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 混合系白血病是急性白血病的一个侵袭性亚群，临床结局差，严重的白血病并发症是白血病的一个重要特征。 需要改进治疗方案。混合系白血病的一个遗传标志是染色体易位 导致MLL融合蛋白的形成，该融合蛋白通过错误定位 基本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Dot 1 L，将其激活甲基化标记误认为是发育调控基因， 是MLL在发育过程中的天然靶点。Dot 1 L的催化活性对于白血病的发生和发展都是必不可少的。 转化和维持，使Dot 1 L抑制的主要策略之一，目前 治疗发展确定Dot 1 L活性的分子基础是实现合理的 Dot 1 L靶向治疗的发展，但我们对Dot 1 L靶向治疗的结构和机制基础的理解， Dot 1 L在其天然核小体底物上的活性仍然不完全。 靶组蛋白H3赖氨酸79残基的Dot 1 L甲基化依赖于其核小体的在先泛素化 组蛋白H2 B上赖氨酸120处的底物。然而，这种跨组蛋白串扰的结构基础还没有 确立了习阐明Dot 1 L在其核小体底物上的活性机制， Dot 1 L抑制的新策略，我们解决了Dot 1 L的3.9kb cryo-EM结构结合位点特异性 泛素化的核小体，提供了第一个高分辨率的洞察Dot 1 L如何与其核小体接合 底物，并由泛素调节。在这种结构的指导下，我们鉴定了Dot 1 L中对以下蛋白质必需的残基： 其核小体特异性活性和通过泛素的上调。在目标1中，我们将突变这些Dot 1 L残基 观察它们对白血病细胞活力和已知癌基因的Dot 1 L活性的影响。这项工作将定义 Dot 1 L在白血病发生中的核小体特异性和泛素依赖性活性， Dot 1 L-核小体和Dot 1 L-泛素界面作为治疗靶点。 许多MLL融合伴侣蛋白也是超级延伸复合物（SEC）的组分， 一种转录调节蛋白复合物，通过释放RNA促进转录的延伸期 启动子近端暂停的聚合酶II。SEC对于白血病转化也是必不可少的， 混合谱系白血病，通过以下途径进一步促进MLL融合靶基因的基因表达失调： 转录延伸的过度激活。尽管如此，SEC的结构组织和MLL的影响- 关于SEC结构和功能的融合尚未被探索。在目标2中，我提出了一个完整的结构和 SEC的功能表征，以确定SEC驱动的白血病发生的机制基础 在MLL融合蛋白的背景下的进展。 总之，这些目标将确定新的目标沿着分子途径驱动白血病形成 合理治疗开发的临床前基础。