Modulation of Runx2 activity by arginine methylation

项目概要/摘要 蛋白质精氨酸甲基化是一个快速发展的领域，其在癌症中的重要性日益得到认可，但是 关于骨骼中的这种机制几乎一无所知。该提案基于以下方面的新发现： Runx2 的精氨酸甲基化。我们已经鉴定出两种可甲基化的蛋白质精氨酸甲基转移酶 对四个定义的精氨酸残基进行 Runx2。通过将这些残基突变为模拟甲基化的丙氨酸- 在缺乏状态下，我们已经证明了 Runx2 介导的 Runx2 精氨酸甲基化的需要 癌细胞的运动性。在这里，我们将研究 Runx2 精氨酸甲基化在其典型作用的背景下 成骨细胞分化和骨形成。目标 1 是表征时间精氨酸甲基化 成骨细胞培养模型中的 Runx2。我们还将比较 WT Runx2 与 不可甲基化突变体 Runx2，以及操纵介导 Runx2 的甲基转移酶的后果 甲基化。目标 2 阐述了 Runx2 精氨酸甲基化在调节身体和功能方面的作用。 与 BMP-Smads 通路的相互作用。最后，目标 3 是定义 Runx2 的精氨酸甲基化，以及 体内颅骨发育过程中介导 Runx2 甲基化的甲基转移酶的表达，以及 比较 WT 与未甲基化 Runx2 在颅骨器官培养物中的成骨潜力。总体而言，这 该提案提出了这样的假设：成骨过程中 Runx2 活性需要精氨酸甲基化 分化并对 BMP-Smad 信号反应至关重要。此外，我们在研究中纳入了 设计基因组和蛋白质组分析，以促进 Runx2 蛋白质-蛋白质的公正发现 相互作用、Runx2 基因组目标和受精氨酸甲基化影响的 Runx2 调节基因。 从长远来看，我们设想将这个探索性项目中获得的知识转化为改进 了解骨骼发育、稳态和再生，并提供新的治疗方法 解决骨骼疾病的方法。

PROJECT SUMMARY / ABSTRACT Protein arginine methylation is a rapidly growing field with increasingly recognized importance in cancer, but almost nothing is known about this mechanism in bone. This proposal is based on novel discoveries on the arginine methylation of Runx2. We have identified two protein arginine methyltransferases that methylate Runx2 on four defined arginine residues. By mutating these residues to alanine that mimic the methylation- deficient state, we have demonstrated the requirement for Runx2 arginine methylation in Runx2-mediated cancer cell motility. Here, we will investigate Runx2 arginine methylation in the context of its canonical role in osteoblast differentiation and bone formation. Aim 1 is to characterize the temporal arginine methylation of Runx2 in osteogenic cell culture models. We will also compare the osteogenic potentials of WT Runx2 versus unmethylatable mutant Runx2, and the consequences of manipulating methyltransferases that mediate Runx2 methylation. Aim 2 addresses the role of Runx2 arginine methylation in modulating the physical and functional interaction with BMP-Smads pathway. Finally, Aim 3 is to define the arginine methylation of Runx2, and the expression of methyltransferases that mediate Runx2 methylation during skull development in vivo, and to compare the osteogenic potential of WT versus unmethylatable Runx2 in calvarial organ cultures. Overall, this proposal addresses the hypothesis that arginine methylation is required for Runx2 activity during osteogenic differentiation and essential for BMP-Smad signaling response. Additionally, we have incorporated in the study design genomic and proteomic analyses to facilitate unbiased discoveries of Runx2 protein-protein interactions, Runx2 genomic targets and Runx2-regulated genes that are impacted by its arginine methylation. In the long run, we envision translation of knowledge acquired in this exploratory project to improve the understanding of bone development, homeostasis and regeneration, and provide novel therapeutic approaches to tackle skeletal disorders.