Mechanisms of molecular machines that regulate the neuronal cytoskeleton

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(Garnham和Roll-Mecak，2012)。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。我们正在积极地从生物物理量上纯化几种微管蛋白修饰酶以研究它们的作用机制。此外，我们在破译微管蛋白酪氨酸连接酶的生物物理作用机制方面也取得了重大进展。我的实验室确定了微管蛋白酪氨酸连接酶的第一个结构，该酶负责翻译后将酪氨酸添加到α-微管蛋白的C末端，作为微管蛋白去酪氨酸/酪氨酸化循环的一部分(Szyk等人，2011年)。α-微管蛋白中的C-末端酪氨酸是微管动力学调节因子招募的开/关信号。微管蛋白酪氨酸连接酶缺失与多种癌症的侵袭性肿瘤进展和转移有关，包括神经母细胞瘤、乳腺癌和前列腺癌。此外，微管蛋白酪氨酸连接酶与微管蛋白修饰酶家族中的一些酶同源，微管蛋白酪氨酸连接酶家族包括多谷氨酸酶和多甘氨酸酶，它们在微管运动调节中起重要作用，最近参与了神经退行性变。我们的微管蛋白酪氨酸连接酶的晶体结构揭示了微管蛋白酪氨酸连接酶支架是如何支持微管蛋白翻译后修饰酶库的扩展的，这些酶识别α-或β-微管蛋白尾巴，并解决了一个长期存在的难题，即为什么微管蛋白酪氨酸连接酶只修改单体微管蛋白，而不修改已经整合到微管中的微管蛋白。我们还发现，微管蛋白酪氨酸连接酶使用一种新的策略来防止微管蛋白掺入微管，方法是将微管蛋白二聚体封顶在原本参与微管晶格相互作用的界面上(Szyk等人，2011年)。这一发现开启了一种有趣的可能性，即在微管蛋白酪氨酸连接酶水平低的癌细胞中观察到的微管动力学和形态的变化(耐药肿瘤的标志)可能不仅是由于微管蛋白酪氨酸化下调，而且是由于聚合能力微管蛋白库的增加。 我们正在使用TTL和其他微管蛋白修饰酶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以及马达和微管结合蛋白募集的影响。

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 (Garnham and Roll-Mecak, 2012). 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. We are actively working on purifying to homogeneity and in biophysical quantities several tubulin modification enzymes to investigate their mechanism of action. In addition, we have made significant progress on deciphering the biophysical mechanism of action of tubulin tyrosine ligase. My laboratory determined the first structure of tubulin tyrosine ligase, the enzyme responsible for the post-translational addition of a tyrosine to the C-terminus of alpha-tubulin as part of the tubulin detyrosination/tyrosination cycle (Szyk et al., 2011). The C-terminal tyrosine in alpha-tubulin serves as an ON/OFF signal for the recruitment of microtubule dynamics regulators. Tubulin tyrosine ligase loss is associated with aggressive tumor progression and metastasis in several cancers, including neuroblastomas, breast and prostate cancers. Moreover, tubulin tyrosine ligase is homologous to enzymes that are part of the larger family of tubulin modifying enzymes, the tubulin tyrosine ligase-like family that includes polyglutamylases and polyglycylases that are important in microtubule motor regulation and have been recently involved in neurodegeneration. Our crystal structure of tubulin tyrosine ligase reveals how the tubulin tyrosine ligase scaffold supported the expansion of the repertory of tubulin post-translational modification enzymes that recognize either the alpha- or beta-tubulin tail and resolves a long-standing puzzle as to why tubulin tyrosine ligase modifies only monomeric tubulin and not tubulin that is already incorporated into microtubules. We also discovered that tubulin tyrosine ligase uses a novel strategy to prevent tubulin incorporation into microtubules by capping the tubulin dimer on an interface that would otherwise be involved in microtubule lattice interactions (Szyk et al., 2011). This finding opens the interesting possibility that changes in microtubule dynamics and morphology observed in cancer cells with low tubulin tyrosine ligase levels (a hallmark of drug resistant tumors) may be due not only to down-regulation of tubulin tyrosination, but also an increase in the polymerization competent tubulin pool. We are now using TTL and other tubulin modification enzymes to modify microtubules in vitro in order to investigate the effects of the introduced tubulin modification on microtubule dynamics and the recruitment of motors and microtubule association proteins.