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Readout of the tubulin code by cellular effectors

通过细胞效应器读出微管蛋白代码

基本信息

批准号：
10915999
负责人：
Antonina Roll-Mecak
金额：
$ 145.2万
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 post-translational modifications. Disruption of tubulin modification levels and patterns leads to cancers, neuropathologies and defective axonal regeneration. Our long-term goal is to understand how cells use tubulin isoform diversity and posttranslational modifications to regulate the structure and dynamics of microtubules as well as their interactions with molecular motors and microtubule associated proteins (MAPs). Although discovered over thirty years ago, an understanding of the roles of the chemical and genetic complexity of tubulin has remained elusive. My group integrates techniques and concepts from biophysics, proteomics, structural and cell biology to address this fundamental problem in microtubule cell biology. My laboratory has made significant progress towards these goals. These include: (1) development of novel methods for generating homogenous engineered single isoform recombinant unmodified human tubulin (Vemu et al., J. Biol. Chem., 2016); (2) determination of the first structure and dynamic instability parameters of recombinant isotopically pure recombinant neuronal tubulin (Vemu et al., J. Biol. Chem., 2016; Vemu et al. 2020); (3) demonstration that microtubules with different isoform compositions exhibit different dynamic properties and that these properties can be proportionally tuned by varying tubulin isoform composition (Vemu et al., Mol. Biol. Cell, 2017).(4) development of a biochemical platform for obtaining tubulin with quantitatively defined levels of posttranslational modifications (Valenstein and Roll-Mecak, Cell 2016) and use of this platform to (5) showing the graded response of an important microtubule regulator, the hereditary spastic paraplegia protein spastin, to tubulin glutamylation (Valenstein and Roll-Mecak, Cell 2016) thus furnishing strong support for the tubulin code hypothesis. Using our platform for generating quantitatively defined modified microtubules as well recombinant engineered human microtubules, we are currently investigating how the tubulin code, both through genetic variation and posttranslational modifications, regulates the basic biophysical properties of microtubules as well as molecular motors and neuronal MAPs with strong involvement in neurodegenerative disorders. Specifically, this year we elucidated how glutamylation, a modification long associated with microtubule stability in cells affects microtubule dynamics. Cell biologists from diverse fields use antibodies against this modification as a proxy for stable microtubules in cells. Using our recent advances in obtaining differentially glutamylated tubulin coupled with microtubule dynamics reconstitution we showed that, surprisingly, glutamylation acts as a negative regulator of microtubule growth (Chen and Roll-Mecak, 2023)and does not stabilize microtubules. Thus, the higher stability of glutamylated microtubules in cells must be due to trans effects. Our future efforts will focus on identifying the proteome that is recruited to glutamylated microtubules. Taken together, our most recent work on glutamylation, as well as our previous work on tyrosination, detyrosination and Delta2 tubulin established that none of the posttranslational modifications long associated with increased microtubule stability in cells, confer increased stability directly. Thus, these modifications function as signals to recruit effectors in a temporally and spatially controlled manner to the microtubule.

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。我们的长期目标是了解细胞如何使用微管蛋白异构体多样性和翻译后修饰来调节微管的结构和动力学，以及它们与分子马达和微管相关蛋白（MAP）的相互作用。虽然在三十多年前发现，但对微管蛋白的化学和遗传复杂性的作用的理解仍然难以捉摸。我的团队整合了生物物理学、蛋白质组学、结构和细胞生物学的技术和概念，以解决微管细胞生物学中的这一基本问题。我的实验室在实现这些目标方面取得了重大进展。这些包括：（1）开发用于产生同质工程化单一同种型重组未修饰人微管蛋白的新方法（Vemu et al.，J. Biol. Chem.，2016年）;（2）重组同位素纯的重组神经元微管蛋白的第一结构和动态不稳定性参数的测定（Vemu等，J. Biol. Chem.，二〇一六年; Vemu等人，2020）;（3）证明具有不同同种型组成的微管表现出不同的动态特性，并且这些特性可以通过改变微管蛋白同种型组成来成比例地调节（Vemu等人，摩尔Cell，2017）。(4)开发用于获得具有定量限定水平的翻译后修饰的微管蛋白的生物化学平台（Valenstein和Roll-Mecak，Cell 2016）和使用该平台来（5）显示重要的微管调节剂，遗传性痉挛性截瘫蛋白spastin，对微管蛋白谷氨酰化的分级反应（Valenstein和Roll-Mecak，Cell 2016），从而为微管蛋白编码假说提供了强有力的支持。使用我们的平台，用于产生定量定义的修饰微管以及重组工程化的人微管，我们目前正在研究如何微管蛋白代码，通过遗传变异和翻译后修饰，调节微管的基本生物物理特性以及分子马达和神经元MAP与神经退行性疾病的强烈参与。具体来说，今年我们阐明了谷氨酰化，一种长期与细胞中微管稳定性相关的修饰如何影响微管动力学。来自不同领域的细胞生物学家使用针对这种修饰的抗体作为细胞中稳定微管的代表。利用我们最近在获得差异谷氨酰化微管蛋白与微管动力学重建结合方面的进展，我们发现，令人惊讶的是，谷氨酰化作为微管生长的负调节剂（Chen和Roll-Mecak，2023），并且不稳定微管。因此，细胞中谷氨酰化微管的更高稳定性必须归因于反式效应。我们未来的工作将集中在确定蛋白质组，招募到谷氨酰化微管。综上所述，我们最近对谷氨酰化的研究，以及我们之前对酪氨酸化、去酪氨酸化和Delta 2微管蛋白的研究表明，长期以来与细胞中微管稳定性增加相关的翻译后修饰都没有直接增加稳定性。因此，这些修饰作为信号以时间和空间控制的方式将效应物募集到微管。