Molecular mechanisms underlying the assembly of the human proteasome and endogenous protein complexes

人类蛋白酶体和内源蛋白复合物组装的分子机制

• 批准号: 10500937
• 负责人: Jianhua Zhao
• 金额: $ 48.75万
• 依托单位: SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2027-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10500937
• 关键词: Area; Biochemical; Biological; Biological Models; Biological Process; Cell Line; Cell Proliferation; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Cryoelectron Microscopy; Disease; Electron Microscopy; Eligibility Determination; Environment; Epithelial Cells; FRAP1 gene; Genes; Goals; Health; Hematopoietic; Human; Immunodeficiency and Cancer; In Vitro; Insecta; Light; Lysosomes; Macromolecular Complexes; Mammalian Cell; Mass Spectrum Analysis; Membrane Proteins; Metabolic Pathway; Methodological Studies; Methodology; Methods; Molecular; Molecular Chaperones; Molecular Machines; Nerve Degeneration; Nucleic Acids; Pathway interactions; Preparation; Production; Proteins; Ribonucleoproteins; Sampling; Signal Transduction; System; Techniques; Work; biological systems; cell growth; cell type; cryogenics; detection of nutrient; fighting; insight; interest; macromolecular assembly; macromolecule; multicatalytic endopeptidase complex; novel therapeutics; overexpression; particle; protein complex; protein degradation; protein function; protein purification; structural biology

Project Summary Advances in structural biology techniques including single particle cryogenic electron microscopy (cryo-EM) have enabled unprecedented molecular insights into the function of biological macromolecules. However, the study of many proteins and ribonucleoprotein complexes remains challenging due to current limitations in sample preparation approaches. Traditionally, proteins of interest are produced in over-expression systems within bacterial, insect, and mammalian cell lines. While this approach can allow the production and purification of proteins with high yields, it often requires substantial optimization that can limit the study of biomedically important membrane proteins and large protein complexes, especially where specific chaperones and cellular conditions are required that are difficult to replicate in vitro. To overcome these limitations, we are developing methodology to efficiently tag and purify endogenous proteins by leveraging advances in CRISPR/Cas gene editing. This approach enables us to investigate macromolecular complexes and their intricate assembly pathways under native and context-specific conditions that are relevant to human health and disease. We are interested in developing and applying the approach in three major areas of study: constitutive protein complexes, cell-type specific macromolecular assemblies, and cell state dependent membrane protein complexes. Using the proteasome as a model system, we will investigate the assembly pathway of proteasomal complexes by cryo-EM and mass spectrometry. This work will provide mechanistic insights into critical protein degradation machinery and help to establish important methodology for the study of endogenous protein assemblies. Next, we will expand our approach to the study of protein complexes in different cell types, including hematopoietic and epithelial cells. This goal will be achieved by developing efficient strategies to optimize CRISPR/Cas gene editing in specialized cell types, which will constitute an important step towards the study of proteins in their native states. Finally, we will examine the conditional assembly of protein complexes and membrane protein assemblies. For this direction, we will investigate proteins involved in nutrient sensing at the lysosome in conjunction with mTOR signaling. This work will provide molecular insights into the mechanisms regulating key metabolic pathways and shed light on how mTOR integrates different signals to promote cell growth and proliferation. Additionally, we will establish protocols for screening cellular and biochemical conditions to acquire context-specific protein assemblies. Altogether, these studies will provide mechanistic insights into remarkable molecular machines and develop important methodology that can be applied to the study of other biological systems. These methods will enable us to unravel the molecular mechanisms underlying the function of proteins in specific cellular environments and help advance structural biology towards understanding how biological macromolecules work in their native contexts.

项目摘要 结构生物学技术的进展，包括单粒子低温电子显微镜（cryo-EM） 使我们对生物大分子的功能有了前所未有的深入了解。但 许多蛋白质和核糖核蛋白复合物的研究仍然具有挑战性， 样品制备方法。传统上，感兴趣的蛋白质在过表达系统中产生 在细菌、昆虫和哺乳动物细胞系中。虽然这种方法可以允许生产和纯化 蛋白质产量高，它往往需要大量的优化，可以限制生物医学的研究 重要的膜蛋白和大的蛋白复合物，特别是当特定的伴侣和细胞 需要难以在体外复制的条件。为了克服这些限制，我们正在开发 利用CRISPR/Cas基因的进展有效标记和纯化内源蛋白的方法 编辑.这种方法使我们能够研究大分子复合物及其复杂的组装 在与人类健康和疾病相关的天然和特定环境条件下的途径。我们 有兴趣在三个主要研究领域开发和应用该方法：组成蛋白 复合物、细胞类型特异性大分子组装体和细胞状态依赖性膜蛋白 配合物以蛋白酶体为模型系统，我们将研究蛋白酶体的组装途径。 蛋白酶体复合物的冷冻电镜和质谱。这项工作将提供机械的见解， 关键的蛋白质降解机制，并帮助建立重要的方法学研究 内源性蛋白质组装。接下来，我们将扩大我们的方法来研究蛋白质复合物， 不同的细胞类型，包括造血细胞和上皮细胞。这一目标将通过发展 在专门的细胞类型中优化CRISPR/Cas基因编辑的有效策略，这将构成一个 这是研究天然状态蛋白质的重要一步。最后，我们将检查条件 蛋白质复合物的组装和膜蛋白组装。对于这个方向，我们将进行调查 与mTOR信号传导相关的溶酶体营养感测蛋白质。这项工作将 提供分子的见解调节关键代谢途径的机制，并阐明如何 mTOR整合不同的信号以促进细胞生长和增殖。此外，我们将建立 用于筛选细胞和生物化学条件以获得环境特异性蛋白质组装体的方案。 总之，这些研究将为非凡的分子机器提供机理上的见解， 这是一种重要的方法，可以应用于其他生物系统的研究。这些方法将使 我们要解开蛋白质在特定的细胞环境中的功能的分子机制 并帮助推进结构生物学，以了解生物大分子如何在其自然环境中发挥作用， contexts.