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Abstract：
Proper protein arginine methylation by protein arginine methyltransferase 1 (PRMT1) is critical for maintaining cellular health, while dysregulation is often associated with disease. How the activity of PRMT1 is regulated is therefore paramount, but is not clearly understood. Several studies have observed higher order oligomeric species of PRMT1, but it is unclear if these exist at physiological concentrations and there is confusion in the literature about how oligomerization affects activity. We therefore sought to determine which oligomeric species of PRMT1 are physiologically relevant, and quantitatively correlate activity with specific oligomer forms. Through quantitative western blotting, we determined that concentrations of PRMT1 available in a variety of human cell lines are in the sub-micromolar to low micromolar range. Isothermal spectral shift binding data were modeled to a monomer/dimer/tetramer equilibrium with an EC50 for tetramer dissociation of ~20 nM. A combination of sedimentation velocity and Native polyacrylamide gel electrophoresis experiments directly confirmed that the major oligomeric species of PRMT1 at physiological concentrations would be dimers and tetramers. Surprisingly, the methyltransferase activity of a dimeric PRMT1 variant is similar to wild type, tetrameric PRMT1 with some purified substrates, but dimer and tetramer forms of PRMT1 show differences in catalytic efficiencies and substrate specificity for other substrates. Our results define an oligomerization paradigm for PRMT1, show that the biophysical characteristics of PRMT1 are poised to support a monomer/dimer/tetramer equilibrium in vivo, and suggest that the oligomeric state of PRMT1 could be used to regulate substrate specificity.
摘要：
通过蛋白质精氨酸甲基转移酶1（PRMT1）进行适当的蛋白质精氨酸甲基化对于维持细胞健康至关重要，而失调通常与疾病有关。因此，如何调节PRMT1的活性是至关重要的，但不清楚。一些研究已经观察到PRMT1的高阶寡聚物种,但是不清楚这些物种是否以生理浓度存在,并且文献中关于寡聚化如何影响活性存在混淆。因此，我们试图确定PRMT1的哪些寡聚物种是生理相关的，并将活性与特定的寡聚体形式定量相关。通过定量的蛋白质印迹,我们确定在多种人细胞系中可用的PRMT1浓度在亚微摩尔至低微摩尔范围内。将等温光谱位移结合数据建模为单体/二聚体/四聚体平衡,其中四聚体解离的EC50为〜20nM。沉降速度和天然聚丙烯酰胺凝胶电泳实验的组合直接证实，生理浓度下PRMT1的主要寡聚物将是二聚体和四聚体。令人惊讶的是,二聚PRMT1变体的甲基转移酶活性与野生型相似，四聚体PRMT1与一些纯化的底物，但是PRMT1的二聚体和四聚体形式在催化效率和对其他底物的底物特异性方面显示出差异。我们的结果定义了PRMT1的寡聚化范例，表明PRMT1的生物物理特征有望在体内支持单体/二聚体/四聚体平衡，并表明PRMT1的寡聚状态可用于调节底物特异性。
