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.

项目总结/摘要 组蛋白翻译后修饰在核小体上的动态写入和擦除调控真核生物 通过调节染色质组织和募集染色质结合蛋白来调节基因表达。的甲基化 丝氨酸可以激活或抑制转录，这取决于组蛋白残基及其甲基化状态。蛋白 精氨酸甲基转移酶5（PRMT 5），沿着其专性结合配偶体甲基体蛋白50（MEP 50）， 是所有真核生物中精氨酸对称二甲基化的主要复合物。此外，PRMT 5- MEP 50可以激活或抑制几个基因的转录，这取决于酶的哪个残基。 修改。PRMT 5-MEP 50催化四个组蛋白残基上的甲基化，即组蛋白H2 A-Arg 3（H2 AR 3）， H3-Arg 2（H3 R2）、H3-Arg 8（H3 R8）和H4-Arg 3（H4 R3）。由于PRMT 5-MEP 50在转录中的不同作用， 调节，PRMT 5在几种癌症中过表达，因为它调节几种转移的转录。 抑制基因和上皮-间质转化激活基因。尽管PRMT 5-MEP 50的重要性 在基因表达中，对PRMT 5-MEP 50如何甲基化组蛋白和/或核小体知之甚少 印刷受体.然而，最近的工作已经揭示，PRMT 5的特异性受以下调节：（1）识别 细胞溶质H2 A-H2 B二聚体甲基化H2 AR 3和（2）能够优先甲基化H2 AR 3中的组蛋白H4。 PRMT 5（COPR 5）底物适配器协调员的存在。尽管有这些发现， 这种特异性仍然是未知的。使用生物化学和结构方法相结合，我将 研究PRMT 5-MEP 50复合物的组蛋白特异性和活性的机制。 在目标1中，我将通过定量测定H2 A-H2 B二聚体对PRMT 5-MEP 50识别的贡献， PRMT 5-MEP 50对各种含组蛋白H2 A底物的活性和结合。提供分子 这种识别的细节，我将确定PRMT 5-MEP 50结合H2 A-H2 B二聚体的结构，使用冷冻-凝胶电泳。 电子显微镜（cryo-EM）。在筛选H2 A甲基化的底物时，我发现PRMT 5- MEP 50活性通过组蛋白H2 BK 120（H2 BK 120-Ub）的泛素化来刺激。我会在体内探测 siRNA敲除的干扰。然后，我将通过量化活动来揭示这种激活的机制 以及在H2 BK 120-Ub存在下PRMT 5-MEP 50的结合和解析PRMT 5-MEP 50的EM结构。 MEP 50与H2 A-H2 BK 120-Ub二聚体结合。在目标2中，我将阐明COPR 5和PRMT 5的功能。 MEP 50-COPR 5复合物。我的初步数据显示，COPR 5不与核小体结合， 招募PRMT 5-MEP 50到核小体，与先前对COPR 5功能的推测相矛盾。所以我 将确定COPR 5的首选含组蛋白的底物，并量化COPR 5的结合和贡献， PRMT 5-MEP 50的酶活性。最后，我将解出PRMT 5-MEP 50-COPR 5的结构， 其组蛋白底物。总之，该提议将构建PRMT 5的分子框架- MEP 50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。