Cell signaling through O-GlcNAc reader proteins

项目总结/摘要 O-连接的b-N-乙酰葡糖胺（O-GlcNAc）是一种普遍存在的翻译后修饰（PTM）， 哺乳动物，装饰数以千计的细胞核和细胞质蛋白质。O-GlcNAc循环是一种重要的调节剂 细胞生理学的无数方面，并在许多人类疾病中失调，如癌症，糖尿病， 和神经退化尽管有这种广泛的病理生理学意义，O-GlcNAc信号传导的主要方面 仍然不完全了解，包括O-GlcNAc转导的生化机制 信息.在我们的项目开始时，一些研究表明，O-GlcNAc化介导蛋白质-蛋白质 在染色质重塑、去泛素化和核被膜等多种过程中， 组装件.这些结果表明，O-GlcNAc可能通过PPI的保守模式进行信号传导。不过小 这些胞内糖蛋白-蛋白质复合物的结构或功能是已知的。 我们假设哺乳动物的“阅读器”蛋白可能存在，并且可能是GIO-GlcNAc信号。在 在第一个项目期间，我们设计并部署了一种方法来识别特异性和直接结合O- 体外和人细胞中的GlcNAc酰化肽和蛋白质。其中包括14-3-3家庭，这是很好的- 已知与磷蛋白结合。我们的研究结果表明14-3-3蛋白可能是信号整合子，介导 广泛记录的O-GlcNAc和O-磷酸盐之间的串扰，具有广泛的影响， 胞内信号传导此外，我们在核输入中鉴定和表征了O-GlcNAc介导的PPI， 受体、中间丝（IF）细胞骨架蛋白和泛素E3连接酶复合物组分。建筑 在这些结果的基础上，我们将在下一个项目期间重点研究一组不同的模型O-GlcNAc结合蛋白。 这一策略将产生新的见解，个别O-GlcNAc介导的PPI的功能，在特定的细胞 生物过程，同时也提供了跨阅读器蛋白质的比较视角，使我们能够辨别 O-GlcNAc介导的PPI在生物物理、生物化学和生理水平的一般主题。 我们工作的长期目标是了解O的原理、机制和功能， 哺乳动物中GlcNAc介导的PPI。在下一个项目期间，我们将通过三个具体的项目来推进这一目标。 目标。在目的1中，我们将通过14-3-3定义O-GlcNAc结合的生化范围和表型效应。 proteins.在目的2中，我们将确定O-GlcNAc/核转运受体相互作用在细胞器间的作用， 贩卖人口在目标3中，我们将剖析O-GlcNAc介导的相互作用在两种模型IF中的功能重要性。 proteins.我们相信，我们的工作将大大推进细胞内信号传导领域， 阐明单个O-GlcNAc介导的PPI的机制和功能，并通过揭示保守的 O-GlcNAc跨阅读器蛋白识别的生物化学原理。

Project Summary/Abstract O-linked b-N-acetylglucosamine (O-GlcNAc) is a ubiquitous post-translational modification (PTM) in mammals, decorating thousands of nuclear and cytoplasmic proteins. O-GlcNAc cycling is an essential regulator of myriad aspects of cell physiology and is dysregulated in numerous human diseases, such as cancer, diabetes and neurodegeneration. Despite this broad pathophysiological significance, major aspects of O-GlcNAc signaling remain incompletely understood, including the biochemical mechanisms through which O-GlcNAc transduces information. At the start of our project, several studies had shown that O-GlcNAcylation mediates protein-protein interactions (PPIs) in processes as diverse as chromatin remodeling, deubiquitination and nuclear envelope assembly. These results suggested that O-GlcNAc might signal through conserved modes of PPI. However, little was known about either the structure or function of these intracellular glycoprotein-protein complexes. We hypothesized that mammalian “reader” proteins might exist and transduce O-GlcNAc signals. In the first project period, we devised and deployed a method to identify proteins that bind specifically and directly to O- GlcNAcylated peptides and proteins in vitro and in human cells. These include the 14-3-3 family, which are well- known to bind to phosphoproteins. Our results indicate that 14-3-3 proteins may be signal integrators, mediating the extensively documented crosstalk between O-GlcNAc and O-phosphate, with broad implications for intracellular signaling. In addition, we identified and characterized O-GlcNAc-mediated PPIs among nuclear import receptors, intermediate filament (IF) cytoskeleton proteins and ubiquitin E3 ligase complex components. Building on these results, we will study a focused set of diverse model O-GlcNAc-binding proteins in the next project period. This strategy will generate new insight into the functions of individual O-GlcNAc-mediated PPIs in particular cell biological processes, while also providing a comparative perspective across reader proteins, allowing us to discern general themes of O-GlcNAc-mediated PPIs at the biophysical, biochemical and physiological levels. The long-term objective of our work is to understand the principles, mechanisms and functions of O- GlcNAc-mediated PPIs in mammals. In the next project period, we will advance this goal through three Specific Aims. In Aim 1, we will define the biochemical scope and phenotypic effects of O-GlcNAc binding by 14-3-3 proteins. In Aim 2, we will determine the role of O-GlcNAc/nuclear transport receptor interactions in inter-organelle trafficking. In Aim 3, we will dissect the functional importance of O-GlcNAc-mediated interactions in two model IF proteins. We believe our proposed work will significantly advance the field of intracellular signaling both by elucidating the mechanisms and functions of individual O-GlcNAc-mediated PPIs and by revealing conserved biochemical principles of O-GlcNAc recognition across reader proteins.