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步，包括单个颗粒低温电子显微镜（Cryo-EM） 已经实现了对生物大分子功能的前所未有的分子见解。但是， 由于目前的局限性，许多蛋白质和核糖核蛋白复合物的研究仍然具有挑 样品准备方法。传统上，感兴趣的蛋白质是在过表达系统中生产的 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净化 产量高的蛋白质，通常需要实质性优化，以限制生物医学研究的研究 重要的膜蛋白和大蛋白质复合物，尤其是在特定的伴侣和细胞的情况下 需要在体外​​复制难以复制的条件。为了克服这些限制，我们正在发展 通过利用CRISPR/CAS基因的进步来有效标记和纯化内源性蛋白质的方法论 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 与人类健康和疾病相关的本地和上下文特异性条件下的途径。我们是 有兴趣在三个主要研究领域开发和应用该方法：构成蛋白 复合物，细胞类型的特异性大分子组件和细胞状态依赖性膜蛋白 复合物。使用蛋白酶体作为模型系统，我们将研究的组装途径 冷冻EM和质谱法的蛋白酶体复合物。这项工作将提供机械洞察力 关键蛋白质降解机制，并有助于建立重要的方法来研究 内源性蛋白质组件。接下来，我们将扩展我们在研究蛋白质复合物的方法 不同的细胞类型，包括造血细胞和上皮细胞。这一目标将通过开发实现 在专门细胞类型中优化CRISPR/CAS基因编辑的有效策略，这将构成一种 迈向研究蛋白质的重要​​步骤。最后，我们将检查条件 蛋白质复合物和膜蛋白组装的组装。对于这个方向，我们将调查 与MTOR信号传导结合的溶酶体中养分传感的蛋白质。这项工作将 提供有关调节关键代谢途径的机制的分子见解，并阐明了如何 MTOR整合了不同的信号以促进细胞的生长和增殖。此外，我们将建立 筛选细胞和生化条件的方案，以获取上下文特异性蛋白质组件。 总的来说，这些研究将提供机械洞察力，以对出色的分子机器进行发展并发展 可以应用于其他生物系统的重要方法。这些方法将启用 我们要揭示蛋白质在特定细胞环境中蛋白质功能的基础机制 并有助于推进结构生物学，以了解生物大分子如何在其本地中起作用 上下文。