Molecular Basis of Histone Methylation by PRMT5

PRMT5 组蛋白甲基化的分子基础

• 批准号: 10607316
• 负责人: Sanim Rahman
• 金额: $ 4.77万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10607316
• 关键词: ARG2 gene; Acute Myelocytic Leukemia; Affinity; Arginine; Binding; Binding Proteins; Biochemical; Biological Assay; Biological Process; Biophysics; Cell Nucleus; Chromatin; Chromatin Modeling; Colon Carcinoma; Complex; Cryoelectron Microscopy; Cytoplasm; Cytosol; Data; Deposition; Development; Drug Design; Enzyme Kinetics; Enzymes; Epithelium; Eukaryota; Gene Expression; Genes; Genetic Transcription; Histone H2A; Histone H2B; Histone H3; Histone H4; Histones; Human; In Vitro; Knowledge; Lysine; Malignant Neoplasms; Malignant neoplasm of lung; Malignant neoplasm of ovary; Malignant neoplasm of prostate; Malignant neoplasm of urinary bladder; Mesenchymal; Metastasis Suppressor Genes; Methylation; Methyltransferase; Modification; Molecular; Molecular Chaperones; Nucleosomes; Post-Translational Protein Processing; Protein-Arginine N-Methyltransferase; Proteins; Research; Research Personnel; Role; Small Interfering RNA; Specificity; Structure; Substrate Specificity; Techniques; Testing; Training; Transcriptional Regulation; Ubiquitination; Work; Writing; career; design; dimer; gene repression; histone methylation; histone methyltransferase; histone modification; in vivo; inhibitor; insight; knock-down; malignant stomach neoplasm; overexpression; protein function; reconstitution; recruit; screening

Project Summary/Abstract The dynamic writing and erasing of histone post-translational modifications on nucleosomes regulate eukaryotic gene expression by tuning chromatin organization and recruiting chromatin-binding proteins. The methylation of arginines can activate or repress transcription depending on the histone residue and its methylation state. Protein arginine methyltransferase 5 (PRMT5), along with its obligate binding partner Methylosome protein 50 (MEP50), is the primary complex for the symmetric dimethylation of arginine across all eukaryotes. In addition, PRMT5- MEP50 can either activate or repress the transcription of several genes, depending on which residue the enzyme modifies. PRMT5-MEP50 catalyzes methylation on four histone residues, namely histone H2A-Arg3 (H2AR3), H3-Arg2 (H3R2), H3-Arg8 (H3R8), and H4-Arg3 (H4R3). Due to PRMT5-MEP50’s diverse roles in transcription regulation, PRMT5 is overexpressed in several cancers as it regulates the transcription of several metastasis suppressor genes and epithelial-mesenchymal transition activating genes. Despite PRMT5-MEP50’s importance in gene expression, very little is known of how PRMT5-MEP50 methylates histone and/or nucleosome substrates. However, recent work has revealed that PRMT5’s specificity is regulated by (1) recognition of cytosolic H2A-H2B dimers to methylate H2AR3 and (2) being able to preferentially methylate histone H4 in the presence of substrate adaptor Coordinator of PRMT5 (COPR5). Despite these findings, molecular determinants towards this specificity are still unknown. Using a combination of biochemical and structural approaches, I will investigate the mechanism of histone specificity and activity by the PRMT5-MEP50 complex. In Aim 1, I will determine contributions towards PRMT5-MEP50’s recognition of H2A-H2B dimers by quantifying the activity and binding of PRMT5-MEP50 on various histone H2A-containing substrates. To provide molecular detail of this recognition, I will determine the structure of PRMT5-MEP50 bound to H2A-H2B dimers using cryo- electron microscopy (cryo-EM). While screening substrates of H2A methylation, I discovered that PRMT5- MEP50 activity is stimulated by ubiquitination of histone H2BK120 (H2BK120-Ub). I will probe in vivo relevance of this crosstalk by siRNA knockdowns. I will then reveal the mechanism of this activation by quantifying activity and binding of PRMT5-MEP50 in the presence of H2BK120-Ub and resolving the EM structure of PRMT5- MEP50 bound to H2A-H2BK120-Ub dimers. In Aim 2, I will elucidate the function of COPR5 and the PRMT5- MEP50-COPR5 complex. My preliminary data revealed that COPR5 does not bind to nucleosomes and cannot recruit PRMT5-MEP50 to the nucleosome, conflicting previous speculations of COPR5’s function. Therefore, I will identify COPR5’s preferred histone-containing substrate and quantify COPR5’s binding and contribution to the enzymatic activity of PRMT5-MEP50. Finally, I will solve the structure of PRMT5-MEP50-COPR5 bound to its histone substrate by cryo-EM. Together, this proposal will construct a molecular framework of PRMT5- MEP50’s substrate specificity to aid in structure-based drug design, by revealing substrate-specific interactions.

项目摘要/摘要 组蛋白对核小体的翻译后修饰的动态写作和擦除，调节真核。 通过调整染色质组织和募集染色质结合蛋白来表达基因表达。甲基化 精氨酸可以根据组蛋白居住及其甲基化状态激活或反映转录。蛋白质 精氨酸甲基转移酶5（PRMT5），以及其强制性结合伴侣甲基菌蛋白50（MEP50），， 是在所有真核生物中精氨酸对称二甲基化的主要复合物。此外，PRMT5- MEP50可以激活或反映几种基因的转录，具体取决于酶的居住 修饰符。 PRMT5-MEP50催化甲基化的四个Hisstone残留物，即组蛋白H2A-ARG3（H2AR3）， H3-ARG2（H3R2），H3-ARG8（H3R8）和H4-ARG3（H4R3）。由于PRMT5-MEP50在转录中的作用 调节，PRMT5在几种癌症中过表达，因为它调节了几种转移的转录 抑制基因和上皮 - 间质转变激活基因。尽管PRMT5-MEP50的重要性 在基因表达中，很少知道PRMT5-MEP50如何甲基化组蛋白和/或核小体 基材。但是，最近的工作表明，PRMT5的特异性受（1）认可的调节 胞质H2A-H2B二聚体甲基化H2AR3和（2）能够优先将甲基化组蛋白H4在 PRMT5的底物适配器协调员的存在（COPR5）。尽管有这些发现，分子决定剂 朝向这种特殊性仍然未知。使用生化和结构方法的结合，我将 研究PRMT​​5-MEP50复合物的组蛋白特异性和活性的机制。 在AIM 1中，我将通过量化来确定对PRMT5-MEP50对H2A-H2B二聚体的认可的贡献 PRMT5-MEP50对各种Hisstone H2A底物的活性和结合。提供分子 此识别的详细信息，我将确定使用Cryo-与H2A-H2B二聚体结合的PRMT5-MEP50的结构 电子显微镜（冷冻EM）。在筛选H2A甲基化的底物时，我发现Prmt5- MEP50活性通过组蛋白H2BK120（H2BK120-UB）的泛素化而刺激。我将探测体内相关性 sirna敲除的串扰。然后，我将通过量化活动来揭示这种激活的机制 在H2BK120-UB存在下PRMT5-MEP50的结合并解决PRMT5-的EM结构 MEP50绑定到H2A-H2BK120-UB二聚体。在AIM 2中，我将阐明copr5和prmt5-的功能 MEP50-COPR5复合物。我的初步数据表明，copr5不与核小体结合，不能 将PRMT5-MEP50招募到核小体，对COPR5功能的先前猜测冲突。因此，我 将确定COPR5首选的含Hisstone的底物，并量化COPR5的约束力和贡献 PRMT5-MEP50的酶活性。最后，我将解决与 它的组蛋白底物通过冷冻EM。总之，该建议将构建一个PRMT5-的分子框架 MEP50的底物特异性通过揭示底物特异性相互作用来帮助基于结构的药物设计。