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: 10551852
• 负责人: MARK D DISTEFANO
• 金额: $ 35.28万
• 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/10551852
• 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; Penetration; Peptides; Play; Post-Translational Protein Processing; Prevalence; Process; Progeria; Protein Isoprenylation; Proteins; Proteomics; RAS driven cancer; Reaction; Research; Role; Sampling; Signal Transduction; Site; Therapeutic Agents; Therapeutic Intervention; Time; Virus Diseases; cancer therapy; experimental study; improved; inhibitor; isoprenoid; isoprenylation; 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 蛋白质前化的特征在于添加Farnesyl（C15）或黄烷基果仁酰基（C20）类异on- 半胱氨酸残基位于不同蛋白质的C末端。虽然最初被认为是罕见的 修改，现在很明显，蛋白质前化在桉树中普遍存在，并且至关重要。 参与肿瘤发生，分泌，核结构和信号转导的多种蛋白质。它一直 据估计，哺乳动物细胞中所有蛋白质的多达2％都是异源肾上腺素化的。这种流行率，耦合 这些修饰蛋白中许多在细胞信号传导中起着的核心作用，强调了 这种翻译后修改的意义。原先性抑制剂最初是作为治疗性开发的 癌症治疗的药物。通过开发精确的医学方法，以特定的ras- 驱动的癌症，正在进行一些临床试验。这些抑制剂也正在研究以进行治疗 在各种其他疾病中，包括疟疾，病毒感染，帕金森氏病和后代。 在婚前化研究领域的两个紧密相连的关键问题涉及哪些蛋白质是蛋白质的 它们如何改变疾病？显然有必要的是可以比较的全球方法 正常和疾病状态下的婚前类，允许那些水平变化的蛋白质蛋白 确定。如果可能的话，它将揭示用于治疗干预的新目标 疾病。为了解决这个问题，将合成用于改进代谢标签的新的类化探针，并将合成 将开发降低样品复杂性和改善探针输送的方法。这些将用于 细胞培养和疾病小鼠模型中的定量蛋白质组学实验。另一组关键集 问题是控制原始化效率的问题，是如何调节前化的？解决这些 问题，需要的是允许实际测定蛋白质前化过程的策略 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前原化反应 相关的细胞成分。它还可以开放其他治疗干预途径 可以揭示可能针对的新监管相互作用。为此，细胞穿透肽 将准备掩盖其原始化位点的笼中半胱氨酸残留物，然后将其随后 以时间控制的方式分解后，通过显微镜监测的前烯基化。补充 通过分类酶连接制备的全长蛋白的实验，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 基于肽的模型。