Chemical Approaches to 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蛋白底物中特定精氨酸残基的甲基化。PRMTs的异常表达已在多种人类疾病中被观察到。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明确。我们的长期研究目标是阐明参与癌症和心血管疾病发病机制的关键PRMT介导的生化途径，并开发有效的PRMT抑制剂。在本课题中，我们计划通过探索和应用新的化学生物学方法，开展关键PRMT酶的机制、调控和功能研究。将追求两个具体目标：a)设计独特的生化分析来阐明PRMT1调节底物特异性的机制。调控PRMTs底物特异性的机制尚不清楚。还需要确定精氨酸甲基化的动态调控。我们将引入环境敏感的荧光团来探索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，我们将利用生物物理探针，利用表达蛋白连接对PRMT1进行位点特异性标记，研究PRMT1在催化过程中的构象变化。此外，我们将创建一个半活性的PRMT1异聚物，以确定PRMT1寡聚在调节底物结合和甲基化中的催化作用。拟议的研究将产生新的PRMT催化非放射性分析，提供对PRMT底物特异性的分子理解，并为设计特异性PRMT1抑制剂提供关键见解；b)开发化学探针，梳理前列腺癌细胞中PRMT1的底物特异性。PRMTs在前列腺癌发病机制中的重要性日益被认识。为了揭示PRMT1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来鉴定PRMT1-相互作用蛋白，从中确定底物候选蛋白。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，用于前列腺癌细胞精氨酸甲基化底物的全球定位。本研究的结果将对理解prmt催化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。这项工作的完成还将为基础的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有关的疾病的治疗药物的开发。

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.