Spatiotemporal tools to interrogate O-GlcNAc functions in cellular signaling

探究细胞信号传导中 O-GlcNAc 功能的时空工具

• 批准号: 10605263
• 负责人: Charlie Fehl
• 金额: $ 36.42万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10605263
• 关键词: Address; Awareness; Biology; Breast Cancer Risk Factor; Cell Nucleus; Cells; Cellular Metabolic Process; Chemicals; Collaborations; Communities; Cytosol; Development; Diabetes Mellitus; Disease; Disease Pathway; Drug Targeting; Endoplasmic Reticulum; Ensure; Event; Funding; Glucose; Glycobiology; Goals; Hour; Human; Hyperglycemia; Incidence; Industrialization; Inflammation; Insulin; Knowledge; Label; Laboratories; Lead; Link; Location; Malignant Neoplasms; Maps; Metabolic; Metabolic Diseases; Metabolism; Methods; Modification; Molecular; Molecular Probes; Morphologic artifacts; Movement; NF-kappa B; Non-Insulin-Dependent Diabetes Mellitus; Nutrient; Oncogenic; Output; Pathology; Patients; Photochemistry; Physiologic pulse; Physiological; Physiology; Post-Translational Protein Processing; Process; Proteins; Publishing; Real-Time Systems; Reporting; Research; Resolution; Scientist; Sialic Acids; Signal Transduction; Specialist; Stress; Subcellular Spaces; System; Therapeutic; Time; United States National Institutes of Health; Work; cancer risk; diabetic patient; disorder risk; human disease; innovation; insulin signaling; link protein; malignant breast neoplasm; programs; public health relevance; spatiotemporal; success; sugar; technology development; tool; tumor metabolism

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.

抽象的 菲尔实验室的总体目标是开发化学生物学策略来确定功能 信号传导过程中蛋白质修饰的影响。具体来说，细胞代谢和压力各自导致 使用 O-连接的 N-乙酰氨基葡萄糖 (O-GlcNAc) 进行多种蛋白质修饰，但目前还没有工具 能够以定义的时间和空间分辨率捕获高度动态和瞬态的 O-GlcNAc 事件。缺乏 “时间和空间”的严格性阻碍了科学界将新陈代谢与疾病生理学联系起来， 包括观察到患有乳腺癌等恶性肿瘤的糖尿病患者的癌症风险显着升高。 在此 MIRA 应用程序中，我们提出了通过开发实时技术来解决这一关键差距的策略 以及以 O-GlcNAc 为基石连接细胞代谢和癌症过程的空间分子工具。 NIH 资助的优秀研究已在人体细胞的蛋白质中发现了 2000 多种 O-GlcNAc，但目前的工具 依赖于破坏的生理学，导致伪影，或错过之前发生的关键 GlcNAc 驱动的信号事件 全球代谢再平衡在不到一个小时内发生。我们假设高血糖的关键驱动因素 新陈代谢和病理学发生在营养和信号刺激的最初几分钟内，迄今为止这是 无法在活细胞中观察到。我们在光化学和系统糖生物学支持方面发表的工作 我们独特的策略可在 O-GlcNAc 重新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 在细胞质或细胞质之间移动 内质网进入细胞核，模拟可能将异常的胰岛素和 糖尿病中的葡萄糖释放与乳腺癌风险有关。我们的实时系统可用于跟踪 O-GlcNAc 事件 在患病胰岛素生理机能的 15 分钟快速脉冲期间，胰岛素信号首次发出。 另一种用于靶向细胞内 O-GlcNAc 靶向邻近标记的工具能够追踪 O-GlcNAcyllated 亚细胞空间中的蛋白质，目前还没有报道的工具能够在活细胞中特异性标记这些蛋白质。我们 建议在未来5年内开发我们的“时间和空间”分子工具并将其应用于独特的领域 通过 NFkB 靶向进行疾病生物学机制研究。我们积极与代谢疾病合作 和癌症专家确保疾病相关性，以及工业科学家的技术 开发以扩大 O-GlcNAc 生物学在代谢驱动疾病途径中的工业认识。 该提案的研究成果包括分子探针、时空策略和目标 将细胞代谢与信号传导联系起来。我们的有利化学战略具有广泛影响的潜力 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，例如调节唾液酸修饰 癌症代谢和炎症的界面。成功将建立一个持久的独立研究领域。