CHEMICAL BIOLOGY APPROACHES TO STUDY O-GLCNAC IN MAMMALIAN CELLS

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 O-连接的β-N-乙酰葡糖胺（O-GlcNAc）对细胞内蛋白质的动态修饰是一种新的研究方法。 关键的和普遍存在的细胞信号传导范式，调节底物的功能，定位和稳定性。蛋白质OGlcNAc化控制多种哺乳动物组织中的许多过程，并且其失调与人类疾病如II型糖尿病和神经变性有关。尽管其广泛的功能意义，O-GlcNAc信号转导的主要方面知之甚少。具体来说，三个关键问题仍然没有答案：1。在O-GlcNAc调节的途径中，功能上重要的底物是什么？2. O-GlcNAc对这些底物有什么生化作用？3.蛋白质如何靶向信号依赖性O-GlcNAc酰化？由于O-GlcNAc是一种不受直接遗传控制的瞬时翻译后修饰，因此仅使用传统的分子生物学技术来回答这些问题具有挑战性。 因此，我们开创了利用非天然糖试剂和同源检测探针研究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，O-GlcNAc靶标上糖基化位点的鉴定对于理解修饰在两个过程中的作用至关重要。UCSF质谱设施将提供重要的专业知识和协作资源，用于确定感兴趣的蛋白质上的O-GlcNAcylation位点。总之，该项目将利用新的化学方法和质谱法来剖析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. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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.