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Mechanism of tubulin modification enzymes

微管蛋白修饰酶的机制

基本信息

批准号：
10915998
负责人：
Antonina Roll-Mecak
金额：
$ 136.91万
依托单位：
NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

项目摘要

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.

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。它们具有高度多样性、丰富性和进化上保守的翻译后修饰。微管蛋白修饰水平和模式的破坏导致癌症、神经病理学和有缺陷的轴突再生。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产生细胞中观察到的复杂微管修饰模式。具体来说，我们的目标是（1）确定关键微管蛋白修饰酶的分离以及与微管复合的高分辨率结构，以了解其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;（三）研究微管蛋白修饰酶之间的生物化学相互作用，以及这如何引起时间和空间调节的修饰模式。该项目利用我们的能力，使未修饰的和重组的单一亚型工程人微管蛋白，再加上我们的专业知识与一系列的结构技术（X射线晶体学，cryo-EM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。我们继续在实现这些目标方面取得进展。具体而言，我们专注于TTLL谷氨酰化酶和甘氨酰化酶，微管蛋白修饰酶的最大家族。谷氨酰化和甘氨酰化涉及翻译后ATP-添加谷氨酸链到微管蛋白C-末端尾部。它是人类大脑中最丰富的微管蛋白修饰。我们继续致力于识别TTLL酶的基于机制的抑制剂，并通过结构分析对其进行表征。我们与我们的合作者一起确定了几种基于机制的微管糖基化酶抑制剂。在过去的一年中，我们的特点是底物特异性和调控机制的小管甘氨酰化酶TTLL 3，8和10和阐明的机制，聚甘氨酸链长控制。我们还开发了微管的组合修饰方法，现在用于发现这些修饰如何调节微管马达和微管相关蛋白的活性。