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

基本信息

批准号：
10641861
负责人：
Steven Glynn
金额：
$ 33.85万
依托单位：
STATE UNIVERSITY NEW YORK STONY BROOK
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-07-15 至 2025-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10641861
关键词：
ATP Hydrolysis ATP phosphohydrolase Active Sites Address Affinity Apoptosis Apoptotic Architecture Automobile Driving Binding Binding Sites Biochemical Biological Assay Catalytic Domain Cell physiology Cells Collection Complex Crowding Dangerousness Development Environment Enzymes Equilibrium Eukaryotic Cell Event Family Fluorescence Polarization Functional disorder Future Genome Goals Grant Human Individual Inner mitochondrial membrane Kinetics Knowledge Link Maps Mass Spectrum Analysis Measures Membrane Metabolic Mitochondria Mitochondrial Proteins Modeling Molecular Molecular Chaperones Mutation Mutation Analysis Names Neurodegenerative Disorders OPA1 gene Organelles Oxidative Phosphorylation Peptide Fragments Peptide Hydrolases Peptide Signal Sequences Peptides Phospholipid Metabolism Physiological Protein Engineering Protein Isoforms Proteins Proteolysis Proteome Quality Control Reactive Oxygen Species Regulation Regulation of Proteolysis Role Series Set protein Side Signal Transduction Site Specificity Spinocerebellar Ataxias Structure Surface System Therapeutic Transmembrane Domain Yeasts crosslink experimental study human disease in vivo in vivo monitoring oxidative damage preference protein degradation proteostasis small molecule

项目摘要

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.

线粒体内膜是重要细胞功能的场所，例如氧化磷酸化、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，需要不断重塑才能做出反应细胞不断变化的代谢需求以及由细胞引起的损伤的出现活性氧。这种重塑是由两种线粒体 AAA+ 蛋白酶完成的，它利用 ATP 的能量来识别、展开和降解来自在内膜之内和周围。在人类中，这些蛋白酶的功能障碍已被与严重神经退行性疾病（如脊髓小脑共济失调）的发展有关。 AAA+ 蛋白酶组装成六聚体，形成内部蛋白水解室，其中底物被 ATP 酶环强制移位。线粒体 AAA+ 的研究蛋白酶长期以来因其多个可溶性催化结构域的组合而受到阻碍用于锚定到内膜的不溶性跨膜结构域。我们利用一种蛋白质—— 将先前膜限制的六聚体蛋白酶组装在可溶的活性形式。我们的目标是使用这些重建的蛋白酶来执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。这该提案的第一个目标是定义如何在无数的基质中选择用于降解的基质线粒体蛋白。降解信号序列将从生理学中鉴定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常见的蛋白酶识别。第二个目的是检查识别复合体这些蛋白酶和特定底物之间形成。一系列补充生化方法将绘制蛋白酶底物结合位点并识别互补接触点用于促进选择和降解。最后，我们将研究该架构如何降解室内的蛋白水解位点实现肽键切割的特异性特异性，导致一类底物的位点特异性裂解，包括调节剂线粒体裂变。这些实验将共同提供严格的机制分析线粒体 AAA+ 蛋白酶并提供帮助开发的基础知识小分子调节剂作为未来的治疗方法。

项目成果

期刊论文数量（5）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Quality-control mechanisms targeting translationally stalled and C-terminally extended poly(GR) associated with ALS/FTD.

针对与 ALS/FTD 相关的翻译停滞和 C 末端延伸聚 (GR) 的质量控制机制。

DOI：
发表时间：
2020
期刊：
影响因子：
11.1
作者：
Li, Shuangxi;Wu, Zhihao;Tantray, Ishaq;Li, Yu;Chen, Songjie;Dong, Jason;Glynn, Steven;Vogel, Hannes;Snyder, Michael;Lu, Bingwei
通讯作者：
Lu, Bingwei

Multifunctional Mitochondrial AAA Proteases.

多功能线粒体 AAA 蛋白酶。