Spatiotemporal tools to interrogate O-GlcNAc functions in cellular signaling

摘要 FEHL实验室的总体目标是开发化学生物学策略来确定功能 信号传递过程中蛋白质修饰的影响。具体地说，细胞新陈代谢和压力分别导致 用O-连接的N-乙酰氨基葡萄糖(O-GlcNAc)修饰多种蛋白质，但目前还没有工具 能够以定义的时间和空间分辨率捕捉高度动态和瞬时的O-GlcNAc事件。缺乏 “时空”的严谨性阻碍了科学界将新陈代谢与疾病生理联系起来， 包括在糖尿病患者中观察到的乳腺癌等恶性肿瘤的癌症风险显著增加。 在这个Mira应用程序中，我们提出了我们的策略，通过开发实时 以及空间分子工具，以O-GlcNAc为基石连接细胞新陈代谢和癌症过程。 美国国立卫生研究院资助的优秀研究已经发现了2000多种O-GlcNAc关于人类细胞中的蛋白质，但目前的工具 依赖被破坏的生理，导致伪影，或者错过之前发生的关键的GlcNAc驱动的信号事件 全球新陈代谢再平衡将在不到一小时内完成。我们假设高血糖的主要驱动因素 新陈代谢和病理存在于营养和信号刺激的最初几分钟内，到目前为止 不可能在活细胞中观察到。我们在光化学和系统糖生物学方面发表的工作支持 我们独特的策略，在O-GlcNAc再平衡发生之前，在几分钟内触发O-GlcNAc过程。我们的 光笼糖工具能够触发致癌转录因子NFkB在胞浆或胞浆之间的移动 内质网进入细胞核，模拟可能将异常的胰岛素和 伴有乳腺癌风险的糖尿病患者的葡萄糖释放。我们的实时系统可以用来跟踪O-GlcNAc事件 在快速、15分钟的病态胰岛素生理脉冲期间第一次发送胰岛素信号。 另一种靶向细胞内O-GlcNAc靶向邻近标记的工具能够跟踪O-GlcNacylated 亚细胞空间中的蛋白质，没有报道的工具能够在活细胞中专门标记这些蛋白质。我们 建议在未来5年内开发我们的“时空”分子工具，并将其应用于独特的 通过NFkB靶向在疾病生物学中的机制研究。我们积极与代谢性疾病合作 和癌症专家，以确保疾病的相关性，以及与工业科学家的技术 开发以扩大O-GlcNAc生物学在新陈代谢驱动的疾病途径中的工业意识。 该方案的研究成果包括分子探针、时空策略和靶向 将细胞新陈代谢与信号联系起来。我们的促成化学战略有可能产生广泛的影响 在科学界通过建立时间和空间方法来研究蛋白质修饰。我们的 平台可以扩展到其他PTM和药物靶点，例如调节 肿瘤代谢与炎症的界面。成功将建立一个持久的独立研究利基市场。

Abstract The overall goal of the Fehl laboratory is to develop chemical biology strategies to determine the functional impact of protein modifications during signaling processes. Specifically, cellular metabolism and stress each lead to diverse protein modifications with O-linked N-acetylglucosamine sugar (O-GlcNAc) but no tools are currently able to capture highly dynamic and transient O-GlcNAc events with defined time and spatial resolution. Lack of “time and space” rigor hinders the scientific community from connecting metabolism with disease physiology, including significantly elevated cancer risk in diabetic patients observed for malignancies like breast cancer. In this MIRA application, we pose our strategies to address this critical gap through the development of real-time and space molecular tools that bridge cell metabolism and cancer processes using O-GlcNAc as the keystone. Excellent NIH-funded research has discovered over 2000 O-GlcNAc on proteins in human cells, but current tools rely on disrupted physiology, leading to artifacts, or miss key GlcNAc-driven signaling events that occur before global metabolic rebalancing occurs in less than an hour. We hypothesize that the key drivers of hyperglycemic metabolism and pathology lie within the first few minutes of nutrient and signaling stimulation, which to date is not possible to observe in living cells. Our published work in photochemistry and systems glycobiology support our unique strategies to trigger O-GlcNAc processes in minutes, before O-GlcNAc rebalancing occurs. Our photocaged sugar tool is able to trigger the oncogenic transcription factor NFkB movement between cytosol or endoplasmic reticulum into the nucleus, simulating physiological events that potentially link aberrant insulin and glucose release in diabetes with breast cancer risk. Our real time system can be used to track O-GlcNAc events during insulin signaling for the first time during the rapid, 15-minute pulses of diseased insulin physiology. Another tool for targeted intracellular O-GlcNAc-targeted proximity labeling is able to track O-GlcNAcylated proteins in subcellular space, which no reported tool has the capability to specifically label in live cells. We propose in the next 5 years to develop our “time and space” molecular tools and apply them for unique mechanistic studies in disease biology through NFkB targeting. We actively collaborate with metabolic disease and cancer specialists to ensure disease relevance, as well as with industrial scientists for technology development to expand industrial awareness of O-GlcNAc biology in metabolism-driven disease pathways. The research outputs of this proposal include molecular probes, spatiotemporal strategies, and targets to connect cellular metabolism with signaling. Our enabling chemical strategies have the potential for broad impact in the scientific community by establishing temporal and spatial methods to study protein modifications. Our platforms can be extended to other PTMs and drug targets, such as sialic acid modifications that regulate the interface of cancer metabolism and inflammation. Success will establish a lasting independent research niche.