Cell signaling through O-GlcNAc reader proteins

 描述(申请人提供)：O-连接β-N-乙酰氨基葡萄糖(O-GlcNAc)是哺乳动物中普遍存在的一种翻译后修饰，修饰成千上万的核蛋白、细胞质和线粒体蛋白。O-GlcNAc循环是细胞新陈代谢、细胞周期进程和细胞死亡的重要调节因子，在癌症、糖尿病和心律失常等多种人类疾病中处于失调状态。尽管O-GlcNAc具有广泛的病理生理意义，但O-GlcNAc信号的主要方面仍然不清楚，包括O-GlcNAc如何在细胞内传递生物信息。最近的一些研究表明，O-GlcNAc在染色质重塑、去泛素化和核膜组装等不同的过程中诱导蛋白质-蛋白质相互作用，提示O-GlcNAc可能通过保守的蛋白质-蛋白质相互作用模式来传递信号。然而，人们对这些细胞内糖蛋白-蛋白复合体的结构或功能知之甚少。我们假设哺乳动物的“阅读器”蛋白可能存在，并识别O-GlcNAc部分以用于信号传递。通过类比其他翻译后修饰的阅读器蛋白质，我们推断专用的O-GlcNAc阅读器--或许多阅读器共有的蛋白质结构域--可能传递糖基化编码的信号。在初步研究中，我们使用一种新的生化方法在体外和细胞中鉴定了几种与O-GlcNacylated(但不是未糖化)的多肽和蛋白质特异和直接结合的蛋白质。有趣的是，一组可能的O-GlcNAc阅读器蛋白也与磷酸蛋白结合，表明它们可能是信号整合者，介导了先前描述的O-GlcNAc和O-磷酸之间的串扰。这一结果可能对我们通过翻译后修饰理解细胞信号具有广泛的意义。本项目的目的是鉴定我们鉴定的O-GlcNAc阅读器蛋白的生化和细胞生物学功能。我们将通过三个具体目标实现这一目标。在目标1中，我们将由候选读者定义O-GlcNAc结合的生化范围。在目标2中，我们将确定O-GlcNAc阅读器蛋白的内源性糖蛋白结合伙伴，并建立这些相互作用在细胞信号转导中的作用。在目标3中，我们将通过求解与糖基化配体结合的阅读器蛋白的晶体结构来确定O-GlcNAc介导的蛋白质-蛋白质相互作用的生物物理基础。我们的工作将通过在细胞生物学、生化和原子水平上表征O-GlcNAc阅读器蛋白来显著推进细胞信号转导领域。

 DESCRIPTION (provided by applicant): O-linked β-N-acetylglucosamine (O-GlcNAc) is a ubiquitous post-translational modification in mammals, decorating thousands of nuclear, cytoplasmic and mitochondrial proteins. O-GlcNAc cycling is an essential regulator of cell metabolism, cell cycle progression and cell death, and is dysregulated in numerous human diseases, such as cancer, diabetes and cardiac arrhythmia. Despite its broad pathophysiological significance, major aspects of O-GlcNAc signaling remain obscure, including how O-GlcNAc transduces biological information inside the cell. Recently, several studies showed that O-GlcNAcylation induces protein-protein interactions in processes as diverse as chromatin remodeling, deubiquitination and nuclear envelope assembly, suggesting that O-GlcNAc may signal through conserved modes of protein-protein interaction. However, little is known about either the structure or function of these intracellular glycoprotein-protein complexes. We hypothesized that mammalian "reader" proteins might exist and recognize O-GlcNAc moieties for signaling purposes. By analogy to reader proteins for other post-translational modifications, we reasoned that dedicated O-GlcNAc readers - or protein domains common to many readers - might relay glycosylation-encoded signals. In preliminary studies, we used a new biochemical approach to identify several proteins that bind specifically and directly to O-GlcNAcylated (but not unglycosylated) peptides and proteins in vitro and in cells. Interestingly, one group of putative O-GlcNAc reader proteins also binds to phosphoproteins, suggesting that they may be signal integrators, mediating the previously described crosstalk between O-GlcNAc and O- phosphate. This result may have wide-ranging implications for our understanding of cell signaling through post- translational modifications. The objective of this project is to characterize the biochemical and cell biological functions of the O- GlcNAc reader proteins that we identified. We will accomplish this goal through three specific aims. In Aim 1, we will define the biochemical scope of O-GlcNAc binding by the candidate readers. In Aim 2, we will identify the endogenous glycoprotein binding partners of O-GlcNAc reader proteins, and establish the role of these interactions in cell signaling. In Aim 3, we will determine the biophysical basis of an O-GlcNAc-mediated protein- protein interaction by solving the crystal structure of a reader protein bound to a glycosylated ligand. Our work will significantly advance the field of cell signaling by characterizing O-GlcNAc reader proteins at the cell biological, biochemical and atomic levels.