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

 描述(申请人提供)：线粒体内膜是细胞基本功能的场所，如氧化磷酸化、磷脂代谢和细胞凋亡调节。内膜不断受到呼吸副产物--活性氧的侵袭。以限制这种损害的影响，并保持 在整个线粒体的蛋白稳定中，AAA+蛋白水解酶利用ATP的能量从内膜内和周围识别、展开和降解蛋白质底物。AAA+蛋白水解酶以六聚体的形式聚集在一起，形成一个内部蛋白水解室，底物被ATPase模块强行转移到里面。ATPase活性部位是由相邻亚基的相互作用产生的，因此寡聚化是活性的要求。线粒体AAA+蛋白水解酶YME1L和AFG3L2主要是锚定在内膜上的可溶性酶，是一类在膜界面工作的蛋白水解酶。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的发展有关，如脊髓小脑性共济失调。由于在体外研究膜锚定酶的困难，阻碍了对这些重要酶的分子机制的理解。我已经开发了一种新的方法来组装以前受膜限制的六聚体蛋白酶，使其成为一种可溶的、活性的形式。这一突破首次允许将已建立的溶液生化和生物物理技术应用于研究线粒体内膜的蛋白稳定。该提案的第一个目的是确定如何在内膜中容纳的无数蛋白质中选择底物蛋白质进行降解。模型蛋白质和已知的生理底物都将被用来确定哪些特征是必要的，并且足以驱动降解。第二个目的是分析ATP水解和拉力产生的协调，以了解这些分子机器如何能够从内膜中提取底物。最后，将利用产生大量可溶性活性蛋白酶的能力来确定处于活性状态的蛋白酶的晶体结构，以及与核苷酸和蛋白质底物的复合体。总之，这些实验将形成第一个严格的线粒体AAA+蛋白水解酶的机制分析，并为小分子调节剂作为未来治疗药物的开发提供基础知识。

 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.