Molecular dissection of the C-terminal tails of tubulin and the effect of their polyglycylation on binding and microtubule assembly

微管蛋白 C 末端尾部的分子解剖及其多糖基化对结合和微管组装的影响

• 批准号: 9142795
• 负责人: LOREN E HOUGH
• 金额: $ 35.84万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9142795
• 关键词: Affect; Behavior; Binding; Binding Proteins; Biological Assay; C-terminal; CLIP-170 gene; Cell Proliferation; Cell division; Cells; Cellular biology; Charge; Cilia; Crystallography; Disease; Dissection; Electron Microscopy; Environment; Excision; Glutamates; Glycine; Isotope Labeling; Isotopes; Label; Length; Malignant Neoplasms; Mechanics; Methods; Microtubule Polymerization; Microtubules; Molecular; Molecular Probes; Motor; Mutation; Poly G; Polymers; Principal Investigator; Property; Protein Family; Proteins; Reading; Regulation; Role; Side; Site; System; Tail; Techniques; Testing; Text; Tubulin; Work; cell motility; dimer; insight; overexpression; polymerization; programs; simulation; tau Proteins

Principal Investigator/Program Director(Last, First, Middle): Hough, Loren E. Microtubules (MTs) made from ↵- tubulin heterodimers are important for cell migration, long range transport, and cell division. A primary site of tubulin regulation is the C-terminal tails (CTTs). Major questions remain about the molecular mechanism of CTT function and regulation. CTTs affect microtubule length dynamics and mechanical properties even though they contribute only a small percentage of the binding interface between adjacent dimers. CTTs are a major site of tubulin post-translational modification (PTM) which regulates tubulin's binding interactions. PTM of the CTTs alters the processivity of motor proteins1, 2 and the affinity of proteins which affect MT stability (e.g. MCAK, CLIP-170)3, 4 and proteins which stabilize MTs (e.g. tau).5 Despite their importance, there are few molecular probes of CTT behavior. Because CTTs are flexible, they are typically undetectable in electron microscopy or x-ray crystallography studies. NMR is the best approach for determining the molecular mechanism of CTT regulation of MT polymerization, mechanics, binding and regulation by PTMs. However, standard techniques for incorporation of heavy isotopes have not worked for tubulin—the proteins have not yet been made in prokaryotic systems, and tubulin typically down regulates its own expression, making over-expression difficult in eukaryotic systems. We developed a method to produce heavy isotope labeled tubulin for study by NMR, allowing for a detailed study of the molecular mechanism of CTT function. Building on this breakthrough, we will study the role of CTT polyglycylation in MT regulation. The addition of glycine residues to glutamate side chains occurs on both tubulin CTTs. First identified as one of the two major poly-modifications on tubulin, polyglycylation is associated with particularly stable MTs, especially axonemes in mobile cilia.6 Mutations that decrease polyglycylation are associated with reduced stability of cilia, reduced numbers of cilia, increases in cell proliferation, and cancer. However, it is not known why polyglycylation causes these effects. PTMs can operate through a variety of mechanisms: modification of the overall charge distribution,7 alteration of binding interfaces,8 induction of structural changes through allosteric mechanisms,9 and modulation of disordered protein ensembles.10 We will combine NMR, binding assays, molecular simulation, and cell biology to test potential mechanisms of CTT function. We will study two focused questions: 1. How does polyglycylation affect MT polymerization dynamics and stiffness? The presence of the CTT affects MT polymerization dynamics and mechanical properties, suggesting that the CTTs of one dimer interact with other dimers in the MT lattice.11,12 We will purify heavy isotope-labeled tubulin with varying degrees of polyglycylation using mutations in TTLL3-family proteins and mutations in the tubulin CTT at polyglycylation sites. We will use MT assembly assays to determine the effect of polyglycylation on MT nucleation and polymerization dynamics. We will study the effects of polyglycylation on CTT properties by NMR for both dimers and polymer- ized MTs. In conjunction with molecular simulations, we will determine and refine hypotheses for the molecular mechanisms by which CTT polyglycylation affects MT properties. We will test these hypotheses through mutation. 2. How does polyglycylation affect MT binding interactions? Removal of the CTTs alters the binding dy- namics of MT-stabilizing and destabilizing binding partners, suggesting that CTT PTM may regulate MTs through effects on MT-interacting proteins. However, little structural information is available on how MT binding partners recognize the CTTs, even those whose interaction is PTM dependent. For example, PTM of the CTTs alters the processivity of motor proteins1, 2 and the affinity of proteins which affect MT stability (e.g. MCAK, CLIP-170).3, 4 We will determine how the binding affinity of MT-binding proteins varies with the degree of CTT polyglycyla- tion. By NMR, we will determine the binding interface between the CTT and MT-binding protein, and determine whether and how the binding interface changes when the extent of polyglycylation is altered. We will determine the residues most affected in environment upon binding. This work will allow us to generate and test hypotheses about the molecular mechanism by which poly-G tails affect MT-binding properties. Answering these questions will give unique insight into how tubulin CTT post-translational modification regulates MTs. We are uniquely poised to do this work because we have a direct read-out of CTT average environment and binding interface through the use of NMR. This project will be a detailed characterization of the mechanisms of cellular MT regulation by tubulin CTT polyglycylation and provide a framework for dissecting cellular protein regulation through PTM of disordered domains. Beyond tubulin specifically, the T. thermophila heavy labeling, overexpression, and secretion systems will allow our approach to enable NMR structural studies of a wide variety of eukaryotic proteins. Program (Date) Page 0 Proposal Text

主要研究者/项目负责人（最后，第一，中间）：Hough，Loren E。 由β-微管蛋白异源二聚体组成的微管（MT）对于细胞迁移、长距离运输， 和细胞分裂。微管蛋白调节的主要位点是C-末端尾（CTT）。主要问题仍然存在 关于CTT功能和调节的分子机制。CTTs影响微管长度动力学， 机械性能，即使它们只占粘合界面的一小部分， 相邻二聚体。CTTs是微管蛋白翻译后修饰（PTM）的主要位点，其调节微管蛋白的 结合相互作用。CTTs的PTM改变了运动蛋白的持续合成能力1，2和蛋白质的亲和力， 影响MT稳定性（例如MCAK、CLIP-170）3、4和稳定MT的蛋白质（例如tau）。 尽管它们的重要性，有几个CTT行为的分子探针。因为CTT是灵活的，他们 通常在电子显微镜或X射线晶体学研究中检测不到。核磁共振是最好的方法， 确定了CTT调控MT的分子机制，包括MT的聚合、力学、结合和调控 通过PTM。然而，用于掺入重同位素的标准技术对微管蛋白不起作用， 蛋白质尚未在原核系统中制备，并且微管蛋白通常下调其自身的表达， 使得在真核系统中过表达困难。我们开发了一种方法， 微管蛋白的NMR研究，允许CTT功能的分子机制的详细研究。 基于这一突破，我们将研究CTT多聚甘氨酰化在MT调控中的作用。添加 甘氨酸残基与谷氨酸侧链的连接发生在两种微管蛋白CTT上。首先被艾德为两大 微管蛋白的多聚修饰，多聚甘氨酰化与特别稳定的MT，特别是轴丝相关 在移动的纤毛中。6减少多聚甘氨酰化的突变与纤毛稳定性降低， 纤毛的数量，细胞增殖的增加和癌症。然而，尚不清楚为什么多聚甘氨酰化会导致 这些影响。PTM可以通过多种机制进行操作：修改整体电荷分布，7 改变结合界面，8通过变构机制诱导结构变化，9和调节 10我们将结合联合收割机核磁共振、结合分析、分子模拟和细胞生物学 以测试CTT功能的潜在机制。我们将重点研究两个问题： 1.聚甘氨酰化如何影响MT聚合动力学和刚度？CTT的存在 影响MT聚合动力学和机械性能，表明一个二聚体的CTTs相互作用 11，12我们将纯化重同位素标记的微管蛋白， 使用TTLL 3家族蛋白中的突变和微管蛋白CTT中的突变在多聚甘氨酰化位点处进行多聚甘氨酰化。 我们将使用MT组装测定来确定多聚甘氨酰化对MT成核和聚合的影响。 动力学我们将通过NMR研究聚甘氨酰化对二聚体和聚合物的CTT性质的影响。 化MT。结合分子模拟，我们将确定和完善分子的假设， CTT多聚甘氨酰化影响MT性质的机制。我们将通过突变来检验这些假设。 2.多聚甘氨酰化如何影响MT结合相互作用？去除CTT改变了结合方式， MT稳定和不稳定结合伴侣的动力学，表明CTT PTM可能通过以下途径调节MT： 对MT相互作用蛋白的影响。然而，很少有结构信息是如何MT结合伙伴， 识别CTT，即使是那些相互作用依赖于PTM的CTT。例如，CTT的PTM改变了 运动蛋白的持续合成能力1，2和影响MT稳定性的蛋白质的亲和力（例如MCAK，CLIP-170）。3，4 我们将确定MT结合蛋白的结合亲和力如何随CTT多聚甘氨酰化程度而变化。 是的。通过NMR，我们将确定CTT和MT结合蛋白之间的结合界面，并确定 当多聚甘氨酰化程度改变时，结合界面是否以及如何改变。我们将确定 在结合后对环境影响最大的残留物。这项工作将使我们能够产生和测试假设 关于聚G尾影响MT结合特性的分子机制。 对这些问题的探讨将使我们对微管蛋白CTT的翻译后修饰有一个独特的认识。 规范MT。我们为开展这项工作做好了独特的准备，因为我们可以直接读出CTT平均值 环境和结合界面通过使用NMR。该项目将详细描述 微管蛋白CTT多聚糖基化调节细胞MT的机制，并提供了一个框架，解剖 通过无序结构域的PTM调节细胞蛋白质。除微管蛋白外，T.热毁 重标记，过表达和分泌系统将使我们的方法，使核磁共振结构研究 真核生物蛋白质的合成 计划（日期）第0页提案文本