Chemical Approaches to Protein Arginine Methylation

蛋白质精氨酸甲基化的化学方法

• 批准号: 8693069
• 负责人: Y. George Zheng
• 金额: $ 14.24万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8693069
• 关键词: Chemical; Approaches; Protein; Arginine; Methylation

DESCRIPTION (provided by applicant): Protein arginine methyltransferases (PRMTs) are a relatively new type of chromatin-modifying enzymes that catalyze the methylation of specific arginine residues in histone and nonhistone substrates. Aberrant expression of PRMTs has been observed in various human diseases. However, the biological impact of protein arginine methylation and the molecular basis of PRMT catalysis are poorly defined. Our long-term research goal is to elucidate the biochemical pathways mediated by key PRMTs that contribute to the pathogenesis of cancer and cardiovascular disorders, and to develop effective PRMT inhibitors. In this proposal, we plan to carry out studies on mechanism, regulation, and function of key PRMT enzymes, by exploring and applying new chemical biology approaches. Two specific aims will be pursued: a) Design unique biochemical assays to elucidate the mechanism of substrate specificity regulation by PRMT1. The mechanisms that govern the substrate specificity of PRMTs are not well understood. It also remains to determine the dynamic regulation of arginine methylation. We will introduce environmentally sensitive fluorophores to probe how key motifs in the substrates dynamically regulate arginine recognition and methylation. Also, we will use expressed protein ligation to site specifically label PRMT1 with biophysical probes to investigate the conformational changes of PRMT1 during the catalytic process. Further, we will create a semi-active hetero-oligomer of PRMT1 to determine the catalytic role of PRMT1 oligomerization in regulating substrate binding and methylation. The proposed study will yield new non-radioactive assays of PRMT catalysis, provide molecular understanding of PRMT substrate specificity, and offer critical insight for designing specific PRMT1 inhibitors; b) Develop chemical probes to sort out the substrate specificity of PRMT1 in prostate cancer cells. The importance of PRMTs in prostate cancer pathogenesis is increasingly recognized. To reveal the function of PRMTs in the disease, we propose a series of unique chemical biology approaches to investigate the substrate specificity of PRMT1 in both androgen-dependent and androgen-refractory prostate cancer cells. First, we will prepare biotin-labeled PRMT1 to identify PRMT1- interactive proteins, from which substrate candidates will be determined. Second, we will design, synthesize and evaluate AdoMet analogs as chemical probes to investigate cellular substrates of PRMT1. Third, we will create new chemical probes for global mapping of arginine-methylated substrates in prostate cancer cells. The results of the proposed research will be essential for understanding the mechanism and the biological impact of PRMT-catalyzed methylation in gene regulation and signal transduction. Accomplishment of the proposed work will also provide new chemical tools for both basic PRMT biology research and facilitate the development of therapeutic agents for the treatment of carcinoma, cardiovascular disorders, and other diseases related to the deregulation of protein arginine methylation. PUBLIC HEALTH RELEVANCE: Malfunctioning of protein arginine methyltransferases (PRMTs) is closely associated with the pathogenesis of various human diseases. We propose a series of chemical biology strategies to elucidate the substrate specificity and enzymatic functions of key PRMTs. This effort is of great significance for the development of potent PRMT inhibitors to treat prostate carcinoma and other diseases associated with the deregulation of protein arginine methylation.

描述（由申请人提供）：蛋白精氨酸甲基转移酶（PRMTS）是一种相对较新的染色质修饰酶，可催化组蛋白和非组织蛋白底物中特定精氨酸残基的甲基化。在各种人类疾病中已经观察到PRMT的异常表达。然而，蛋白质精氨酸甲基化和PRMT催化的分子基础的生物学影响很差。我们的长期研究目标是阐明由关键PRMT介导的生化途径，这些途径有助于癌症和心血管疾病的发病机理，并开发有效的PRMT抑制剂。在此提案中，我们计划通过探索和应用新的化学生物学方法来进行关键PRMT酶的机制，调节和功能的研究。将追求两个具体目标：a）设计独特的生化测定，以阐明PRMT1底物特异性调节的机制。尚不清楚控制PRMT的底物特异性的机制。还需要确定精氨酸甲基化的动态调节。我们将引入对环境敏感的荧光团，以探测底物中的关键基序如何动态调节精氨酸识别和甲基化。此外，我们将使用表达的蛋白质连接将其专门用生物物理探针标记PRMT1来研究催化过程中PRMT1的构象变化。此外，我们将创建PRMT1的半活性异寡聚体，以确定PRMT1低聚在调节底物结合和甲基化中的催化作用。拟议的研究将产生有关PRMT催化的新的非放射性测定，提供对PRMT底物特异性的分子理解，并为设计特定的PRMT1抑制剂提供关键的见解； b）开发化学探针以整理前列腺癌细胞中PRMT1的底物特异性。 PRMT在前列腺癌发病机理中的重要性越来越多。为了揭示PRMT在该疾病中的功能，我们提出了一系列独特的化学生物学方法，以研究PRMT​​1在雄激素依赖性和雄激素难治性前列腺癌细胞中的底物特异性。首先，我们将准备生物素标记的PRMT1来识别PRMT1互动蛋白，从中确定底物候选者。其次，我们将设计，合成和评估Adomet类似物作为化学探针研究PRMT​​1的细胞底物。第三，我们将创建新的化学探针，用于在前列腺癌细胞中全球精氨酸 - 甲基化底物的全球映射。拟议研究的结果对于理解PRMT催化甲基化基因调节和信号转导的机制和生物学影响至关重要。拟议工作的完成还将为基本的PRMT生物学研究提供新的化学工具，并促进治疗癌，心血管疾病和其他与蛋白质精氨酸甲基化管制有关的癌症治疗的治疗剂。 公共卫生相关性：蛋白质精氨酸甲基转移酶（PRMT）的故障与各种人类疾病的发病机理密切相关。我们提出了一系列化学生物学策略，以阐明关键PRMT的底物特异性和酶促功能。这项工作对于开发有效的PRMT抑制剂来治疗前列腺癌和与蛋白精氨酸甲基化管制有关的其他疾病具有重要意义。