Chemical Approaches to Protein Arginine Methylation

描述（由申请人提供）：蛋白质精氨酸甲基转移酶（PRMT）是一种相对新型的染色质修饰酶，其催化组蛋白和非组蛋白底物中特定精氨酸残基的甲基化。已在多种人类疾病中观察到 PRMT 的异常表达。然而，蛋白质精氨酸甲基化的生物学影响和 PRMT 催化的分子基础尚不清楚。我们的长期研究目标是阐明关键 PRMT 介导的生化途径，从而促进癌症和心血管疾病的发病机制，并开发有效的 PRMT 抑制剂。在本提案中，我们计划通过探索和应用新的化学生物学方法，对关键PRMT酶的机制、调控和功能进行研究。我们将追求两个具体目标： a) 设计独特的生化检测来阐明 PRMT1 底物特异性调节的机制。控制 PRMT 底物特异性的机制尚不清楚。精氨酸甲基化的动态调节还有待确定。我们将引入环境敏感的荧光团来探究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，我们将使用表达蛋白连接技术，用生物物理探针对PRMT1进行位点特异性标记，以研究PRMT​​1在催化过程中的构象变化。此外，我们将创建 PRMT1 的半活性异源寡聚体，以确定 PRMT1 寡聚化在调节底物结合和甲基化中的催化作用。拟议的研究将产生新的 PRMT 催化非放射性测定方法，提供对 PRMT 底物特异性的分子理解，并为设计特异性 PRMT1 抑制剂提供重要见解； b) 开发化学探针来筛选前列腺癌细胞中 PRMT1 的底物特异性。 PRMT 在前列腺癌发病机制中的重要性日益得到认识。为了揭示 PRMT 在疾病中的功能，我们提出了一系列独特的化学生物学方法来研究 PRMT1 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，我们将制备生物素标记的 PRMT1 以鉴定 PRMT1 相互作用蛋白，从中确定候选底物。其次，我们将设计、合成和评估 AdoMet 类似物作为化学探针来研究 PRMT1 的细胞底物。第三，我们将创建新的化学探针，用于前列腺癌细胞中精氨酸甲基化底物的全局图谱。本研究的结果对于理解 PRMT 催化的甲基化在基因调控和信号转导中的机制和生物学影响至关重要。拟议工作的完成还将为基础 PRMT 生物学研究提供新的化学工具，并促进治疗癌症、心血管疾病和其他与蛋白质精氨酸甲基化失调相关疾病的治疗药物的开发。 公共健康相关性：蛋白质精氨酸甲基转移酶 (PRMT) 的功能障碍与各种人类疾病的发病机制密切相关。我们提出了一系列化学生物学策略来阐明关键 PRMT 的底物特异性和酶功能。这项工作对于开发有效的 PRMT 抑制剂来治疗前列腺癌和其他与蛋白质精氨酸甲基化失调相关的疾病具有重要意义。

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.