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: 10728390
• 负责人: Charlie Fehl
• 金额: $ 7万
• 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/10728390
• 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 FEHL实验室的总体目标是制定化学生物学策略以确定功能 信号过程中蛋白质修饰的影响。具体而言，细胞代谢和应力每个铅 用O连接的N-乙酰葡萄糖糖（O-GLCNAC）修饰的潜水蛋白质修饰，但目前尚无工具 可以捕获具有定义时间和空间分辨率的高度动态和瞬态O-GLCNAC事件。缺乏 严格的“时间和空间”阻碍了科学界的新陈代谢与疾病生理学的联系， 包括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应用中，我们采取了通过实时发展解决这一关键差距的策略 以及使用O-GLCNAC作为Keystone桥接细胞代谢和癌症过程的空间分子工具。 由NIH资助的出色研究发现了2000年人类细胞中蛋白质的O-GLCNAC，但是当前的工具 依靠破坏的生理学，导致工件或错过以前发生的密钥GLCNAC驱动的信号事件 全球代谢重新平衡发生在不到一个小时的时间内。我们假设高血糖的关键驱动因素 代谢和病理位于营养素的前几分钟和信号刺激之内，迄今为止 在活细胞中无法观察。我们发表的光化学和系统糖生物学支持工作 在O-GLCNAC重新平衡之前，我们在几分钟内触发O-GLCNAC过程的独特策略。 光封糖工具能够触发细胞质转录因子NFKB的运动或在细胞质之间的运动 内质网进入细胞核，模拟物理事件，这些事件可能将异常胰岛素和 患有乳腺癌风险的糖尿病中的葡萄糖释放。我们的实时系统可用于跟踪O-GLCNAC事件 在胰岛素信号传导的过程中，在快速的15分钟胰岛素生理脉冲期间。 针对靶向细胞内O-GlCNAC靶向的另一个工具，能够跟踪O-Glcnacylated 亚细胞空间中的蛋白质，该蛋白质没有报道的工具能够在活细胞中专门标记。我们 未来5年的提议开发我们的“时间和空间”分子工具，并将其应用于独特 通过NFKB靶向疾病生物学的机理研究。我们积极与代谢疾病合作 和癌症专家，以确保疾病相关，以及与技术的工业科学家 开发以扩大新陈代谢驱动疾病途径中O-GLCNAC生物学的工业意识。 该提案的研究产出包括分子问题，空间时间策略以及目标 将细胞代谢与信号连接。我们的化学策略有可能产生广泛影响 在科学界，通过建立临时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和药物靶标，例如调节的唾液酸修饰 癌症代谢和炎症的界面。成功将建立持久的独立研究利基市场。