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.

项目概要 蛋白质异戊二烯化的特征是添加法尼基 (C15) 或香叶基香叶基 (C20) 类异戊二烯 半胱氨酸残基位于不同蛋白质的 C 末端附近。虽然最初被认为是罕见的 修饰，现在很清楚蛋白质异戊二烯化在真核生物中广泛存在，并且对于 多种参与肿瘤发生、分泌、核结构和信号转导的蛋白质。它一直 据估计，哺乳动物细胞中多达 2% 的蛋白质被异戊二烯化。这种普遍性，加上 许多这些修饰蛋白在细胞信号传导中发挥着核心作用，强调了 这种翻译后修饰的重要性。异戊二烯化抑制剂最初是作为治疗药物而开发的 癌症治疗剂。随着针对特定 Ras 的精准医学方法的发展 驱动癌症，多项临床试验正在进行中。这些抑制剂也正在研究用于治疗 多种其他疾病，包括疟疾、病毒感染、帕金森病和早衰症。 异戊二烯化研究领域两个密切相关的关键问题涉及哪些蛋白质被异戊二烯化 它们在疾病中如何变化？显然需要的是可以比较的全局方法 正常和疾病状态下的异戊烯基化，并允许那些水平发生变化的异戊烯化蛋白 确定。如果这是可能的，它将揭示针对这些使人衰弱的疾病进行治疗干预的新目标。 疾病。为了解决这个问题，将合成用于改进代谢标记的新类异戊二烯探针，并 将开发降低样品复杂性和改善探针输送的方法。这些将被用于 细胞培养和疾病小鼠模型中的定量蛋白质组学实验。另一组关键的 问题是什么控制着异戊二烯化效率以及如何调节异戊二烯化？为了解决这些 问题，我们需要的是能够真实分析蛋白质异戊二烯化过程的策略 在活细胞中的时间。这将允许在所有存在的情况下以整体方式研究异戊二烯化反应。 相关的细胞成分。它还可以为治疗干预开辟更多途径，因为它 可以揭示新的有针对性的监管互动。为了实现这一目标，细胞穿透肽 将制备带有笼状半胱氨酸残基以掩盖其异戊二烯化位点的笼状半胱氨酸残基，并随后制备它们 以时间控制的方式解笼后通过显微镜监测异戊二烯化。补充 使用通过分选酶连接制备的全长蛋白质进行实验，包含相同的光激活触发器 将用于探索与伴侣蛋白的潜在相互作用，而这些相互作用可能无法用更简单的方法观察到 基于肽的模型。