Mechanisms of molecular machines that regulate the neuronal cytoskeleton

调节神经元细胞骨架的分子机器机制

• 批准号: 9157559
• 负责人: Antonina Roll-Mecak
• 金额: $ 149.73万
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9157559
• 关键词: Affect; Alzheimer' s Disease; Amyotrophic Lateral Sclerosis; Architecture; Area; Bacterial Infections; Behavior; Binding; Biochemical; Biomechanics; Biophysics; C-terminal; Cardiovascular Diseases; Cell Nucleus; Cell division; Cell physiology; Cells; Cellular biology; Code; Complex; Cryoelectron Microscopy; Cues; Cytoskeleton; Development; Disease; Dissection; Ensure; Enzymes; Etiology; Eukaryotic Cell; Excision; Family; Fluorescence; Focus Groups; Generations; Genes; Geometry; Goals; Growth; Human; Hybrids; In Vitro; Individual; Intracellular Transport; Malignant Neoplasms; Mass Spectrum Analysis; Mechanics; Methods; Microtubule-Associated Proteins; Microtubules; Modification; Molecular Biology; Molecular Machines; Morphogenesis; Motor; Movement; Mutation; Mycoses; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Parkinson Disease; Play; Polymers; Post-Translational Protein Processing; Preparation; Property; Protein Isoforms; Radial; Recombinants; Recruitment Activity; Research; Role; Site; Structure; Tail; Techniques; Time; Tubulin; Virus Diseases; Work; X-Ray Crystallography; alpha Tubulin; beta Tubulin; human disease; in vitro Assay; katanin; novel; operation; programs; response; single molecule; spastin; structural biology; three dimensional structure; tyrosyltubulin ligase

Microtubules are polymers essential for cell morphogenesis, cell division and intracellular transport. Microtubules execute their diverse cellular roles by forming suprastructures with highly distinctive geometries: the radial cytoplasmic array, the short, highly parallel axonemal array, the spindle array or the tiled long axonal array. The microtubule cytoskeleton is a complex function of many unit operations, the individual actions of cytoskeletal regulators: nucleation, growth and shrinkage, severing and motor movement. Moreover, the microtubule itself is more than just a naive roadway for cellular components to transit along. Alpha and beta tubulins have multiple isoforms and are subject to highly diverse, abundant and evolutionarily conserved post-translational modifications that mark subpopulations of microtubules (Yu et al, 2015). Given the central role microtubules play in basic cellular processes, it is not surprising that microtubule regulators have been implicated in many human diseases, including cancers, cardiovascular disease, fungal, bacterial and viral infections, as well as neurodegenerative disorders such as Parkinson's, Alzheimer's and Amyotrophic lateral sclerosis. Our efforts concentrate on two families of microtubule regulators: microtubule severing enzymes and enzymes that post-translationally modify tubulin. Our research plan is highly interdisciplinary, integrating techniques and concepts from biophysics, structural, molecular and cell biology to answer two closely interdigitated questions: how is the structure of the microtubule locally perturbed when it is engaged by these regulators and how do these regulators affect microtubule architecture and dynamics at the cellular level? Perturbation of microtubule dynamics has emerged as a common theme in a variety of neurodegenerative diseases and our work has implications for the etiologies of all these disorders. In the last year we initiated several studies aimed at understanding the mechanistic underpinnings of the functions of microtubule post-translational modifications as well as continued our work on the mechanism of microtubule severing by spastin. Recent work from my group focused on the mechanism of action of the neuronal tubulin glutamylase TTLL7 (Garnham et al., 2015). Using a hybrid approach combining X-ray crystallography, cryo-electron microscopy, mass spectrometry and single molecule fluorescence we were able to visualize for the first time how a glutamylase of the TTLL superfamily of tubulin modification enzymes recognizes the microtubule and discriminates between soluble and polymeric tubulin as well as between α- and β-tubulin, ensuring preferential glutamylation of the β-tubulin tail. The structure of the TTLL7 bound to the microtubule also allowed us to visualize for the first time the elusive C-terminal tails of α- and β-tubulin which are both engaged by TTLL7 as part of a tripartite microtubule recognition strategy that involves also a cationic microtubule binding domain that we found is universal in all tubulin glutamylases with autonomous activity. Removal of this domain in all glutamylases with autonomous activity reduces their activity to background level. My group has also been responsible in the last year for the development of novel methods for generating recombinant human tubulin as well as homogenous acetylated, glutamylated, or tyrosinated human tubulin and microtubules for in vitro assays (Vemu et al., 2014). The generation of differentially modified microtubules now enables a mechanistic dissection of the effects of tubulin post-translational modifications on the dynamics and mechanical properties of microtubules as well as the recruitment and behavior of motors and microtubule-associated proteins. Lastly, we are actively working on purifying to homogeneity and in biophysical quantities several other tubulin modification enzymes, both glutamylases and glycylases, to investigate their mechanism of action. We are currently also using these enzyme preparations to modify microtubules in vitro in order to investigate the effects of the introduced tubulin modification on microtubule dynamics and the recruitment and activity of motors and the microtubule severing enzymes spastin and katanin.

微管是细胞形态发生、细胞分裂和细胞内运输所必需的聚合物。微管通过形成具有非常独特的几何形状的超结构来执行其不同的细胞角色：放射状细胞质阵列、短的、高度平行的轴丝阵列、纺锤体阵列或平铺的长轴突阵列。微管细胞骨架是许多单位操作的复杂功能，是细胞骨架调节因子的个体作用：成核、生长和收缩、断裂和运动。此外，微管本身不仅仅是细胞成分传递的幼稚道路。α微管蛋白和β微管蛋白具有多种异构体，并受到高度多样化、丰富和进化保守的翻译后修饰的影响，这些修饰标志着微管亚群(Yu等人，2015)。鉴于微管在基本细胞过程中发挥的核心作用，微管调节器与许多人类疾病有关也就不足为奇了，这些疾病包括癌症、心血管疾病、真菌、细菌和病毒感染，以及帕金森氏症、阿尔茨海默氏症和肌萎缩侧索硬化症等神经退行性疾病。 我们的工作集中在微管调节的两个家族：微管切断酶和翻译后修饰微管蛋白的酶。我们的研究计划是高度跨学科的，整合了生物物理学、结构生物学、分子生物学和细胞生物学的技术和概念，以回答两个紧密交错的问题：当微管结构被这些调节器参与时，微管的结构是如何局部扰动的，以及这些调节器如何在细胞水平上影响微管的结构和动力学？微管动力学的扰动已经成为各种神经退行性疾病的共同主题，我们的工作对所有这些疾病的病因都有影响。 在过去的一年里，我们启动了几项旨在了解微管翻译后修饰功能的机制基础的研究，并继续我们对痉挛蛋白切断微管的机制的研究。 我的小组最近的工作重点是神经元微管蛋白谷氨酰胺酶TTLL7的作用机制(Garnham等人，2015年)。使用结合X射线结晶学、冷冻电子显微镜、质谱学和单分子荧光的混合方法，我们首次能够可视化TTLL超家族微管蛋白修饰酶的谷氨酸酶如何识别微管并区分可溶性和聚合微管蛋白以及-微管蛋白和-微管蛋白，确保-微管蛋白尾部的优先谷氨酰化。TTLL7结合到微管的结构也使我们第一次能够看到难以捉摸的微管蛋白的C末端尾巴，这两个尾巴都被TTLL7作为三方微管识别策略的一部分，该策略还包括一个阳离子微管结合域，我们发现它在所有具有自主活性的微管蛋白谷氨酰胺酶中都是普遍存在的。去除所有具有自主活性的谷氨酰胺酶中的这个结构域，使它们的活性降低到本底水平。 去年，我的团队还负责开发新的方法来产生重组人微管蛋白，以及用于体外分析的同质乙酰化、谷氨酸化或酪氨酸化的人微管蛋白和微管(Vemu等人，2014年)。现在，差异修饰微管的产生使得对微管蛋白翻译后修饰对微管的动力学和机械性能的影响以及对发动机和微管相关蛋白的招募和行为的机械剖析成为可能。 最后，我们正在积极地进行纯化以获得均一性，并在生物物理量上纯化其他几种微管蛋白修饰酶，包括谷氨酸酶和甘氨酸酶，以研究它们的作用机制。我们目前还在使用这些酶制剂对微管进行体外修饰，以研究引入的微管蛋白修饰对微管动力学、运动和微管切断酶spastin和katanin的招募和活性的影响。