Structural and dynamic insights into the mechanisms of mitochondrial AAA proteases

线粒体是真核细胞的能量源泉，在许多重要的代谢和信号通路中发挥着重要作用。由于线粒体内存在着强烈的代谢活动，有效的、有针对性的蛋白质质量控制系统对细胞器的功能完整性至关重要，对细胞的整体生存也至关重要。线粒体蛋白平衡的失衡与神经系统疾病、心血管疾病和癌症有关。线粒体蛋白质质量控制的核心是线粒体蛋白酶网络，该网络以受监管的方式降解不需要的和受损的蛋白质。我研究的长期目标是从结构和动态的角度了解各种线粒体蛋白水解酶的功能，并揭示它们如何以专门和协调的方式在正常和应激条件下维持线粒体蛋白恒定。在拟议的研究中，我们将重点关注两种保守的依赖于ATP的线粒体蛋白水解酶，m-AAA和i-AAA，称为线粒体AAA蛋白水解酶，它们靶向线粒体膜间、基质和膜间间隙的底物，在维持线粒体蛋白平衡方面发挥重要作用。这些蛋白水解酶的缺陷与神经和神经退行性疾病以及代谢功能障碍有关。该提案描述了以溶液核磁共振(核磁共振)光谱为主要手段，结合其他生物物理和生化技术，对线粒体AAA蛋白酶的结构和动力学进行彻底的表征，旨在从机制上深入了解蛋白酶如何执行和调节其功能。为了实现这一目标，我们提出了以下具体目标：(1)确定AAA酶与其底物之间的相互作用部位，并确定底物识别的机制；(2)确定和表征重要的功能结构特征和构象动力学；(3)确定连接结构基序的变构途径并表征结构域通讯；(4)从结构角度了解不同亚基之间的协作性及其在ATP水解和展开/蛋白分解功能中的作用。这项拟议的研究计划将提供对AAA蛋白酶如何发挥作用以及疾病突变如何导致其酶活性受损的基本理解，并最终阐明如何在线粒体中实现蛋白质质量控制。将为HQP提供广泛的培训机会，不仅获得蛋白质核磁共振光谱学方面的专门知识，而且还精通使用各种其他生化和生物物理工具对蛋白质的结构和功能进行表征。我们希望培养下一代科学家，使他们在学术和工业生涯中都能蓬勃发展。

Mitochondria are the energy powerhouses of the eukaryotic cells and play prominent roles in a number of essential metabolic and signaling pathways. Due to the intense metabolic activities occurring inside mitochondria, an efficient and targeted protein quality control system in mitochondria is vital for the functional integrity of the organelle and critical for overall cell survival. Imbalances in the mitochondrial proteostasis have been implicated in neurological diseases, cardiovascular disorders, and cancer. Central to mitochondrial protein quality control is a network of mitochondrial proteases which degrade undesired and damaged proteins in a regulated fashion. The long-term objective of my research is to understand how various mitochondrial proteases function from a structural and dynamic perspective and to uncover how they work in a specialized and concerted manner to maintain mitochondrial proteostasis under normal and stressed conditions.     In the proposed research, we will focus on two conserved ATP-dependent mitochondrial proteases, m-AAA and i-AAA, referred to as mitochondrial AAA proteases, which target substrates in the mitochondrial intermembrane, the matrix and the intermembrane space, and play essential roles in maintaining mitochondrial proteostasis. Defects in these proteases have been implicated in neurological and neurodegenerative disorders as well as metabolic dysfunctions. The proposal describes a thorough structural and dynamical characterization of the mitochondrial AAA proteases using solution nuclear magnetic resonance (NMR) spectroscopy as the main approach in conjunction with other biophysical and biochemical techniques, designed to gain mechanistic insights into how the proteases carry out and regulate their functions. To achieve this goal, we propose the following specific aims: (1) identify interaction sites between the AAA proteases and their substrates, and determine the mechanism of substrate recognition; (2) identify and characterize functionally important structural features and conformational dynamics; (3) determine allosteric pathways connecting structural motifs and characterize domain communications; (4) understand cooperativity between different subunits from a structural perspective and its roles in ATP hydrolysis and unfolding/proteolytic functions. This proposed research program will provide a fundamental understanding of how the AAA proteases function and how disease mutations may lead to impairment of their enzymatic activities, and ultimately shed light into how protein quality control is achieved in mitochondria. Extensive training opportunities will be provided for HQPs not only to acquire expertise in protein NMR spectroscopy but also to be proficient in structural and functional characterization of proteins using a variety of other biochemical and biophysical tools. We hope to nurture a future generation of scientists to flourish in both academic and industrial careers.