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

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的调节。这些活动是 由复合线粒体蛋白质组执行，需要不断重新计算才能做出反应 对细胞不断变化的新陈代谢需求和由 活性氧物种。这种重新计算是由两种线粒体AAA+蛋白酶执行的， 它利用ATP的能量来识别、展开和降解蛋白质底物 在内膜内和周围的。在人类中，这些蛋白水解酶的功能失调 与严重的神经退行性疾病，如脊髓小脑性共济失调的发展有关。 AAA+蛋白水解酶以六聚体的形式聚集，形成一个内部蛋白水解室，进入 底物被一环ATPase强制移位。线粒体AAA+的研究 长期以来，蛋白酶一直受到其多个可溶催化结构域与 不可溶的跨膜结构域，用于锚定到内膜。我们利用一种蛋白质- 在一种工程方法中组装先前膜受限的六聚体蛋白酶 可溶的、活性的形式。我们的目标是用这些重建的蛋白水解酶对 线粒体内膜能量依赖性蛋白分解的驱动机制。这个 该提案的第一个目标是定义如何在无数种底物中选择降解底物 线粒体蛋白质。降解信号序列将从生理上识别 底物询问这些信号是否在不同的线粒体蛋白中保守 能够识别常见的蛋白水解酶。第二个目标是检查识别复合体 在这些酶和特定底物之间形成的。一系列互补的生化 方法将绘制蛋白酶底物结合部位图，并确定互补接触 用于促进选择和退化。最后，我们将研究 降解室内的蛋白水解点实现了多肽键断裂的专一性 特异性，导致一类底物的位点特异性切割，包括 线粒体分裂。总之，这些实验将提供一个严格的机械分析 线粒体AAA+蛋白水解酶，并提供基础知识，以帮助发育 小分子调节剂作为未来的治疗药物。

The mitochondrial inner membrane is the site of essential cellular functions such as oxidative phosphorylation, phospholipid metabolism, and the regulation of apoptosis. These activities are performed by a composite mitochondrial proteome that requires constant resculpting to respond to both the changing metabolic demands of the cell and the emergence of damage driven by reactive oxygen species. This resculpting is performed by two mitochondrial AAA+ proteases, which harness the energy of ATP to recognize, unfold and degrade protein substrates both from within and surrounding the inner membrane. In humans, dysfunction of these proteases has been linked to the development of severe neurodegenerative disorders such as spinocerebellar ataxia. AAA+ proteases assemble as hexamers to form an internal proteolytic chamber into which substrates are forcibly translocated by a ring of ATPases. The study of the mitochondrial AAA+ proteases has been long hampered by their combination of multiple soluble catalytic domains with insoluble transmembrane domains for anchoring into the inner membrane. We utilize a protein- engineering approach to assemble previously membrane-constrained hexameric proteases in a soluble, active form. Our goal is to use these rebuilt proteases to perform a rigorous analysis of the mechanisms driving energy-dependent proteolysis at the mitochondrial inner membrane. The first aim of the proposal is to define how substrates are selected for degradation among the myriad mitochondrial proteins. Degradation signal sequences will be identified from physiological substrates to ask whether these signals are conserved across diverse mitochondrial proteins to enable recognition by common proteases. The second aim is to examine the recognition complex formed between these proteases and specific substrates. A series of complementary biochemical approaches will map the protease substrate binding sites and identify the complementary contacts used to promote selection and degradation. Finally, we will examine how the architecture of the proteolytic sites within the degradation chamber achieves specificity of peptide-bond cleavage specificity, resulting in site-specific cleavage of a class of substrates, including the regulator of mitochondrial fission. Together, these experiments will provide a rigorous mechanistic analysis of the mitochondrial AAA+ proteases and provide foundational knowledge to aid the development of small molecule modulators as future therapeutics.