Characterization of native AMPA receptor structure and function in glioblastoma

胶质母细胞瘤中天然 AMPA 受体结构和功能的表征

• 批准号: 10554656
• 负责人: Catherine Jeanette Spangler
• 金额: $ 9.25万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-01 至 2026-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10554656
• 关键词: AMPA Receptors; Acute leukemia; Automobile Driving; Cell Differentiation process; Cell Line; Cell Survival; Cells; Chimeric Proteins; Chromosomal translocation; Clinical; Clinical Treatment; Complex; Cryoelectron Microscopy; Deposition; Development; Disease; Drug Targeting; Enzymes; Epigenetic Process; Foundations; Gene Expression; Genes; Genetic; Genetic Transcription; Glioblastoma; Histone H2B; Histone H3; Histones; Homeobox Genes; In Vitro; Knowledge; Lead; Leukemic Cell; Lysine; MLL gene; Maintenance; Malignant Neoplasms; Mammalian Cell; Methylation; Methyltransferase; Mixed-Lineage Leukemia; Modification; Molecular; Mutate; Mutation; Normal Cell; Nucleosomes; Oncogene Activation; Oncogenes; Oncogenic; Outcome; Pathway interactions; Phase; Post-Translational Protein Processing; Postdoctoral Fellow; Protein Biochemistry; Protein Chemistry; Proteins; RNA Polymerase II; Regulator Genes; Reporting; Research; Resolution; Role; Site; Site-Directed Mutagenesis; Structure; Transcription Elongation; Transcription Regulatory Protein; Ubiquitin; Up-Regulation; Variant; Work; combat; epigenetic regulation; genetic regulatory protein; histone methylation; histone methyltransferase; histone modification; improved; insight; leukemia; leukemogenesis; member; mixed lineage leukemia cell; new therapeutic target; novel; novel strategies; overexpression; pre-clinical; promoter; protein complex; receptor structure function; recruit; structural biology; targeted treatment; therapeutic development; therapeutic target; tool; tumorigenesis

PROJECT SUMMARY Mixed lineage leukemia is an aggressive subset of acute leukemias with poor clinical outcome and a severe need for improved treatment options. A genetic hallmark of mixed lineage leukemia is chromosomal translocation of the MLL gene, resulting in the formation of MLL-fusion proteins that drive leukemogenesis by mislocalizing essential cellular epigenetic machinery. Many MLL-fusion partner proteins interact with the histone methyltransferase Dot1L, mistargeting its activating methylation mark to developmental regulatory genes that are native targets of MLL during development. Dot1L catalytic activity is essential for both leukemogenic transformation and maintenance, making Dot1L inhibition one of the major strategies underlying current therapeutic development. Defining the molecular basis of Dot1L activity is an essential step towards rational development of Dot1L-targeted therapeutics, but our understanding of the structural and mechanistic basis for Dot1L activity on its native nucleosome substrate is still incomplete. Dot1L methylation of its target histone H3 lysine 79 residue is dependent on prior ubiquitylation of its nucleosome substrate on histone H2B at lysine 120. However, the structural basis for this trans-histone crosstalk has not been established. To elucidate the mechanistic basis for Dot1L activity on its nucleosome substrate and identify novel strategies for Dot1L inhibition, we solved a 3.9Å cryo-EM structure of Dot1L bound to a site-specifically ubiquitylated nucleosome, providing the first high-resolution insight into how Dot1L engages with its nucleosome substrate and is regulated by ubiquitin. Guided by this structure, we identified residues in Dot1L essential for both its nucleosome-specific activity and upregulation by ubiquitin. In Aim 1, we will mutate these Dot1L residues to observe their effect on leukemia cell viability and Dot1L activity at known oncogenes. This work will define the nucleosome-specific and ubiquitin-dependent activities of Dot1L in leukemogenesis and probe the potential of the Dot1L-nucleosome and Dot1L-ubiquitin interfaces as therapeutic targets. Many MLL-fusion partner proteins are also components of the super elongation complex (SEC), a large transcriptional regulatory protein complex that promotes the elongation phase of transcription by releasing RNA polymerase II from promoter-proximal pausing. The SEC is also essential for leukemogenic transformation in mixed lineage leukemia, further promoting misregulation of gene expression at MLL-fusion target genes through overactivation of transcriptional elongation. Still, the structural organization of the SEC and the influence of MLL- fusions on SEC structure and function has not been explored. In Aim 2, I propose a complete structural and functional characterization of the SEC to determine the mechanistic basis for SEC-driven leukemogenic progression in the context of MLL-fusion proteins. Together, these aims will identify novel targets along the molecular pathways driving leukemogenesis to form the pre-clinical foundation for rational therapeutic development.

项目摘要 混合谱系白血病是急性白血病的积极子集，临床结果差，严重 需要改善治疗选择。混合谱系白血病的遗传标志是染色体易位 MLL基因的of，导致形成MLL融合蛋白，这些蛋白通过错误定位而驱动白血病生成 必需的细胞表观遗传机制。许多MLL融合伴侣蛋白与组蛋白相互作用 甲基转移酶dot1l，将其激活甲基化标记错误定位到发育调节基因 是开发过程中MLL的本地靶标。 DOT1L催化活性对于两种白血病必不可少 转换和维护，使dot1l抑制当前的主要策略之一 治疗发展。定义DOT1L活性的分子基础是迈向理性的重要步骤 开发doT1L靶向疗法，但我们对结构和机械基础的理解 DOT1L对其天然核底物的活性仍然不完整。 其靶组蛋白H3赖氨酸79居住的DOT1L甲基化取决于其核小体的泛素化 赖氨酸120的组蛋白H2B的底物。但是，这种反式串扰的结构基础尚未 建立了。阐明DOT1L活性在其核小体底物上的机理基础并鉴定 DOT1L抑制的新型策略，我们求解了与位点特定于位点特定的DOT1L的3.9Å冷冻EM结构 泛素化的核小体，为DOT1L如何与其核小体互动提供了第一个高分辨率的见解 底物，由泛素调节。在这种结构的指导下，我们确定了DOT1L中的残差 其核小体特异性活性和泛素的更新。在AIM 1中，我们将突变这些dot1l残差 观察它们对已知肿瘤基因的白血病细胞活力和DOT1L活性的影响。这项工作将定义 DOT1L在白血病发生中的核小体特异性和泛素依赖性活性，并探测了潜在的潜力 DOT1L核体和DOT1L-泛素接口作为治疗靶标。 许多MLL融合伴侣蛋白也是超级延伸络合物（SEC）的组成部分，一个大的 转录调节蛋白复合物通过释放RNA促进转录的伸长阶段 聚合酶II来自启动子暂停。 SEC对于白血病转化也是必不可少的 混合谱系白血病，通过 转录伸长过度活化。尽管如此，SEC的结构组织和MLL的影响 尚未探索有关SEC结构和功能的融合。在AIM 2中，我提出了一个完整的结构和 SEC的功能表征以确定SEC驱动的白血病的机理基础 在MLL融合蛋白的背景下进展。 这些目标共同确定沿着分子途径的新靶标，驱动白血病形成形成 理性治疗发展前临床前基础。