Dynamics of Arginine Methylation in Cellular Processes

背景。蛋白质精氨酸n-甲基转移酶（PRMTs）对蛋白质中精氨酸残基的甲基化是一种主要存在于染色质相关蛋白和rna结合蛋白中的修饰（PTM），已被证明在许多重要的细胞过程中起调节作用。一旦蛋白水解释放，游离二甲基化精氨酸可以竞争性地抑制一氧化氮合酶，并阻止其调节血管张力、疼痛、神经传递和对微生物的炎症反应的功能。这种PTM最近成为越来越多疾病的生物标志物，包括癌症、心血管疾病和炎症性肺部疾病。然而，与其他瞬时PTMs不同，精氨酸甲基化是蛋白质上的稳定标记。在过去的几年里，我的实验室已经建立了研究精氨酸甲基化的技术，以便我们可以确定PRMTs如何对细胞编程做出贡献。最近，我们能够使用蛋白质组学技术观察到，用肿瘤坏死因子- α或脂多糖处理的培养人肺上皮细胞的蛋白质与未经处理的细胞的蛋白质之间精氨酸甲基化的巨大变化。这些实验还揭示了炎症反应的蛋白质介质作为新的PRMT底物。研究项目目标。我们实验室的长期目标是了解PRMTs对细胞增殖、分化和死亡的独立和联合贡献。我们的短期目标之一是在不同的炎症反应途径中建立prmt特异性精氨酸甲基化网络，我们正在请求NSERC的支持。科学的方法。我们将对培养的人类细胞系进行治疗，引起nf - kappab介导的炎症反应。来自处理过和未处理过的对照细胞的蛋白质将被收集，要么用二甲基精氨酸特异性抗体进行印迹，要么用于蛋白质组学分析，这是与dr。来自UBC高通量生物学中心的Leonard Foster和nicholas Scott。我们的蛋白质组学方法利用了最近报道的甲基化肽富集策略，在质谱分析之前使用亲水相互作用液相色谱。然后将对照组和治疗组的甲基化蛋白进行相对定量比较，使我们能够观察PTMs的变化，从而建立炎症中的精氨酸甲基化网络。下一步将是确定特定的PRMT参与炎症途径，要么采用基于细胞的测定，其中特定的PRMT表达水平将通过siRNA降低，要么通过药物抑制小分子培养细胞中的特定PRMT。我们还将从细胞中共免疫沉淀甲基化蛋白，通过Western blot和质谱分析确定哪些PRMTs与其底物相关。然后用体外甲基化试验对这些底物进行测试，以证明PRMT底物的特异性。的影响。精氨酸甲基化已被证明在重要的细胞过程中发挥特定的功能，但这种PTM尚未在更广泛的细胞背景下进行研究。通过观察蛋白质组在全球范围内的精氨酸甲基化变化，我们将确定其修饰可能在炎症事件中起关键作用的底物，并展示该PTM如何参与各种信号通路。这项工作将为PRMT活动的整合如何促进细胞编程并最终决定细胞命运提供机制见解。我们的发现将影响癌症生物学、炎症和生物化学领域。

Background. The methylation of arginine residues within proteins by enzymes called protein arginine N-methyltransferases (PRMTs), is a modification (PTM) found predominantly in chromatin-associated and RNA-binding proteins, and has been shown to play regulatory roles in many important cellular processes. Once proteolytically liberated, free dimethylated arginines can competitively inhibit nitric oxide synthases and prevent their functions in regulating vascular tone, pain, neurotransmission, and the inflammatory response to microorganisms. This PTM has recently emerged as a biomarker for a growing list of diseases including cancer, cardiovascular, and inflammatory lung diseases. Unlike other transient PTMs, however, arginine methylation is a stable mark on proteins. For the past few years my laboratory has established techniques to study arginine methylation so that we may determine how PRMTs contribute to cellular programming. Recently, we were able to see using proteomic techniques large changes in arginine methylation between proteins from cultured human lung epithelial cells treated with tumor necrosis factor-alpha or lipopolysaccharide and proteins from untreated cells. These experiments also revealed protein mediators of the inflammatory response as new PRMT substrates. Research Program Objectives. The long-term objective of our lab is to understand the independent and combined contributions of PRMTs toward cell proliferation, differentiation, and death. One of our short-term goals, for which we are requesting NSERC support, is to establish PRMT-specific arginine methylation networks in different inflammatory response pathways. Scientific Approach. We will subject cultured human cell lines to treatments that cause an NF-kappaB-mediated inflammatory responses. Proteins from treated and untreated control cells will be harvested and either blotted with dimethylarginine-specific antibodies or used for proteomic analysis as part of an ongoing collaboration with Drs. Leonard Foster and Nichollas Scott at the UBC Centre for High-Throughput Biology. Our proteomic approach takes advantage of a recently reported enrichment strategy for methylated peptides using hydrophilic interaction liquid chromatography prior to sample analysis by mass spectrometry. Methylated proteins from control and treatment groups will then be compared for relative quantitation, allowing us to observe changes in PTMs as a way to establish arginine methylation networks in inflammation. The next step will be to determine specific PRMT involvement in inflammatory pathways either by employing cell-based assays in which specific PRMT expression levels will be decreased via siRNA, or by pharmacologically inhibiting specific PRMTs in cultured cells with small molecules. We will also co-immunoprecipitate methylated proteins from cells to determine which PRMTs associate with their substrates by Western blot and mass spectrometry. These substrates will then be tested with in vitro methylation assays to demonstrate PRMT substrate specificity. Impact. Arginine methylation has been demonstrated to perform specific functions in important cellular processes, but this PTM has yet to be studied in the broader context of the cell. By observing arginine methylation changes on the global scale of the proteome, we will identify substrates whose modifications may serve critical roles in inflammatory events, and demonstrate how this PTM participates in various signaling pathways. This work will provide mechanistic insight into how the integration of PRMT activities contributes to cellular programming and ultimately determines cell fate. Our findings should impact the fields of cancer biology, inflammation, and biochemistry.