Using rebuilt AAA+ enzymes to uncover the mechanisms of proteolysis at the mitochondrial inner membrane

使用重建的 AAA 酶揭示线粒体内膜的蛋白水解机制

• 批准号: 8944505
• 负责人: Steven Glynn
• 金额: $ 30.82万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-15 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8944505
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Active Sites; Address; Affect; Apoptosis; Apoptotic; Binding; Binding Sites; Biochemical; Cell physiology; Communication; Complex; Crystallization; Development; Diabetes Mellitus; Enzymes; Eukaryotic Cell; Foundations; Functional disorder; Future; Goals; Grant; Housing; Human; In Vitro; Inner mitochondrial membrane; Kinetics; Knowledge; Label; Link; Malignant Neoplasms; Maps; Measures; Membrane; Membrane Proteins; Methods; Mitochondria; Modeling; Molecular; Molecular Machines; Motion; Mutate; Mutation; Neurodegenerative Disorders; Nucleotides; Operating System; Organelles; Oxidative Phosphorylation; Peptide Hydrolases; Peptides; Phospholipid Metabolism; Physiological; Process; Production; Proteins; Proteolysis; Reactive Oxygen Species; Regulation; Resolution; Respiration; Signal Transduction; Site; Solutions; Spinocerebellar Ataxias; Structure; System; Therapeutic; Time; assault; biophysical techniques; disease-causing mutation; genetic regulatory protein; human disease; novel; novel strategies; novel therapeutics; nucleotide analog; protein degradation; public health relevance; research study; small molecule

 DESCRIPTION (provided by applicant): The mitochondrial inner membrane is the site of essential cellular functions such as oxidative phosphorylation, phospholipid metabolism, and the regulation of apoptosis. The inner membrane is under constant assault from reactive oxygen species, inevitable by-products of respiration. To limit the effects of this damage and to maintain proteostasis throughout mitochondria, AAA+ proteases harness the energy of ATP to recognize, unfold and degrade protein substrates both from within and surrounding the inner membrane. AAA+ proteases assemble as hexamers to form an internal proteolytic chamber into which substrates are forcibly translocated by an ATPase module. The ATPase active site is created by interactions from adjacent subunits such that oligomerization is a requirement for activity. The mitochondrial AAA+ proteases YME1L and AFG3L2 are largely soluble enzymes that are anchored in the inner membrane and represent a significantly understudied class of proteolytic system that operate at the membrane interface. In humans, dysfunction of these proteases has been linked to the development of severe neurodegenerative disorders such as spinocerebellar ataxia. Understanding the molecular mechanisms of these important enzymes has been hampered by the difficulty in studying membrane-anchored enzymes in vitro. I have developed a novel approach to assemble previously membrane-constrained hexameric proteases in a soluble, active form. This breakthrough allows for the application of established solution biochemical and biophysical techniques to the study of proteostasis at the mitochondrial inner membrane for the first time. The first aim of the proposal is to define how substrate proteins are selected for degradation among the myriad proteins housed in the inner membrane. Both model proteins and known physiological substrates will be used to determine what features are necessary and sufficient to drive degradation. The second aim is analyze the coordination of ATP hydrolysis and the production of pulling forces to understand how these molecular machines are capable of extracting substrates from within the inner membrane. Finally, the ability to produce large quantities of soluble active protease will be leveraged to determine crystal structures of the proteases in their active state, and in complex with nucleotide and protein substrates. Together, these experiments will form the first rigorous mechanistic analysis of the mitochondrial AAA+ proteases and provide foundational knowledge to aid the development of small molecule modulators as future therapeutics.

 描述（由应用提供）：线粒体内膜是必需细胞功能的位点，例如氧化物磷酸化，磷脂代谢和凋亡的调节。内膜受到反应性氧的不断攻击，不可避免的呼吸副产品。限制这种损害的影响并维持 在整个线粒体中，AAA+蛋白酶利用ATP的能量识别，展开和降解蛋白质底物。 AAA+蛋白酶作为六聚体形成一个内部蛋白水解腔，由ATPase模块强制翻译成内部蛋白水解腔。 ATPase活动位点是由相邻亚基的相互作用创建的，因此寡聚是活动的要求。线粒体AAA+蛋白酶YME1L和AFG3L2是固定在内膜中的很大程度上的固体酶，代表了在膜界面上运行的一类众所周知的蛋白水解系统。在人类中，这些蛋白酶的功能障碍与严重的神经退行性疾病（如脊髓脑性共济失调）的发展有关。在体外研究膜锚定酶时，很难理解这些重要酶的分子机制。我开发了一种新型的方法，以固体活性形式组装以前膜约束的六聚体蛋白。这一突破允许将已建立的溶液生化和生物物理技术应用于第一次在线粒体内膜的蛋白质抑制症中。该提案的第一个目的是定义如何选择底物蛋白在内膜中含有的无数蛋白质中降解。模型蛋白质和已知的物理基材都将用于确定哪些特征是必要的，足以驱动降解。第二个目的是分析ATP水解的协调和拉力的产生，以了解这些分子机器如何从内膜内提取底物。最后，将利用生产大量固体活性蛋白的能力来确定其活性状态下蛋白酶的晶体结构，并与核丁基和蛋白质底物复杂化。总之，这些实验将构成线粒体AAA+蛋白的首次严格机械分析，并提供基础知识，以帮助开发小分子调节剂作为未来的治疗。