Structural Basis of Phosphorylation and Alternative Splicing in Dynein Regulation

标题：Dynein调控中磷酸化和选择性剪接的结构基础生命系统使用生物马达将材料沿着被称为微管的火车状轨道沿特定方向输送到细胞中。动力蛋白是一种这样的马达，它对许多细胞过程都是必不可少的，包括细胞分裂期间染色体的运输，含有营养物质的小泡的运动，以及神经元的迁移。动力蛋白如何知道何时以及运输什么货物需要与控制动力蛋白功能的其他蛋白质相互作用。这些相互作用由动力蛋白区域的蛋白质序列改变来调节，动力蛋白区域缺乏规则的独特三维结构，被称为固有无序蛋白质。预期结果将具体显示这些修饰如何帮助选择结合伙伴，以及随后运输什么货物。了解这一系统最终将成为设计能够按需运输材料的合成纳米机器的基础。这项工作将促进本科生和研究生在前沿研究实践中的培养。此外，PI和她的团队将影响俄勒冈州立大学和该地区其他学科的进展，提供获得生物物理仪器的途径，发起和组织一年一度的核磁共振研讨会和一年一度的以生物物理学为重点的地区性一天会议，并向K-12地区的学校实施不同复杂性的动手演示和科学活动，其中包括蛋白质无序的模型/动画和俄勒冈科学博物馆大规模演示的结构变化。这项研究解决了动力蛋白的一个亚基中的磷酸化和内在无序如何调节动力蛋白与包括动力蛋白在内的多种调节蛋白质的相互作用，动力蛋白的相互作用在所有真核细胞中都存在，并在神经元中发挥特别重要的作用，以及裸、裸、一种核分布蛋白，在包括动粒和中心体迁移在内的各种过程中都是必不可少的。动力蛋白中间链(IC)的N末端300个氨基酸残基的无序结构域负责这些相互作用，也是大多数动力蛋白活性的枢纽。虽然有充分的证据表明磷酸化和异构体表达是动力蛋白调节的关键，但还没有关于它们影响的分子机制的研究。这个项目将特别关注：1)IC磷酸化如何影响组织特异性IC亚型与dynactin的相互作用，2)IC磷酸化如何在dynactin和nude调节蛋白之间进行选择，以及3)IC调控在哺乳动物、果蝇和酵母中的不同。这些关于动力蛋白调节的悬而未决的问题将结合最先进的核磁共振、恒温滴定量热法、合成生物学、计算方法和基于活体的分析来解决，并将阐明内在无序蛋白质的磷酸化和选择性剪接通常如何控制它们的分子功能，并最终影响整个蛋白质相互作用网络的行为。值得注意的是，使用来自酵母、果蝇和哺乳动物来源的蛋白质，评估了为诱导高度无序区域的结构变化而进化的不同IC过程，强调了蛋白质无序和IC亚型在动力蛋白功能调节中的战略作用。

TITLE: Structural Basis of Phosphorylation and Alternative Splicing in Dynein RegulationLiving systems use biological motors to transport materials into cells in specific directions along train-like tracks called microtubules. One such motor is dynein which is essential for many cellular processes including transport of chromosomes during cell division, movement of vesicles containing nutrients, and in neuronal migration. How dynein knows when and what cargo to transport requires interactions with other proteins that control dynein function. These interactions are regulated by modification in the protein sequence in regions of dynein that lack regular unique three-dimensional structure and are referred to as intrinsically disordered proteins. Expected results will specifically show how these modifications help in selecting the binding partners, and subsequently what cargo to transport. Understanding this system will ultimately be the basis for the design of synthetic nano-machines that can transport materials on demand. This work will facilitate the training of undergraduate and graduate students in the practice of cutting-edge research. In addition, the PI and her team will impact progress in other disciplines at Oregon State University and the region by providing access to biophysical instrumentation, by initiating and organizing an annual NMR workshop and an annual regional one-day conference focused on biophysics, and by implementing hands on demonstrations and science activities of varying complexity to K-12 area schools that include models/animations of protein disorder and structural changes for large-scale demonstrations at the Oregon Museum of Science.This research addresses how phosphorylation and intrinsic disorder in one subunit of dynein modulate the interaction of dynein with multiple regulatory proteins including dynactin, whose interaction occurs in all eukaryotes and plays an especially important role in neurons, and NudE, a nuclear distribution protein which is essential in diverse processes including kinetochore and centrosome migration. The intrinsically disordered N-terminal 300-amino acid domain of dynein intermediate chain (IC) is responsible for these interactions and is also the hub of most dynein activity. While there is ample evidence for phosphorylation and isoform expression as key to dynein regulation, there are no studies of the molecular mechanisms underlying their effects. This project will specifically focus on: 1) How IC phosphorylation affects the interaction of tissue-specific isoforms of IC with dynactin, 2) How IC phosphorylation selects between regulatory proteins dynactin and NudE, and 3) How IC regulation differs among mammals, Drosophila, and yeast. These outstanding questions about dynein regulation will be addressed using a combination of state-of-the-art NMR, isothermal titration calorimetry, synthetic biology, computational methods, and in vivo-based assays and will elucidate how phosphorylation and alternative splicing of intrinsically disordered proteins in general govern their molecular function and ultimately affect the behavior of entire protein interaction networks. Significantly, the use of proteins from yeast, Drosophila and mammalian origin, assesses the different IC processes that evolved for inducing structural changes within highly disordered regions, underscoring the strategic role for protein disorder and IC isoforms in regulation of dynein function.