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

项目总结 需要综合质量控制(QC)系统来感知和管理线粒体应激，并且他们的 调节失调与神经退行性疾病、衰老和癌症有关。人类的三磷酸腺苷依赖性 AAA+蛋白水解酶，LONP1，已成为线粒体功能的主要调节者，是一种不可或缺的 线粒体QC系统的组成部分。LONP1在小鼠中的缺失是胚胎致死的，并改变了LONP1 活动与线粒体疾病、衰老和癌症有关。LONP1的经典功能是降级 氧化损伤和未折叠的基质蛋白，但也调节线粒体生物学的各个方面 通过特异性靶向和降解折叠的蛋白水解靶，如转录因子A(TFAM)和 细胞色素C氧化酶亚基IV(COXIV)。此外，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，对转录和基因组至关重要 复制。LONP1组装成由N-末端底物结合域组成的600 kDa六聚体 (NTD)、AAA+ATPase结构域和C-末端蛋白酶结构域，可作为蛋白酶、伴侣、 或者是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 描述这些活动及其监管，限制外地确定其具体作用的能力 线粒体动态平衡或疾病发病机制。最近的低温电子显微镜(Cryo-EM)研究已经 提供了对LONP1的S保守的AAA+介导的手性底物易位的关键见解 蛋白分解底物的过程接合、展开和降解所需的机构。引人注目的是，穿着这些 结构，即使底物与底物结合，C-末端的蛋白酶结构域也保持不活跃的构象。 LONP1‘S AAA+域。这些发现与最近关于进化相关的细菌Lon蛋白酶的工作形成了对比 并表明LONP1已经进化出额外的调节水平来控制或调节蛋白水解酶活性以满足 手机的需求。此外，这些结果还提出了有关蛋白质降解调节及其 与其他已报道的细胞功能的关系，包括DNA结合。因此，这一行动的长期目标是 建议建立LONP1的S多重功能及其变构调节的机制模型 以确定它们在线粒体维持和疾病中的作用。我假设LONP1采用DISTINCT 受核苷酸状态、底物结合和/或翻译后调控的结构构象 修改以在操作模式之间转换，以调节蛋白水解酶和线粒体DNA结合活性。这就做 结合使用低温EM和生化分析的结构研究，以确定参与的变构机制 调控LONP1‘S蛋白水解酶活性及分子机制和构象状态的研究 LONP1‘S线粒体DNA结合活性所必需的(目的2)。

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).