Dynein light chain as a dimerization hub for natively disordered proteins

动力蛋白轻链作为天然无序蛋白质的二聚化中心

• 批准号: 8137075
• 负责人: ELISAR J BARBAR
• 金额: $ 34.23万
• 依托单位: OREGON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8137075
• 关键词: Affect; Apoptosis; Binding; Binding Sites; Biochemical; Biological; C-terminal; Calorimetry; Cell physiology; Cells; Circular Dichroism; Complement; Complex; Cytoskeleton; Deglutition; Development; Dimerization; Disease; Drosophila genus; Dynein ATPase; Enzymes; Fluorescence Resonance Energy Transfer; Fluorescence Spectroscopy; Foundations; Health; Homeostasis; In Vitro; Intracellular Transport; Lead; Length; Light; Link; Masks; Messenger RNA; Microtubules; Modeling; Molecular; Molecular Motors; Motor; Multiple Partners; Mutation; N-terminal; Nurses; Organism; Phenotype; Physiological Processes; Play; Property; Proteins; Proteolysis; Publishing; Regulation; Role; Solid; Solutions; Stretching; Structure; System; Techniques; Testing; Thermodynamics; Titrations; Variant; Viral Pathogenesis; WD Repeat; Work; X-Ray Crystallography; base; design; dimer; dynein light chain; in vivo; insight; interest; mutant; nephrogenesis; novel; protein complex; protein function; protein protein interaction; research study; structural biology; success

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.

描述（由申请人提供）：动力蛋白轻链 LC8 是基于微管的分子运动动力蛋白的高度保守的重要组成部分。它与许多非动力蛋白的相互作用导致人们普遍认为它起着货物适配器的作用。然而，结构和热力学工作最近表明，LC8 不能充当货物适配器，并导致了范式转变的命题，即 LC8 主要不是动力蛋白亚基，而是在多种蛋白质复合物中发挥作用的重要组成部分。蜂窝系统。该提议的影响深远且统一的假设是，LC8 是一种枢纽蛋白，在动力蛋白及其参与的所有其他复合物中充当二聚化促进剂，促进与其相互作用的蛋白质的二聚化和排序。该提案中的工作将阐明与 LC8 相互作用的两种最著名的蛋白质的结合热力学和结构特征。目标 1 重点关注 LC8 与动力蛋白组装中中间链 (IC) 的相互作用，目标 2 重点关注 LC8 与 Swallow 的相互作用，Swallow 是一种参与果蝇发育过程中 bicoid mRNA 定位的蛋白质，也是LC8 相互作用蛋白与动力蛋白不相关。对于这两个目标，LC8 促进的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和（如果可能的话）全长构建体的体外生物物理学研究以及表达设计变体的细胞和生物体的表型的体内评估来检查IC 和燕子。这些研究将为理解 LC8 在其参与的各种细胞系统中发挥的作用提供坚实的基础，并且还将促进 LC8 领域的总体进展，因为其他 LC8 结合伴侣在生理上很重要。这些研究还具有更广泛的适用性，因为 LC8 可以作为与大量紊乱伙伴相互作用的其他中心蛋白的模型。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，补充主导细胞内运输领域的细胞生物学方法。 Barbar 博士在 LC8 结构与功能关系方面的开创性工作记录，她作为新假设的开发者的角色，即 LC8 是天然无序蛋白质的二聚中心，她在生产有用的蛋白质结构和处理这些复杂和部分无序的方面取得了成功蛋白质，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电池方面的专业知识，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，一小部分被称为“中心”的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（我们认为它是一种枢纽蛋白），以及它在基于微管的运动复合体动力蛋白的组装和调节以及非动力蛋白的组装中作为二聚引擎的作用。参与各种生理过程的复合物。