Mechanism of tubulin modification enzymes

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。它们受到高度多样化，丰富和进化保守的翻译后修饰。微管蛋白修饰水平和模式的破坏导致癌症、神经病变和轴突再生缺陷。破译微管蛋白密码的一个重要方面是了解密码是如何编写的，即，引入这些修饰的酶的机制，以及这些酶之间的合作和竞争如何引起细胞中观察到的复杂的微管修饰模式。具体来说，我们的目标是：(1)确定关键微管蛋白修饰酶的高分辨率结构，以了解它们的底物特异性和催化机制；(2)绘制所有修饰酶的微管蛋白修饰位点；(3)研究微管蛋白修饰酶之间的生化相互作用，以及这种相互作用如何引起时间和空间调节的修饰模式。该项目利用我们制造未经修饰和重组的单异构体工程人微管蛋白的能力，并结合我们的专业知识与一系列结构技术（x射线晶体学，低温电镜和SAXS），高分辨率质谱，经典动力学和单分子荧光将回答有关微管蛋白修饰酶的机制和调节的基本问题。我们继续朝着这些目标取得进展。具体来说，我们关注的是TTLL谷氨酰酶和糖基化酶，这是微管蛋白修饰酶的最大家族。谷氨酰化和糖基化涉及翻译后谷氨酸链在微管蛋白c端尾部的atp附加。它是人脑中最丰富的微管蛋白修饰。我们继续研究TTLL酶的基于机制的抑制剂，并通过结构分析对其进行表征。与我们的合作者一起，我们确定了几种基于机制的微管蛋白糖基化酶抑制剂。去年，我们研究了管状glyglylases TTLL3、8和10的底物特异性和调控机制，揭示了聚甘氨酸链长调控的机制。我们还开发了微管组合修饰的方法，现在用于发现这些修饰如何调节基于微管的马达和微管相关蛋白的活性。

Microtubules are polymers essential for cell morphogenesis, cell division and intracellular transport. They are subject to highly diverse, abundant and evolutionarily conserved posttranslational modifications. Disruption of tubulin modification levels and patterns leads to cancers, neuropathologies and defective axonal regeneration. An essential aspect of deciphering the tubulin code is to understand how the code is written i.e. the mechanism of the enzymes that introduce these modifications and how cooperation and competition between these enzymes gives rise to the complex microtubule modification patterns observed in cells. Specifically we aim (1) to determine high-resolution structures of key tubulin modification enzymes in isolation as well as in complex with the microtubule to understand their substrate specificity and catalytic mechanism; (2) to map tubulin modification sites for all modification enzymes; (3) to investigate the biochemical interplay between tubulin modification enzymes and how this gives rise to temporally and spatially regulated modification patterns. This project leverages our ability to make unmodified and recombinant single-isoform engineered human tubulin and coupled with our expertise with an array of structural techniques (X-ray crystallography, cryo-EM and SAXS), high-resolution mass spectrometry, classical kinetics and single molecule fluorescence will answer fundamental questions about the mechanism and regulation of tubulin modification enzymes. We have continued to make progress towards these goals. Specifically, we focused on TTLL glutamylases and glycylases, the largest family of tubulin modification enzymes. Glutamylation and glycylation involve the post translational ATP-addition of glutamate chains to the tubulin C-terminal tails. It is the most abundant tubulin modification in the human brain. We have continued our work on identifying mechanism-based inhibitors for TTLL enzymes and characterizing them through structural analysis. Together with our collaborators we identified several mechanism-based inhibitor for tubulin glycylases. In the last year we characterized the substrate specificity and regulation mechanism of tubul glycylases TTLL3, 8 and 10 and shed light on the mechanism of polyglycine chain length control. We we have also developed methods for the combinatorial modification of microtubules to be now used to discover how these modifications regulate the activity of microtubule based motors and microtubule associated proteins.