Cell signaling through O-GlcNAc reader proteins

 描述（申请人提供）：O-连接β-N-乙酰葡萄糖胺（O-GlcNAc）是哺乳动物中普遍存在的翻译后修饰，装饰数千种核、细胞质和线粒体蛋白。O-GlcNAc循环是细胞代谢、细胞周期进展和细胞死亡的重要调节剂，并且在许多人类疾病如癌症、糖尿病和心律失常中失调。尽管O-GlcNAc信号具有广泛的病理生理学意义，但其主要方面仍然不清楚，包括O-GlcNAc如何在细胞内转导生物信息。最近，一些研究表明，O-GlcNAc化诱导蛋白质-蛋白质相互作用的过程中，如染色质重塑，去泛素化和核膜组装，表明O-GlcNAc可能通过保守的蛋白质-蛋白质相互作用模式的信号。然而，这些胞内糖蛋白-蛋白质复合物的结构或功能知之甚少。 我们假设，哺乳动物的“读者”蛋白可能存在，并识别O-GlcNAc部分的信号传导的目的。通过类比其他翻译后修饰的阅读器蛋白，我们推断专用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，我们使用了一种新的生物化学方法来鉴定几种蛋白质，这些蛋白质在体外和细胞中特异性地和直接地与O-GlcNAc酰化（但不是非糖基化）肽和蛋白质结合。有趣的是，一组推定的O-GlcNAc阅读器蛋白也与磷蛋白结合，表明它们可能是信号整合剂，介导先前描述的O-GlcNAc和O-磷酸之间的串扰。这一结果可能对我们理解通过翻译后修饰的细胞信号传导具有广泛的影响。 该项目的目的是表征我们鉴定的O-GlcNAc阅读器蛋白的生物化学和细胞生物学功能。我们将通过三个具体目标来实现这一目标。在目标1中，我们将通过候选读数器定义O-GlcNAc结合的生物化学范围。在目标2中，我们将鉴定O-GlcNAc阅读器蛋白的内源性糖蛋白结合伴侣，并确定这些相互作用在细胞信号传导中的作用。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、生物化学和原子水平上表征O-GlcNAc阅读器蛋白来显著推进细胞信号传导领域。

 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.