Defining Structural and Molecular Mechanisms of The Human Multifunctional Mitochondrial Protease, LONP1

PROJECT SUMMARY Integrated quality control (QC) systems are required to sense and manage mitochondrial stress, and their dysregulation is associated with neurodegenerative disease, aging, and cancer. The human ATP-dependent AAA+ protease, LONP1, has emerged as a master regulator of mitochondrial functions and is an integral component of mitochondrial QC systems. Deletion of LONP1 in mice is embryonically lethal and altered LONP1 activity is associated with mitochondrial diseases, aging, and cancer. LONP1 classically functions by degrading oxidatively damaged and unfolded matrix proteins, but also regulates diverse aspects of mitochondrial biology by specifically targeting and degrading folded proteolytic targets such as transcription factor A (TFAM) and cytochrome C oxidase subunit IV (COXIV). Additionally, LONP1 is a single stranded DNA binding protein that localizes to the non-coding control region of mitochondrial DNA, which is important for transcription and genome replication. LONP1 assembles as a 600 kDa hexamer composed of an N-terminal substrate binding domain (NTD), a AAA+ ATPase domain, and a C-terminal protease domain and can function as a protease, a chaperone, or a DNA binding protein. Despite these diverse critical functions, we lack detailed molecular mechanisms describing these activities and their regulation, limiting the fields capacity to determine their specific roles in mitochondrial homeostasis or disease pathogenesis. Recent cryo-electron microscopy (cryo-EM) studies have provided crucial insights into LONP1's conserved, AAA+-mediated hand-over-hand substrate translocation mechanism required to processively engage, unfold, and degrade proteolytic substrates. Strikingly, in these structures, the C-terminal protease domains remain in an inactive conformation even with substrate bound to LONP1's AAA+ domain. These findings contrast recent work on the evolutionarily related bacterial Lon protease and suggest that LONP1 has evolved additional levels of regulation to control or tune proteolytic activity to meet cellular needs. Moreover, these results raise important questions regarding proteolytic regulation and its relationship to other reported cellular functions, including DNA binding. Therefore, the long-term goal of this proposal will be to establish mechanistic models for LONP1's manifold functions and their allosteric regulation in order to define their role in mitochondrial maintenance and disease. I hypothesize that LONP1 adopts distinct structural conformations allosterically regulated by nucleotide state, substrate binding, and/or posttranslational modifications to shift between operational modes to modulate proteolytic and mtDNA binding activities. I will integrate structural studies using cryo-EM with biochemical assays to identify allosteric mechanisms involved in regulating LONP1's proteolytic activity (Aim 1) and elucidate the molecular mechanism and conformational state required for LONP1's mtDNA binding activity (Aim 2).

项目摘要 需要综合质量控制（QC）系统来感知和管理线粒体压力及其 失调与神经退行性疾病，衰老和癌症有关。人ATP依赖 AAA+蛋白酶，LONP1已成为线粒体功能的主调节器，是一个积分不可或缺的 线粒体QC系统的组成部分。 LONP1在小鼠中的删除是胚胎致死的，改变了LONP1 活性与线粒体疾病，衰老和癌症有关。 LONP1经典通过退化发挥作用 氧化受损和展开的基质蛋白，但也调节线粒体生物学的各个方面 通过专门针对和降解折叠的蛋白水解靶标，例如转录因子A（TFAM）和 细胞色素C氧化酶亚基IV（Coxiv）。另外，LONP1是一种单链DNA结合蛋白 定位于线粒体DNA的非编码控制区，这对于转录和基因组很重要 复制。 LONP1作为由N末端底物结合域组成的600 kDa六聚体组装 （NTD），AAA+ ATPase结构域和C末端蛋白酶域，并且可以用作蛋白酶，伴侣，伴侣， 或DNA结合蛋白。尽管有这些不同的关键功能，但我们缺乏详细的分子机制 描述这些活动及其法规，限制了田野的能力来确定其在 线粒体稳态或疾病发病机理。最近的冷冻电子显微镜（冷冻EM）研究具有 提供了对LONP1保守的AAA+介导的手击底物易位的关键见解 进行过程参与，展开和降解蛋白水解底物所需的机制。令人惊讶的是，在这些中 结构，C末端蛋白酶结构域仍然处于非活动构象中，即使底物结合到 LONP1的AAA+域。这些发现与有关进化相关的细菌lon蛋白酶的最新工作对比 并表明LONP1已经进化了其他调节水平以控制或调整蛋白水解活性以满足 细胞需求。此外，这些结果提出了有关蛋白水解调节及其的重要问题 与其他报道的细胞功能的关系，包括DNA结合。因此，这个长期目标 提案将是为LONP1的流动功能及其变构调节建立机械模型 为了定义它们在线粒体维持和疾病中的作用。我假设LONP1采用了独特的 结构构象在核苷酸状态，底物结合和/或翻译后的变构中 修改操作模式之间转移以调节蛋白水解和mtDNA结合活性。我会 使用冷冻EM与生化测定法整合结构研究，以鉴定涉及的变构机制 调节LONP1的蛋白水解活性（AIM 1）并阐明分子机制和构象状态 LONP1的mtDNA结合活性所需（AIM 2）。