Folding and degradation of membrane proteins

 描述(由申请人提供)：细胞利用选择性降解错误折叠和本质不稳定的蛋白质来维持生理上适当的功能蛋白质水平。细胞内蛋白质折叠和降解之间的不平衡通过突变蛋白质的过度降解(例如囊性纤维化、视网膜色素变性)或错误折叠的蛋白质的有毒积累而超过细胞的降解能力而导致严重的人类疾病(例如阿尔茨海默病)。在理解蛋白质折叠-降解关系和机制方面的一个关键差距是它目前仅限于水溶性(胞浆)蛋白质， 尽管膜蛋白具有重要的生理和致病作用，但人们对其知之甚少。这一知识差距主要是由于分析膜蛋白在其天然脂双层环境中折叠的固有困难。基于我们强大的初步数据和新的空间捕获分子工具，该项目的长期目标是 旨在阐明膜蛋白降解的分子机制和决定因素，通过确定膜蛋白的内在折叠特性与其降解之间的分子和定量关系，包括膜蛋白的整体稳定性和局部稳定性、去折叠速率和跨膜片段的疏水性。我们将使用一个创新的组合模型，包括膜整合的依赖于ATP的大肠杆菌蛋白酶FtsH作为模型降解机，以及来自大肠杆菌的膜内蛋白酶GlpG作为模型底物，两者都在原核和真核细胞中广泛保守。其目的是：1)开发新的方法来确定膜蛋白在其天然双层中的稳定性，通过采用我们先前的空间捕集创新，并得到初步发现的支持。2)由于底物展开是ATP依赖的蛋白水解酶介导的降解的先决条件，利用这些新的方法，我们将阐明FtsH介导的ATP水解力引起的GlpG结构的扰动是如何驱动其展开的，包括协同机制，并鉴定所得到的目标降解的展开状态的构象。3)通过分析一系列突变体的降解情况，确定GlpG的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。本研究的结果将为分析膜蛋白折叠提供定量的方法，将促进对基于折叠特性的膜蛋白细胞质量控制系统的基本认识和当前的概念，并将为蛋白质折叠异常引起的疾病的新疗法的进展提供信息。

 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.