CHEMICAL BIOLOGY APPROACHES TO STUDY O-GLCNAC IN MAMMALIAN CELLS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 O-连接β-N-乙酰氨基葡萄糖(O-GlcNAc)对细胞内蛋白质的动态修饰 关键的和普遍存在的细胞信号模式，调节底物的功能、定位和稳定性。OGlcN酰化蛋白在广泛的哺乳动物组织中控制着许多过程，它的失调与人类疾病有关，如II型糖尿病和神经退行性变。尽管它具有广泛的功能意义，但人们对O-GlcNAc信号的主要方面知之甚少。具体地说，有三个关键问题仍未得到解答：1.O-GlcNAc调控的通路中有哪些重要的功能底物？2.O-GlcNAc对这些底物有什么生化作用？3.蛋白质是如何被信号依赖的O-GlcN酰化作用靶向的？由于O-GlcNAc是一种暂时的翻译后修饰，不受直接基因控制，仅用传统的分子生物学技术来回答这些问题是具有挑战性的。因此，我们开创了利用非天然糖试剂和同源检测探针来研究O-GlcNAc的化学生物学方法。我们将应用这些工具来研究O-GlcNAc在调节两个模型细胞生物学过程中的作用：DNA损伤反应和线粒体代谢。重要的是，识别O-GlcNAc靶标上的糖基化位点对于理解修饰在这两个过程中的作用至关重要。加州大学旧金山分校的质谱学设施将为识别感兴趣蛋白质上的O-GlcN酰化位点提供重要的专业知识和协作资源。总之，该项目将利用新的化学方法和质谱学来剖析O-GlcNAc在哺乳动物细胞生理学中的功能作用。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The dynamic modification of intracellular proteins by O-linked beta-N-acetylglucosamine (O-GlcNAc) is a critical and ubiquitous cell signaling paradigm, regulating substrates' function, localization and stability. Protein OGlcNAcylation controls numerous processes in a wide range of mammalian tissues, and its dysregulation is implicated in human diseases, such as type II diabetes and neurodegeneration. Despite its broad functional significance, major aspects of O-GlcNAc signaling are poorly understood. Specifically, three key questionsremain unanswered: 1. What are the functionally important substrates in pathways that O-GlcNAc regulates? 2.What biochemical effect does O-GlcNAc have on these substrates? 3. How are proteins targeted for signal dependent O-GlcNAcylation? Because O-GlcNAc is a transient post-translational modification not under direct genetic control, these questions are challenging to answer using traditional molecular biology techniques alone. Therefore, we have pioneered chemical biology approaches to studying O-GlcNAc that make use of unnatural sugar reagents and cognate detection probes. We will apply these tools to study the role of O-GlcNAc in regulating two model cell biological processes: the DNA damage response and mitochondrial metabolism. Importantly, the identification of glycosylation sites on O-GlcNAc targets is critical for understanding the role of the modification in both processes. The UCSF Mass Spectrometry Facilty will provide vital expertise and collaborative resources for identifying O-GlcNAcylation sites on proteins of interest. Together, the project will exploit both novel chemical methods and mass spectrometry to dissect the functional role of O-GlcNAc in mammalian cell physiology.