Structural Basis of Phosphorylation and Alternative Splicing in Dynein Regulation

标题：动力蛋白调节中磷酸化和选择性剪接的结构基础生命系统利用生物马达将物质沿沿着火车状轨道（称为微管）以特定方向运送到细胞中。动力蛋白是许多细胞过程所必需的，包括细胞分裂期间染色体的运输、含有营养物质的囊泡的运动以及神经元迁移。动力蛋白如何知道何时以及运输何种货物需要与控制动力蛋白功能的其他蛋白质相互作用。这些相互作用通过动力蛋白区域中的蛋白质序列的修饰来调节，所述动力蛋白区域缺乏规则的独特三维结构，并且被称为固有无序蛋白。预期的结果将具体显示这些修饰如何帮助选择结合伴侣，以及随后运输什么货物。了解这个系统将最终成为设计可以按需运输材料的合成纳米机器的基础。这项工作将有助于培养本科生和研究生从事尖端研究。此外，PI和她的团队将通过提供生物物理仪器，发起和组织年度NMR研讨会和为期一天的年度区域会议，并通过在K-12地区学校实施各种复杂性的动手示范和科学活动，在俄勒冈州科学博物馆的大规模演示中，蛋白质紊乱和结构变化的动画。这项研究讨论了动力蛋白一个亚基的磷酸化和内在紊乱如何调节动力蛋白与多种调节蛋白的相互作用，包括动力蛋白，其相互作用发生在所有真核生物中，在神经元中起着特别重要的作用，和NudE，一种在包括动粒和中心体迁移在内的多种过程中必不可少的核分布蛋白。动力蛋白中间链（IC）的N-末端300个氨基酸的结构域是这些相互作用的原因，也是大多数动力蛋白活性的中心。虽然有足够的证据表明磷酸化和同工型表达是动力蛋白调节的关键，但还没有研究其作用的分子机制。该项目将特别关注：1）IC磷酸化如何影响IC与dynactin的组织特异性亚型的相互作用，2）IC磷酸化如何在调节蛋白dynactin和NudE之间进行选择，以及3）IC调节如何在哺乳动物，果蝇和酵母之间存在差异。这些悬而未决的问题，动力蛋白调节将解决使用国家的最先进的核磁共振，等温滴定量热法，合成生物学，计算方法的组合，并在体内为基础的测定，并将阐明如何磷酸化和选择性剪接的内在无序蛋白质一般管理其分子功能，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，评估了不同的IC过程，演变为诱导高度无序的区域内的结构变化，强调了蛋白质紊乱和IC亚型在调节动力蛋白功能中的战略作用。

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.