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的后代的能力。已经引发了许多努力，以理解干细胞自我更新的分子机制。尽管有最近的进步，但对胚胎茎（ES）细胞的自我更新和分化的机制知之甚少。我们对文献的初步数据和分析促使我们提出了一个假设，这表明（i）细胞内氧化还原状态，（ii）Sirtuin蛋白家族的成员，（III）个体调节蛋白的乙酰化状态，（III）（IV）（iv）自我更新与茎/前代细胞的差异分化决策。在这里，我们提出了开始检验我们的一般假设的实验，即我们将在ES细胞及其分化的后代，胚胎体以及单个Sirtuins在ES细胞中蛋白质乙酰化中的作用之间建立蛋白质乙酰化谱的差异。我们提出，特定蛋白靶标的差异乙酰化 /脱乙酰化是干细胞分化的关键调节剂之一。特别是，我们推测由SIR2家族成员（由细胞内氧化还原状态介导的）介导的蛋白脱乙酰化会调节干细胞自我更新与分化决策。在AIM 1中，我们将建立未分化小鼠（ES）细胞及其分化后代胚胎体的蛋白质乙酰化谱（乙酰化特征）。我们提出了一种新型的质谱法，以比较多能ES细胞及其分化后代的胚胎体中整个细胞蛋白质组的乙酰化。在AIM 2中，我们将将小鼠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。我们将使用RNA干扰技术，系统地将ES细胞中小鼠Sir2家族的成员（个体和组合）击倒，并分析ES细胞的乙酰化谱的变化及其分化潜力。为了操纵其自我更新状态或诱导分化的细胞内氧化还原状态和潜在的体细胞细胞的细胞内氧化还原状态的调节可能具有很大的重要性，并且构成了未来的研究方向。自然，使用小鼠ES细胞的实验将为调节自我更新或诱导人ES细胞的分化的工作奠定基础。