Post-translational regulation of embryonic stem cells: a proteomic study.

胚胎干细胞(ESC)有望通过基于干细胞的疗法的发展为修复医学带来革命性的变化。这种潜力集中在增殖的ES细胞自我更新和成熟为基本上任何类型的细胞的内在能力(多能性)。干细胞移植到受损心肌是治疗终末期心力衰竭的一种新手段。治疗成功的关键在于能够识别和操纵增殖的ESCs，根据需要特异性地分化为心肌细胞。这项建议使用一系列协同和复杂的蛋白质组技术来解决与ESC有关的离散的生物学和临床问题。在这个基于发现的GRANT中，我们专注于4个不同的亚蛋白质组，并包括下游验证和功能分析，以开发新的工具并为ESC的调控提供洞察力。具体目标1侧重于细胞表面蛋白的鉴定，特别是N-连接的糖蛋白组，以创建用于鉴定和监测不同分化阶段的特定生物标志物小组。此外，我们将细胞表面受体的识别与在相同的分化早期阶段发现的分泌调节因子(例如旁分泌因子)联系起来。特定目标2重点分析血清饥饿启动分化后最初24小时内ES细胞的差异磷酸化蛋白质组。这是转录因子B-MYB被磷酸化的同一时间段。B-MYB可能是最早的分化诱因之一。具体目标3，研究B-MYB对其磷酸化的调节，这种蛋白质复合体的变化以及对早期茎干蛋白的功能影响(例如。10月3日和Nanog)。了解胚胎干细胞蛋白质组中复杂和动态的相互关系具有基本的生物学意义，并具有重要的临床意义。

Embryonic stem cells (ESC) hold the promise to revolutionize reparative medicine through the development of stem cell-based therapies. This potential centers on the intrinsic abilities of proliferating ES cells to self-renew and to mature into essentially any cell type (pluripotency). Transplanting stem cells into damaged myocardium is emerging as a novel means for both acute repair and treatment of end-stage heart failure. The crux for therapeutic success will lie in being able to identify and manipulate proliferating ESCs to differentiate specifically into cardiac muscle upon demand. This proposal uses a series of synergistic and sophisticated proteomic technologies to address discrete biological and clinical questions with respect to ESC. In this discovery based grant, we focus on 4 distinctive sub-proteomes and include both downstream validation and functional analysis in order to develop new tools and provide insight into the regulation of ESC. Specific aim 1 focuses on the identification of cell surface proteins, in particular the N-linked glycoproteome to create specific biomarker panel for the identification and monitoring of various stages of differentiation. Furthermore, we link the cell surface receptor identification with the secreted regulatory factors (e.g. paracrine factors) found during the same early stages of differentiation. Specific aim 2 focuses on differential phospho-proteome analysis of ES cells over the initial 24 hours following differentiation initiated by serum starvation. This is the same time period in which the transcription factor, B-myb, is phosphorylated. B-myb is potentially one of the earliest triggers of differentiation. Specific aim 3, investigates B-myb with respect to its regulation by phosphorylation, alterations in this protein complex and the functional consequences on the early stemness proteins (eg. Oct 3 and Nanog). Understanding the complex and dynamic inter-relationships within the ESC proteome is of basic biological interest, and has major clinical implications.