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（由适用提供）：细胞使用错误折叠和本质上不稳定蛋白的选择性降解来维持物理适当的功能蛋白水平。细胞内蛋白质折叠和降解之间的失衡会导致严重的人类疾病，这是由于突变蛋白（例如囊性纤维化，色素性视网膜炎）的过度降解或造成错误折叠的蛋白质积累而使细胞降解能力淹没的蛋白质（例如，alzheimersy's''）。了解蛋白质折叠降解关系和机制的关键差距是其当前仅限制水溶性（胞质）蛋白质， 尽管对膜蛋白的了解知之甚少，但它们的物理和致病性很重要。这种知识差距主要是由于继承了在其天然脂质双层环境中分析膜蛋白折叠的困难。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 通过定义膜蛋白的固有折叠特性之间的分子和定量关系，阐明膜蛋白降解的分子机制和决定剂，包括膜蛋白的固有折叠特性，包括全球稳定性，展开速率，展开速率，以及跨膜片段的疏水性及其降解。我们将使用一个创新的组合模型，该模型由膜整合的ATP依赖性大肠杆菌蛋白FTSH作为模型降解机，以及来自大肠杆菌作为模型底物的膜内蛋白GLPG，它们在原核和真核生物细胞中均广泛保守。目的是：1）开发新方法来确定膜蛋白在其本地双层中的稳定性，通过调整我们先前的空间诱捕创新，并得到初步发现的支持。 2）由于底物展开是由ATP依赖性蛋白酶介导的降解的先决条件，因此使用这些新方法，我们将阐明如何由FTSH介导的ATP水解产生的力引起的GLPG结构的扰动如何驱动其展开机制，并确定所得到的构造。 3）通过分析一系列变体中的降解，定义了GLPG的折叠与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。这项研究的结果将提供用于分析膜蛋白折叠的定量方法，将根据其折叠特性提高对膜蛋白的细胞质量控制系统的基本理解和当前的概念，并将告知因异常蛋白质折叠引起的新疗法的进展。