Regulation of and Target Recognition by Protein Arginine Methyltransferase 1 (PRMT1)

项目总结 精氨酸甲基转移酶1(PRMT1)催化的蛋白质精氨酸甲基化是 目前作为抗癌治疗靶点的强烈兴趣，以及肺，肾， 神经退行性疾病和心血管疾病。到目前为止，大多数研究 表征PRMT1介导的蛋白质甲基化的后果尚未考虑 PRMT1活性改变的生化机制，这是理解的关键 PRMT1如何参与细胞内稳态和上述疾病的进展 状态，以及制定治疗方案以控制依赖PRMT1的细胞事件。这个 在这个项目中检验的两个假说是：1)寡聚体状态和特定部位的变化 PRMT1蛋白的半胱氨酸氧化影响底物靶向性和活性，以及2)一种 PRMT1中的半胱氨酸残基可以被利用来产生/升级异构体特异性的PRMT1 抑制剂。在目标1中，将使用新的基于荧光的生物物理方法来表征 完整细胞中PRMT1寡聚体形成的动力学。经过战略设计的变体 以四聚体、二聚体或单体的形式存在的PRMT1将用于识别结合和 每个寡聚体靶向的催化差异，有助于正在进行的努力了解 PRMT1对其底物的分子识别规律。半胱氨酸的亚磺酰化效应 PRMT1活性部位附近的残基也将被表征，从而允许开发 PRMT1稳定的氧化和还原模拟物将用作人类研究工具 受氧化应激影响的健康。目标1中的目标建立在初步的体外研究结果基础上。 并有望应用于表征机制的长期目标 体内调节PRMT1活性。在目标2中，使用亲核半胱氨酸的策略 将探索PRMT1的活性部位，以实现腺苷衍生物的共价抑制。这 策略将是挽救缺乏异构体特异性的抑制剂的有价值的方法，但是 否则表现良好，以抑制PRMT活性。共价抑制剂代表了一种新的理论基础 开发PRMT1药物和研究工具的途径。本科生和研究生 对未来生物医学研究或医学事业感兴趣的研究人员将参与 项目及接受过实验设计与分析、笔记、科学 写作和演示。

PROJECT SUMMARY Protein arginine methylation catalyzed by Protein arginine methyltransferase 1 (PRMT1) is of intense current interest as an anti-cancer therapeutic target, as well as a mediator of lung, kidney, neurodegenerative and cardiovascular pathologies. To date, the majority of studies characterizing the consequences of PRMT1-mediated protein methylation have not considered the biochemical mechanisms by which PRMT1 activity is altered, which is pivotal in understanding how PRMT1 participates in cellular homeostasis, the progression of the aforementioned disease states, and in developing treatment protocols to control PRMT1-dependent cellular events. The two hypotheses being tested in this project are: 1) changes in oligomeric state and site-specific cysteine oxidation of the PRMT1 protein affect substrate targeting and activity, and 2) that a cysteine residue in PRMT1 can be harnessed to create/upgrade isoform-specific PRMT1 inhibitors. In Aim 1, novel fluorescence-based biophysical methods will be used to characterize the dynamics of PRMT1 oligomer formation in intact cells. Strategically designed variants of PRMT1 which present as tetramers, dimers, or monomers will be used to identify binding and catalytic differences in the targeting by each oligomer, aiding in the ongoing effort to understand molecular recognition rules of PRMT1 for its substrates. The effect of sulfenylation at a cysteine residue near the active site of PRMT1 will also be characterized, allowing for the development of stable oxidized and reduced mimics of PRMT1 to be used as research tools in areas of human health affected by oxidative stress. The objectives in Aim 1 are built upon initial in vitro findings by the PI and are expected to apply to the long-term goal of characterizing mechanisms of regulating PRMT1 activity in vivo. In Aim 2, the strategy of using a nucleophilic cysteine near the active site of PRMT1 to enable covalent inhibition by adenosine derivatives will be explored. This strategy would be a valuable way to salvage inhibitors that show a lack of isoform-specificity, but otherwise perform well to inhibit PRMT activity. Covalent inhibitors represent a novel, rationale avenue for developing PRMT1 drugs and research tools. Undergraduate and graduate researchers with interests in future careers in biomedical research or medicine will be involved in the project and receive training in experimental design and analysis, notebook keeping, scientific writing and presentation.