Dynein light chain as a dimerization hub for natively disordered proteins

描述（由申请人提供）：动力蛋白轻链LC8是一个高度保守的，基于微管的分子马达动力蛋白的重要组成部分。它与许多非动力蛋白的相互作用导致人们普遍认为它是一种货物适配器。然而，最近的结构和热力学研究表明，LC8不能作为货物适配器，并导致了范式转换命题，即LC8主要不是动力蛋白亚基，而是在各种细胞系统中发挥作用的多种蛋白质复合物的重要组成部分。这一提议的深远和统一的假设是LC8是一个枢纽蛋白，在动力蛋白和所有其他参与的复合体中作为二聚化促进者，促进与之相互作用的蛋白质的二聚化和排序。本提案的工作将阐明与LC8相互作用的两种最著名的蛋白质的结合热力学和结构表征。目的1侧重于LC8与动力蛋白组装中间链（IC）的相互作用，目的2侧重于LC8与Swallow的相互作用，Swallow是果蝇发育过程中参与bicoid mRNA定位的蛋白质，也是LC8与动力蛋白无关的相互作用蛋白中研究得最好的例子。为了这两个目的，lc8促进伴侣二聚化的机制和生物学意义将通过代表性蛋白质片段的体外生物物理研究和全长构建，以及表达IC和Swallow设计变体的细胞和生物体的体内表型评估来检验。这些研究将为了解LC8在其参与的各种细胞系统中所起的作用提供坚实的基础，并且由于其他LC8结合伙伴在生理上很重要，因此也将促进LC8领域的总体进展。这些研究也具有更广泛的适用性，因为LC8可以作为与大量紊乱伴侣相互作用的其他枢纽蛋白的模型。此外，这些研究将为动力蛋白运动功能的结构生物学提供新的见解，补充了主导细胞内运输领域的细胞生物学方法。Barbar博士在LC8结构-功能关系方面的开创性工作记录，她作为LC8是天然无序蛋白质二聚化中心的新假设的开发人员的角色，她在生产有用的蛋白质结构和处理这些复杂和部分无序蛋白质方面的成功，以及她在探索这些问题所需的生物物理和生化解决方案技术方面的专业知识，使她非常适合领导这项工作。公共卫生相关性：在蛋白质-蛋白质相互作用网络中，被称为“枢纽”的一小部分蛋白质与许多不同的伙伴相互作用。这些研究将阐明动力蛋白轻链LC8的相互作用机制，以及其作为二聚化引擎在微管运动复合物动力蛋白的组装和调节中的作用，以及参与各种生理过程的非动力蛋白复合物的组装。

DESCRIPTION (provided by applicant): Dynein light chain LC8 is a highly conserved, essential component of the microtubule-based molecular motor dynein. Its interactions with a number of non-dynein proteins led to the widely held view that it functions as a cargo adaptor. However, structural and thermodynamics work has recently shown that LC8 cannot serve as a cargo adaptor and has led to the paradigm-shifting proposition that LC8 is not primarily a dynein subunit, but is an essential component of diverse protein complexes that play roles in a variety of cellular systems. The far-reaching and unifying hypothesis of this proposal is that LC8 is a hub protein and functions as a dimerization facilitator in dynein and in all other complexes in which it participates, promoting the dimerization and ordering of the proteins with which it interacts. Work in this proposal will elucidate binding thermodynamics and structural characterization of the two best-known proteins that interact with LC8. Aim 1 focuses on the interactions of LC8 with the intermediate chain (IC) in dynein assembly and aim 2 focuses on the interactions of LC8 with Swallow, a protein involved in localization of bicoid mRNA during Drosophila development, and the best-studied example of an LC8-interacting protein not associated with dynein. For both aims the mechanism and biological significance of LC8-promoted partner dimerization will be examined by both in vitro biophysical studies of representative protein segments and, when possible, full-length constructs, and in vivo assessment of phenotypes of cells and organisms expressing designed variants of IC and Swallow. These studies will provide a solid foundation for understanding the roles that LC8 plays in the variety of cellular systems in which it participates, and will also promote progress in the LC8 field in general as other LC8 binding partners are physiologically important. These studies also have broader applicability, because LC8 can serve as a model for other hub proteins that interact with a large number of disordered partners. In addition these studies will provide novel insights into the structural biology of dynein motor function, complementing the cell biological approaches that predominate the field of intracellular transport. Dr. Barbar's track record of pioneering work on structure-function relations of LC8, her role as developer of the novel hypothesis that LC8 is a dimerization hub for natively disordered proteins, her success in both producing useful protein constructs and handling these complex and partially disordered proteins, and her expertise in the battery of biophysical and biochemical solution techniques required to probe these questions, make her uniquely suited to lead this effort. PUBLIC HEALTH RELEVANCE: In protein-protein interaction networks, a small proportion of proteins termed "hubs" interact with many diverse partners. These studies will elucidate the mechanism of interaction of dynein light chain LC8, which we propose is a hub protein, and its role as a dimerization engine in the assembly and regulation of the microtubule-based motor complex dynein, and in assembly of non-dynein complexes that are involved in various physiological processes.