Structural and dynamic insights into the mechanisms of mitochondrial AAA proteases

线粒体是真核细胞的能量发源地，在许多重要的代谢和信号传导途径中发挥着重要作用。由于线粒体内部发生强烈的代谢活动，线粒体中高效且有针对性的蛋白质质量控​​制系统对于细胞器的功能完整性至关重要，对于整体细胞的生存也至关重要。线粒体蛋白质稳态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，它以受控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角度了解各种线粒体蛋白酶如何发挥作用，并揭示它们如何以专门且协调的方式发挥作用，以在正常和应激条件下维持线粒体蛋白质稳态。     在本研究中，我们将重点关注两种保守的ATP依赖性线粒体蛋白酶，m-AAA和i-AAA，称为线粒体AAA蛋白酶，其靶向线粒体膜间、基质和膜间空间中的底物，并在维持线粒体蛋白质稳态中发挥重要作用。这些蛋白酶的缺陷与神经系统和神经退行性疾病以及代谢功能障碍有关。该提案描述了线粒体 AAA 蛋白酶的彻底结构和动力学表征，使用溶液核磁共振 (NMR) 光谱作为主要方法，结合其他生物物理和生化技术，旨在获得蛋白酶如何执行和调节其功能的机制见解。为了实现这一目标，我们提出以下具体目标：（1）确定AAA蛋白酶与其底物之间的相互作用位点，并确定底物识别机制； (2) 识别和表征功能上重要的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域通信； (4)从结构角度理解不同亚基之间的协同作用及其在ATP水解和解折叠/蛋白水解功能中的作用。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了解，并最终揭示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，不仅使他们获得蛋白质核磁共振波谱方面的专业知识，而且能够熟练使用各种其他生化和生物物理工具进行蛋白质的结构和功能表征。我们希望培养下一代科学家，使其在学术和工业生涯中蓬勃发展。

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.