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

项目摘要 包括单粒子低温电子显微镜在内的结构生物学技术的进展 使人们对生物大分子的功能有了前所未有的分子洞察。然而， 由于目前的局限性，许多蛋白质和核糖核蛋白复合体的研究仍然具有挑战性 样品制备方法。传统上，感兴趣的蛋白质是在过度表达系统中产生的。 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提纯 对于高产量的蛋白质，它通常需要大量的优化，这可能会限制生物医学的研究 重要的膜蛋白和大的蛋白质复合体，特别是在特定的伴侣和细胞 所需的条件很难在体外复制。为了克服这些限制，我们正在开发 利用CRISPR/Cas基因的进展高效标记和纯化内源蛋白的方法学 正在编辑。这种方法使我们能够研究大分子络合物及其复杂的组装。 在与人类健康和疾病相关的自然和特定环境条件下的通路。我们是 有兴趣在三个主要研究领域开发和应用这种方法：构成蛋白 复合体、细胞类型特定的大分子组件和细胞状态依赖的膜蛋白 复合体。以蛋白酶体为模型系统，研究蛋白酶体的组装途径。 蛋白酶体复合体的冷冻-EM和质谱学分析。这项工作将提供对 关键的蛋白质降解机制，并有助于建立重要的方法学研究 内源性蛋白质组合。接下来，我们将扩展我们的方法来研究蛋白质复合体 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 优化特定细胞类型中CRISPR/Cas基因编辑的有效策略，这将构成一个 朝着研究天然状态的蛋白质迈出了重要的一步。最后，我们将研究条件 蛋白质复合体和膜蛋白质组件的组装。为了这个方向，我们将调查 与mTOR信号一起参与溶酶体营养感知的蛋白质。这项工作将 提供对调节关键代谢途径的机制的分子见解，并阐明如何 MTOR整合不同的信号来促进细胞的生长和增殖。此外，我们还将建立 用于筛选细胞和生化条件以获得特定于上下文的蛋白质组件的协议。 总之，这些研究将为非凡的分子机器提供机械论的见解，并开发 可应用于其他生物系统研究的重要方法。这些方法将使 美国将揭开蛋白质在特定细胞环境中功能的分子机制 有助于推动结构生物学向理解生物大分子如何在其天然环境中发挥作用 上下文。

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.