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: 10383695
• 负责人: MARK D DISTEFANO
• 金额: $ 35.32万
• 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/10383695
• 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; 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.

项目摘要 蛋白质异戊烯化的特征在于将法呢基（C15）或香叶基香叶基（C20）类异戊二烯加成到 位于不同蛋白质的C末端附近的半胱氨酸残基。虽然最初被认为是一种罕见的 由于蛋白质修饰，现在很清楚，蛋白质异戊二烯化在真核生物中广泛存在，并且对于蛋白质修饰至关重要。 参与肿瘤发生、分泌、核结构和信号转导的多种蛋白质。已经 据估计，哺乳动物细胞中多达2%的蛋白质是异戊二烯化的。这种流行，加上 许多这些修饰的蛋白质在细胞信号传导中起着核心作用，强调了 这种翻译后修饰的意义。异戊二烯化抑制剂最初是作为治疗药物开发的 用于癌症治疗的药剂。随着针对特定Ras的精准医学方法的发展， 一些临床试验正在进行中。这些抑制剂也正在研究用于治疗 包括疟疾、病毒感染、帕金森氏症和早衰症在内的各种其他疾病。 异戊二烯化研究领域中两个密切相关的关键问题涉及哪些蛋白质被异戊二烯化 它们在疾病中是如何变化的显然需要的是能够比较 在正常和疾病状态下的异戊二烯基化蛋白质组，并允许那些水平变化的异戊二烯基化蛋白质， 鉴定如果这是可能的，它将揭示新的目标，为治疗干预，在这些衰弱 疾病为了解决这个问题，将合成用于改进代谢标记的新的类异戊二烯探针， 将开发降低样品复杂性和改进探针递送的方法。这些将用于 定量蛋白质组学实验在细胞培养和疾病的小鼠模型。另一组关键的 问题是什么控制异戊二烯化效率，以及异戊二烯化是如何调节的？解决这些 问题，什么是需要的是策略，允许蛋白质异戊二烯化的过程中进行测定，在真实的 活细胞中的时间。这将使异戊烯化反应，以整体的方式进行研究，在存在的所有 相关的细胞成分。它还可以为治疗干预开辟额外的途径，因为它 可以揭示新的监管互动，可以有针对性的。为了实现这一点， 将制备掩蔽其异戊二烯化位点的笼状半胱氨酸残基，并随后 在以时间控制的方式打开后通过显微镜监测异戊烯化。互补 用通过分选酶连接制备的全长蛋白质进行的实验，所述全长蛋白质含有相同的光激活触发物 将用于探索与伴侣蛋白的潜在相互作用，这些相互作用可能不会被观察到， 基于肽的模型