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：
10296122
负责人：
Steven Glynn
金额：
$ 34.36万
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/10296122
关键词：
ATP Hydrolysis ATP phosphohydrolase Active Sites Address Affinity Apoptosis Apoptotic Architecture Automobile Driving Binding Binding Sites Biochemical Biological Biological Assay Catalytic Domain Cell physiology Cells Collection Complex Crowding Dangerousness Development Environment Enzymes Equilibrium Eukaryotic Cell Event Family Fluorescence Polarization Foundations Functional disorder Future Genome Goals Grant Human Individual Inner mitochondrial membrane Kinetics Knowledge Light 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 Time 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.

线粒体内膜是必需细胞功能（例如氧化）的位点磷酸化，磷脂代谢和凋亡的调节。这些活动是由复合线粒体蛋白质组进行的对于细胞的代谢需求不断变化以及造成的损害的出现活性氧。该撤土由两个线粒体AAA+蛋白酶进行，利用ATP的能量识别，展开和降解蛋白质底物都从内部和周围的内膜。在人类中，这些蛋白酶的功能障碍是与严重的神经退行性疾病（例如脊髓脑性共济失调）的发展有关。 AAA+蛋白酶在六聚体形成内部蛋白水解腔中组装底物被ATPases环强行易位。线粒体AAA+的研究多个可溶性催化结构域与不溶性跨膜结构域，用于锚定内膜。我们利用蛋白质工程方法来组装以前膜约束的六聚体蛋白酶可溶性，有源形式。我们的目标是使用这些重建的蛋白酶对在线粒体内膜上驱动能量依赖性蛋白水解的机制。这该提案的第一个目的是定义如何在众多中选择底物降解线粒体蛋白。将从生理中鉴定出降解信号序列基板询问这些信号是否在各种线粒体蛋白上保存到可以通过常见蛋白酶识别。第二个目的是检查识别综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识别互补触点用于促进选择和降解。最后，我们将研究如何降解室内的蛋白水解位点可实现肽键裂解的特异性特异性，导致一类底物的位点特异性切割，包括调节器线粒体裂变。这些实验将共同提供严格的机械分析线粒体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剂。