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

线粒体内膜是重要的细胞功能的场所，如氧化， 磷酸化、磷脂代谢和细胞凋亡的调节。这些活动 由复合线粒体蛋白质组执行， 与细胞不断变化的代谢需求以及由细胞内毒素引起的 活性氧这种重塑由两种线粒体AAA+蛋白酶进行， 它们利用ATP的能量来识别、展开和降解蛋白质底物， 在内膜内部和周围。在人类中，这些蛋白酶的功能障碍已经被发现。 与严重的神经退行性疾病如脊髓小脑共济失调的发展有关。 AAA+蛋白酶组装为六聚体以形成内部蛋白水解室， 底物被ATP酶环强制移位。线粒体AAA+的研究 蛋白酶长期以来受到它们的多个可溶性催化结构域与 用于锚定到内膜中的不溶性跨膜结构域。我们利用蛋白质- 工程化方法来组装以前的膜限制的六聚体蛋白酶， 可溶的活性形式。我们的目标是使用这些重建的蛋白酶来进行严格的分析， 驱动线粒体内膜能量依赖性蛋白水解的机制。的 该提案的第一个目的是定义如何在众多的降解基质中选择降解基质 线粒体蛋白降解信号序列将从生理 底物来询问这些信号是否在不同的线粒体蛋白中保守， 能够被普通蛋白酶识别。第二个目的是检查识别复合体 在这些蛋白酶和特定底物之间形成。一系列互补的生化 方法将绘制蛋白酶底物结合位点，并确定互补接触 用来促进选择和退化。最后，我们将研究 降解室内的蛋白水解位点实现了肽键切割的特异性 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制分析， 线粒体AAA+蛋白酶，并提供基础知识，以帮助发展 小分子调节剂作为未来的治疗方法。

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.