋白家族成员、(iii) 个体调节蛋白的乙酰化状态和 (iv) 自身调节蛋白之间存在联系。 -干/祖细胞的更新与分化决定。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 细胞及其分化后代、胚状体之间蛋白质乙酰化谱的差异以及单个 Sirtuins 在 ES 细胞中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/脱乙酰化是干细胞分化的关键调节剂之一。特别是，我们推测 Sir2 家族成员介导的蛋白质脱乙酰化（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策。在目标 1 中，我们将建立未分化小鼠 (ES) 细胞及其分化后代胚状体的蛋白质乙酰化谱（乙酰化特征）。我们提出了一种新的质谱方法来比较多能 ES 细胞及其分化后代胚状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 家族个体成员的活性与小鼠 ES 细胞的乙酰化特征联系起来。我们将利用RNA干扰技术，系统地敲低ES细胞中的小鼠Sir2家族成员（单个和组合），并分析ES细胞乙酰化谱的变化及其分化潜力。调节胚胎干细胞和潜在成体干细胞的细胞内氧化还原状态以操纵其自我更新状态或诱导分化可能具有非常重要的实际意义，并构成未来的研究方向。当然，小鼠 ES 细胞的实验将为调节人类 ES 细胞的自我更新或诱导分化的工作奠定基础。