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

定义人类多功能线粒体蛋白酶 LONP1 的结构和分子机制

• 批准号: 10549721
• 负责人: Jeffrey Todd Mindrebo
• 金额: $ 6.95万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-03 至 2025-01-02
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10549721
• 关键词: ATPase Domain; Adopted; Affect; Aging; Allosteric Regulation; Automobile Driving; Binding; Biochemical; Biological Assay; Biology; Biophysics; C-terminal; Cell physiology; Cerebrum; Clinical; Complex; Cryoelectron Microscopy; DNA Binding; DNA-Binding Proteins; Dental; Development; Disease; Disparate; Embryo; Genetic Transcription; Genome; Genome Stability; Goals; Hand; Health; Homeostasis; Human; Hypoxia; Knock-out; Length; Maintenance; Malignant Neoplasms; Maps; Mediating; Metabolism; Methodology; Mitochondria; Mitochondrial DNA; Mitochondrial Diseases; Modeling; Molecular; Molecular Chaperones; Molecular Conformation; Morphology; Mus; Mutation; N-terminal; Neurodegenerative Disorders; Nucleotides; Oxidative Phosphorylation; Oxidative Stress; Pathogenesis; Peptide Hydrolases; Post-Translational Protein Processing; Process; Protease Domain; Proteins; Protomer; Publishing; Quality Control; Regulation; Regulation of Proteolysis; Reporting; Respiration; Role; SS DNA BP; Small Interfering RNA; Stress; Structure; Supervision; Syndrome; System; Training; Transcriptional Regulation; Translations; Untranslated RNA; Work; biophysical analysis; cytochrome c oxidase; developmental disease; endopeptidase La; factor A; genomic locus; insight; mimetics; mitochondrial genome; oxidative damage; professor; protein aggregation; proteostasis; response; skeletal; steroidogenic acute regulatory protein; transcription factor

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 需要集成的质量控制（QC）系统来感测和管理线粒体应激，以及它们的代谢。 失调与神经变性疾病、衰老和癌症有关。人类依赖ATP的 AAA+蛋白酶LONP 1已成为线粒体功能的主要调节剂，是线粒体功能的组成部分。 线粒体QC系统的组成部分。小鼠LONP 1的缺失是胚胎致死的， 活性与线粒体疾病、衰老和癌症有关。LONP 1的经典功能是降低 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）。另外，LONP 1是单链DNA结合蛋白， 定位于线粒体DNA的非编码控制区，这对转录和基因组非常重要 复制的LONP 1组装为600 kDa六聚体，由N-末端底物结合结构域组成 (NTD)AAA+ ATP酶结构域和C-末端蛋白酶结构域，并且可以作为蛋白酶，伴侣蛋白， 或DNA结合蛋白。尽管这些不同的关键功能，我们缺乏详细的分子机制 描述这些活动及其管理，限制外地能力，以确定其具体作用， 线粒体稳态或疾病发病机制。最近的冷冻电子显微镜（cryo-EM）研究 提供了LONP 1保守的AAA+介导的手-手底物易位的重要见解 蛋白水解底物前接触、解折叠和降解所需的机制。引人注目的是，在这些 结构，C-末端蛋白酶结构域保持在非活性构象，即使与底物结合， LONP 1的AAA+域。这些发现与最近关于进化相关的细菌Lon蛋白酶的研究形成了对比。 并表明LONP 1已经进化出额外的调节水平，以控制或调节蛋白水解活性， 细胞的需要。此外，这些结果提出了关于蛋白水解调节及其 与其他报道的细胞功能的关系，包括DNA结合。因此，这一长期目标 建议建立LONP 1的多种功能及其变构调节的机制模型 以确定它们在线粒体维护和疾病中的作用。我假设LONP 1采用不同的 由核苷酸状态、底物结合和/或翻译后变构调节的结构构象 修饰以在操作模式之间转换以调节蛋白水解和mtDNA结合活性。我会 利用冷冻电镜结合生化分析进行结构研究，以鉴定 调节LONP 1的蛋白水解活性（Aim 1），阐明其分子机制和构象状态 LONP 1的mtDNA结合活性所必需的（目的2）。