Chemical Approaches for Exploring Protein Prenylation in Living Cells

探索活细胞中蛋白质异戊二烯化的化学方法

• 批准号: 10207169
• 负责人: MARK D DISTEFANO
• 金额: $ 45.31万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-05 至 2026-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10207169
• 关键词: Address; Alzheimer' s Disease; Biological Assay; Cell Culture Techniques; Cells; Chemicals; Clinical Trials; Coupled; Cysteine; Development; Disease; Disease model; Event; Label; Length; Ligation; Light; Link; Malaria; Malignant Neoplasms; Mammalian Cell; Masks; Metabolic; Methods; Microscopy; Modeling; Modification; Molecular Chaperones; Monitor; Nuclear Structure; Parkinson Disease; Peptides; Play; Post-Translational Protein Processing; Prevalence; Process; Progeria; Protein Isoprenylation; Proteins; Proteomics; Reaction; Research; Role; Sampling; Signal Transduction; Site; Therapeutic Agents; Therapeutic Intervention; Time; Virus Diseases; base; cancer therapy; experimental study; improved; inhibitor/antagonist; isoprenoid; mouse model; new therapeutic target; novel therapeutics; precision medicine; prenylation; sortase; tumorigenesis

PROJECT SUMMARY Protein prenylation is characterized by the addition of farnesyl (C15) or geranylgeranyl (C20) isoprenoids to cysteine residues located near the C-termini of different proteins. Although originally considered to be a rare modification, it is now clear that protein prenylation is widespread in eucaryotes and is of critical importance for a variety of proteins involved in oncogenesis, secretion, nuclear structure, and signal transduction. It has been estimated that as many as 2% of all proteins in mammalian cells are isoprenylated. This prevalence, coupled with the central role that many of these modified proteins play in cellular signaling, underscores the significance of this post-translational modification. Prenylation inhibitors were initially developed as therapeutic agents for cancer treatment. With the development of precision medicine approaches that target specific Ras- driven cancers, several clinical trials are ongoing. These inhibitors are also being investigated for the treatment of a wide variety of other disease including malaria, viral infections, Parkinson's disease and progeria. Two closely linked critical questions in the field of prenylation research concern what proteins are prenylated and how do they change in disease? What are clearly necessary are global methods that can compare the prenylomes in normal and disease states and allow those prenylated proteins whose levels change to be identified. If this were possible, it would reveal new targets for therapeutic intervention in these debilitating diseases. To address this, new isoprenoid probes for improved metabolic labeling will be synthesized and methods to decrease sample complexity and improve probe delivery will be developed. These will be utilized in quantitative proteomic experiments in cell culture and mouse models for disease. Another critical set of questions is what controls prenylation efficiency and is how is prenylation regulated? To address these questions, what are needed are strategies that allow the process of protein prenylation to be assayed in real time in live cells. This would allow prenylation reactions to be studied in a holistic manner in the presence of all relevant cellular components. It could open also up additional avenues for therapeutic intervention since it could reveal new regulatory interactions that could be targeted. To accomplish this, cell penetrating peptides with caged cysteine residues that mask their site of prenylation will be prepared and their subsequent prenylation monitored via microscopy after uncaging in a temporally controlled manner. Complementary experiments with full-length proteins, prepared by sortase ligation, containing the same light activated trigger will be used to explore potential interactions with chaperone proteins that may not be observed with the simpler peptide-based models.

项目总结 蛋白质预烯基化的特征是将法尼基(C15)或香叶基(C20)的异戊二烯类加到 半胱氨酸残基位于不同蛋白质的C-末端附近。尽管最初被认为是一种罕见的 修饰，现在清楚的是，蛋白质预烯基化在真核生物中广泛存在，并且对 参与肿瘤发生、分泌、核结构和信号转导的多种蛋白质。一直以来 据估计，哺乳动物细胞中多达2%的蛋白质是异戊二烯基化的。这种流行率，加上 由于这些修饰蛋白中的许多在细胞信号转导中发挥核心作用，强调了 这一翻译后修饰的意义。预基化抑制剂最初是作为治疗药物而开发的 治疗癌症的药物。随着针对特定RAS的精确医学方法的发展- 随着癌症的发展，几项临床试验正在进行中。这些抑制剂也正在被研究用于治疗 各种其他疾病，包括疟疾、病毒感染、帕金森氏症和早衰症。 在前烯基化研究领域中有两个密切相关的关键问题，涉及哪些蛋白质是前烯基化的 它们在疾病中是如何变化的？显然需要的是全局方法，可以比较 在正常和疾病状态下的前烯基化体，并允许那些其水平变化的前烯基化蛋白质 确认身份。如果这是可能的，它将揭示治疗干预这些衰弱的新靶点。 疾病。为了解决这一问题，将合成用于改进代谢标记的新的类异戊二烯探针，并 将开发降低样品复杂性和改进探针传递的方法。这些将用于 细胞培养和小鼠疾病模型中的定量蛋白质组学实验。另一组关键的 问题是什么控制着预烯基化的效率，以及预烯基化是如何调节的？要解决这些问题 问题是，需要的是允许蛋白质预烯基化过程被实时检测的策略 活细胞中的时间。这将允许在所有人在场的情况下以整体的方式研究预烯基化反应 相关的蜂窝组件。它还可以为治疗干预开辟额外的途径，因为它 可能会揭示出可能成为目标的新的监管互动。为了做到这一点，细胞穿透性多肽 用笼状半胱氨酸残基掩蔽它们的预烯基化部位，将制备它们随后的 以时间可控的方式在取消后通过显微镜监测异丙烯基化。互补性 用含有相同光激活触发器的全长蛋白质进行实验，这些蛋白质是由索酸酶连接而成的 将用于探索与伴侣蛋白的潜在相互作用，这些相互作用可能无法用SIMPLE 基于多肽的模型。