Exploring the functional role of tubulin methylation and its regulation by mes-4/NSD in C. elegans

探索秀丽隐杆线虫中微管蛋白甲基化的功能作用及其 mes-4/NSD 的调节

• 批准号: 10752333
• 负责人: Edward Pietryk
• 金额: $ 4.77万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10752333
• 关键词: Address; Alleles; Animals; Architecture; Area; Behavioral; Binding Proteins; Biochemical; Biological Assay; Biology; Caenorhabditis elegans; Cells; Chromatin; Code; Complement; Cytoskeleton; Data; Defect; Disease; Enzymes; Epigenetic Process; Family; Functional disorder; Histone Code; Histones; Human; Hyperactivity; Image; Immunohistochemistry; In Vitro; Individual; Knock-in; Knowledge; Label; Link; Lysine; Malignant Neoplasms; Mass Spectrum Analysis; Methylation; Methyltransferase; Microscopy; Microtubules; Mitosis; Modeling; Modification; Morphology; Mutation; Neurodevelopmental Disorder; Neurons; Organism; Phenotype; Plus End of the Microtubule; Polymers; Post-Translational Protein Processing; Protein Isoforms; Regulation; Research; Resolution; Role; Site; Structure; System; Techniques; Testing; Time; Tissues; Touch sensation; Tubulin; Variant; Western Blotting; Writing; alpha Tubulin; autism spectrum disorder; catalyst; dimer; gain of function; histone methylation; histone methyltransferase; in vivo; loss of function; model organism; mutant; polymerization; response; tool

ABSTRACT The microtubule cytoskeleton serves many critical functions in the cell, and its dysfunction is linked to a plethora of diseases from cancer to neurodevelopmental disorders such as autism. Post-translational modifications (PTM) of the tubulin subunits of microtubules are key regulators of both structure and function of microtubules. Similar to the “Histone Code”, the “Tubulin Code” hypothesis posits microtubule function is tuned through the incorporation of specific tubulin isoforms and PTMs. Methylation is well known as a common PTM on histones, however, the function of tubulin methylation and the enzymatic machinery that “reads, writes and erases” this PTM on microtubules has been largely unexplored. We have identified a new role for the histone- methyltransferase NSD3 as a tubulin methyltransferase that di-methylates -tubulin at lysines 96 and 112 (the K96me2 and K112me2 marks). I am exploring the in vivo role of these new methyl marks on -tubulin utilizing the model organism C. elegans. I have now discovered the NSD3 orthologue in the worm, mes-4, has a somatic role in neurons, leading me to hypothesize loss of mes-4 abrogates α-tubulin methylation at K96 and K112, resulting in defects in organization and function of the neuronal cytoskeleton. To test this hypothesis in Aim1 I will explore the function of K96me2 and K112me2 in the worm utilizing methyl-deficient knockin tubulin mutations to understand how lack of methylation at these sites impacts microtubule structure (Aim1.1) and dynamics (Aim1.2). In Aim 2, I will further determine if loss of mes-4 causes loss of K96me2 and/or K112me2 using imaging and biochemical techniques (Aim2.1-2.2). Studies exploring the role of these methyl marks using loss of function approaches will be complemented by mes-4 gain of function studies (Aim 2.3). Many human cancers are driven by mutations that over/constitutively activate NSD3. I will generate a mes-4 mutant worm carrying the same mutation at the corresponding (conserved) site in C. elegans seen in human cancers, to ask if mes-4 hyperactivity induces cytoskeletal and functional deficits. My doctoral dissertation will thoroughly investigate a new perspective on how epigenetic machinery regulates the cytoskeleton, with far reaching implications for understanding the many diseases linked to cytoskeletal defects.

抽象的 微管细胞骨架在细胞中起许多关键功能，其功能障碍与多个关联链接 从癌症到神经发育疾病（例如自闭症）的疾病。翻译后修改（PTM） 微管的小管蛋白亚基是微管的结构和功能的关键调节剂。相似的 对于“组蛋白代码”，“微管蛋白代码”假设提出了微管功能 特异性小管蛋白同工型和PTM的掺入。甲基化众所周知是组蛋白上的常见PTM， 然而，微管蛋白甲基化和“读，写和擦除”的酶机制的功能 微管上的PTM在很大程度上是出乎意料的。我们已经确定了Hisstone的新角色 - 甲基转移酶NSD3作为微管蛋白甲基转移酶，在赖氨酸96和112处二甲基二甲基盐酸盐-微管蛋白 K96Me2和K112Me2标记）。我正在探索这些新甲基标记在使用微管蛋白上的体内作用 模型有机体秀丽隐杆线虫。我现在发现蠕虫中的NSD3直系同源物Mes-4具有躯体 在神经元中的作用，导致我假设MES-4的丧失消除了K96和K112处的α-微管蛋白甲基化， 导致神经元细胞骨架的组织和功能缺陷。在AIM1 I中检验这一假设 将探索利用甲基缺陷型敲蛋白小管蛋白突变在蠕虫中K96ME2和K112ME2的功能 要了解这些地点缺乏甲基化如何影响微管结构（AIM1.1）和动力学 （AIM1.2）。在AIM 2中，我将进一步确定MES-4的丢失是否导致K96Me2和/或K112Me2的损失 和生化技术（AIM2.1-2.2）。研究使用功能丧失探索这些甲基标记的作用 方法将通过MES-4功能研究获得（AIM 2.3）来完成。许多人类癌症被驱动 通过过度/组成性激活NSD3的突变。我将产生一个携带相同的MES-4突变体蠕虫 在人类癌症中看到的秀丽隐杆线虫中相应（保守的）位点的突变，询问MES-4多动症是否动物 诱导细胞骨架和功能性缺陷。我的博士学位论文将彻底调查新的 表观遗传机械如何调节细胞骨架的观点，对 了解与细胞骨架缺陷有关的许多疾病。