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

胚胎干细胞（ESC）有望通过开发基于干细胞的疗法来彻底改变修复医学。这种潜能集中在增殖的ES细胞自我更新和成熟为基本上任何细胞类型（多能性）的内在能力上。将干细胞移植到受损的心肌中正在成为急性修复和治疗终末期心力衰竭的新手段。治疗成功的关键在于能够识别和操纵增殖的ESC，以根据需要特异性分化为心肌。该建议使用一系列协同和复杂的蛋白质组学技术来解决ESC相关的离散生物学和临床问题。在这项基于发现的资助中，我们专注于4个独特的亚蛋白质组，包括下游验证和功能分析，以开发新的工具并提供对ESC调控的见解。具体目标1侧重于鉴定细胞表面蛋白，特别是N-连接糖蛋白组，以创建用于鉴定和监测不同分化阶段的特异性生物标志物组。此外，我们将细胞表面受体鉴定与在分化的相同早期阶段发现的分泌的调节因子（例如旁分泌因子）联系起来。具体目标2侧重于在血清饥饿引发分化后的最初24小时内对ES细胞进行差异磷酸化蛋白质组分析。这是转录因子B-myb磷酸化的同一时间段。B-myb可能是分化的最早触发因素之一。具体目标3，研究了B-myb通过磷酸化的调节，这种蛋白复合物的改变以及对早期干性蛋白（例如，Oct 3和Nanog）。了解ESC蛋白质组内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.