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物系统使用生物马达沿着称为微管的火车样轨道以特定方向将材料运输到细胞中。动力蛋白就是这样一种马达，它对许多细胞过程至关重要，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和神经元的迁移。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。这些相互作用是通过动力蛋白区域中蛋白质序列的修饰来调节的，这些区域缺乏规则的独特三维结构，被称为内在无序蛋白。预期结果将具体显示这些修改如何帮助选择绑定伙伴，以及随后运输什么货物。了解这个系统将最终成为设计合成纳米机器的基础，这种机器可以按需运输材料。这项工作将有助于培养本科生和研究生从事前沿研究的实践。此外，PI和她的团队将通过提供生物物理仪器，发起和组织年度核磁共振研讨会和年度为期一天的区域生物物理会议，影响俄勒冈州立大学和该地区其他学科的进展。并通过在K-12地区的学校实施不同复杂性的动手演示和科学活动，包括在俄勒冈科学博物馆进行大规模演示的蛋白质紊乱和结构变化的模型/动画。本研究探讨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，以及裸蛋白，一种核分布蛋白，在包括着丝点和中心体迁移在内的多种过程中必不可少。动力蛋白中间链（IC）内在无序的n端300个氨基酸结构域负责这些相互作用，也是大多数动力蛋白活性的中心。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控的关键，但尚未有关于其作用的分子机制的研究。本项目将重点关注：1)IC磷酸化如何影响IC与dynactin组织特异性异构体的相互作用，2)IC磷酸化如何在dynactin和NudE调节蛋白之间进行选择，以及3)哺乳动物、果蝇和酵母之间IC调节的差异。这些关于动力蛋白调控的突出问题将使用最先进的核磁共振、等温滴定量热法、合成生物学、计算方法和基于体内的分析相结合来解决，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，利用酵母、果蝇和哺乳动物来源的蛋白质，评估了在高度紊乱区域内诱导结构变化的不同IC过程，强调了蛋白质紊乱和IC异构体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.