Folding and degradation of membrane proteins

膜蛋白的折叠和降解

• 批准号: 9276014
• 负责人: Heedeok Hong
• 金额: $ 29.52万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-01 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9276014
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; ATP-Dependent Proteases; Affect; Alzheimer' s Disease; Binding; Biotin; Cell membrane; Cell physiology; Cells; Clinical; Couples; Cystic Fibrosis; Data; Disease; Dislocations; Electron Spin Resonance Spectroscopy; Electrons; Environment; Equilibrium; Escherichia coli; Escherichia coli Proteins; Eukaryotic Cell; Gene Mutation; Goals; Human; Hydrophobicity; Knowledge; Lead; Lipid Bilayers; Lipids; Mediating; Membrane; Membrane Proteins; Methodology; Methods; Micelles; Mitochondria; Mitochondrial Membrane Protein; Modeling; Molecular; Molecular Chaperones; Molecular Conformation; Molecular Machines; Monitor; Mutate; Orthologous Gene; Outcomes Research; Pathogenicity; Peptide Hydrolases; Pharmacology; Physiological; Play; Preclinical Drug Evaluation; Predisposition; Process; Prokaryotic Cells; Property; Protease Domain; Protein Conformation; Protein Region; Proteins; Proteolysis; Proteome; Quality Control; Reporting; Research; Retinitis Pigmentosa; Role; Series; Spastic Paraplegia; Spinocerebellar Ataxias; Streptavidin; Structure; System; Testing; Thermodynamics; Translating; Variant; Water; base; design; human disease; improved; innovation; insight; membrane model; misfolded protein; nervous system disorder; novel; novel therapeutics; protein E; protein degradation; protein folding; protein misfolding; public health relevance; rhomboid; small molecule; tool; virtual

 DESCRIPTION (provided by applicant): Cells use selective degradation of misfolded and intrinsically unstable proteins to maintain physiologically appropriate levels of functional proteins. Imbalances between intracellular protein folding and degradation cause severe human diseases, via excessive degradation of mutated proteins (e.g. cystic fibrosis, retinitis pigmentosa), or toxic accumulation of misfolded proteins that overwhelms cellular degradation capacity (e.g. Alzheimer's disease). A key gap in understanding of protein folding-degradation relationships and mechanisms is its current restriction to only water-soluble (cytosolic) proteins, whereas little is known about membrane proteins, despite their major physiological and pathogenic importance. This knowledge gap is mainly due to inherent difficulties of analyzing folding of membrane proteins within their native lipid bilayer environment. Based on our strong preliminary data and novel steric-trapping molecular tools, the long-term objective of this project is to elucidate molecular mechanisms and determinants of membrane protein degradation, by defining the molecular and quantitative relationships between intrinsic folding properties of membrane proteins, including global vs. local stability, unfolding rates, and hydrophobicity of transmembrane segments, and their degradation. We will use an innovative combined model consisting of the membrane- integrated ATP-dependent E. coli protease FtsH as model degradation machine, and the intramembrane protease GlpG from E. coli as model substrate, both of which are widely conserved in prokaryotic and eukaryotic cells. The aims are: 1) To develop new methodologies for determining the stability of membrane proteins in their native bilayers, by adapting our prior steric trapping innovations, supported by preliminary findings. 2) Because substrate unfolding is a prerequisite to degradation mediated by ATP-dependent proteases, using these new methods, we will elucidate how the perturbation of GlpG structure caused by the force generated by FtsH-mediated ATP hydrolysis drives its unfolding, including cooperativity mechanisms, and identify the conformation of the resultant unfolded state that is targeted for degradation. 3) To define the quantitative relationship between folding and degradation rates of GlpG, including the influences of conformational stability and hydrophobicity, by analyzing degradation in a series of variants. Outcomes of this research will provide quantitative methods for analyzing membrane protein folding, will advance fundamental understanding and current concepts of cellular quality control systems for membrane proteins based on their folding properties, and will inform progress towards new therapies for diseases caused by aberrant protein- folding.

 描述（由申请人提供）：细胞利用错误折叠和本质上不稳定的蛋白质的选择性降解来维持功能蛋白质的生理学适当水平。细胞内蛋白质折叠和降解之间的不平衡会通过突变蛋白质的过度降解（例如囊性纤维化、色素性视网膜炎）或错误折叠蛋白质的毒性积累而导致严重的人类疾病，从而压倒细胞降解能力（例如阿尔茨海默病）。理解蛋白质折叠-降解关系和机制的一个关键差距是其目前仅限于水溶性（胞质）蛋白质， 尽管膜蛋白具有重要的生理和致病作用，但人们对它们知之甚少。这种知识差距主要是由于分析膜蛋白在其天然脂质双层环境中的折叠的固有困难。基于我们强大的初步数据和新颖的空间捕获分子工具，该项目的长期目标 的目的是通过定义膜蛋白内在折叠特性之间的分子和定量关系来阐明膜蛋白降解的分子机制和决定因素，包括整体与局部稳定性、解折叠速率和跨膜片段的疏水性及其降解。我们将使用一种创新的组合模型，其中以膜整合的 ATP 依赖性大肠杆菌蛋白酶 FtsH 作为模型降解机，以来自大肠杆菌的膜内蛋白酶 GlpG 作为模型底物，这两种酶在原核和真核细胞中都广泛保守。目标是： 1) 在初步研究结果的支持下，通过采用我们之前的空间捕获创新，开发新的方法来确定膜蛋白在其天然双层中的稳定性。 2）由于底物解折叠是ATP依赖性蛋白酶介导降解的先决条件，因此使用这些新方法，我们将阐明FtsH介导的ATP水解产生的力引起的GlpG结构扰动如何驱动其解折叠，包括协同机制，并确定最终降解目标的解折叠状态的构象。 3）通过分析一系列变体的降解，定义GlpG折叠和降解速率之间的定量关系，包括构象稳定性和疏水性的影响。这项研究的成果将为分析膜蛋白折叠提供定量方法，将促进基于膜蛋白折叠特性的细胞质量控制系统的基本理解和当前概念，并将为异常蛋白折叠引起的疾病的新疗法的进展提供信息。