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.

摘要 微管细胞骨架在细胞中起着许多重要的作用，它的功能障碍与过多的 从癌症到自闭症等神经发育障碍。翻译后修饰（PTM） 微管蛋白亚基的组成是微管结构和功能的关键调节因子。类似 与“组蛋白密码”相比，“微管蛋白密码”假说假定微管功能是通过 特异性微管蛋白同种型和PTM的掺入。甲基化是众所周知的组蛋白上常见的PTM， 然而，微管蛋白甲基化的功能和“读、写和擦除”这种功能的酶机制， 微管上的PTM在很大程度上尚未探索。我们发现了组蛋白的新作用- 甲基转移酶NSD 3作为微管蛋白甲基转移酶，其使α-微管蛋白在赖氨酸96和112处二甲基化（所述甲基转移酶NSD 3是微管蛋白甲基转移酶）。 K96 me 2和K112 me 2标记）。我正在探索这些新的甲基标记在微管蛋白利用中的体内作用， 模式生物C.优美的我现在已经发现了蠕虫中的NSD 3直系同源物，mes-4，具有体细胞 在神经元中的作用，这使我假设mes-4的缺失废除了K96和K112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。为了在Aim 1 I中验证这一假设， 将利用甲基缺陷敲入微管蛋白突变来探索K96 me 2和K112 me 2在蠕虫中的功能 了解这些位点缺乏甲基化如何影响微管结构（Aim1.1）和动力学 （目标1.2）。在目标2中，我将使用成像进一步确定mes-4的丢失是否会导致K96 me 2和/或K112 me 2的丢失 和生物化学技术（目标2.1 -2.2）。利用功能丧失探索这些甲基标记作用的研究 MES-4功能增益研究（目标2.3）将对这些方法进行补充。许多人类癌症是由 通过过度/组成型激活NSD 3的突变。我会制造一个mes-4变异蠕虫携带相同的 在C.在人类癌症中发现的秀丽线虫， 诱导细胞骨架和功能缺陷。我的博士论文将彻底调查一个新的 关于表观遗传机制如何调节细胞骨架的观点， 了解许多与细胞骨架缺陷有关的疾病。