Topological Energetics and the Cellular Quality Control of Integral Membrane Proteins

完整膜蛋白的拓扑能量学和细胞质量控制

• 批准号: 10220073
• 负责人: Jonathan Patrick Schlebach
• 金额: $ 30.46万
• 依托单位: TRUSTEES OF INDIANA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-15 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10220073
• 关键词: Anabolism; Biochemical; Biological; Biological Models; Biological Process; Buffers; Cells; Collection; Complex; Computer Analysis; Conflict (Psychology); Data Analyses; Defect; Disease; Drug Targeting; Endoplasmic Reticulum; Engineering; Environment; Equilibrium; Evolution; Experimental Designs; G-Protein-Coupled Receptors; Genetic Diseases; Housekeeping; Human; Hydrophobicity; In Vitro; Integral Membrane Protein; Investigation; Link; Logic; Measurement; Mediating; Membrane; Membrane Proteins; Molecular; Molecular Chaperones; Molecular Conformation; Monitor; Mutation; Nature; Outcome; Pathogenicity; Play; Point Mutation; Polar Bear; Process; Production; Property; Protein Biosynthesis; Protein Engineering; Proteins; Proteome; Quality Control; Research; Retinitis Pigmentosa; Rhodopsin; Role; Series; Shapes; Side; Structural defect; Structure; Surveys; Testing; Transmembrane Domain; Variant; base; biochemical tools; biophysical techniques; biophysical tools; computerized tools; fitness; innovation; insight; misfolded protein; mutation screening; non-Native; novel; polypeptide; protein degradation; protein folding; protein function; protein structure; protein transport; proteostasis; tool; trafficking; translation assay

ABSTRACT Nearly all biological processes require proper production and degradation of cellular proteins. Maintaining this balance in protein homeostasis (proteostasis) is therefore essential to cellular fitness. Furthermore, a lapse in cellular proteostasis has been linked to the molecular basis of a wide variety of genetic diseases. Nevertheless, the manner by which the cell buffers adaptive swings in proteostasis and the impact of mutations on this process remains poorly understood. This is especially true concerning integral membrane proteins, which account for a quarter of the human proteome and the majority of current drug targets. Emerging evidence suggests the production of folded, functional, and properly localized membrane proteins in the cell is typically inefficient and sensitive to the effects of mutations. The interaction of nascent membrane proteins with molecular chaperones and other components of the cellular quality control (QC) network seems to play a central role in the efficiency of membrane protein biosynthesis and trafficking. However, the structural properties of co-translational folding intermediates as well as the nature of their interactions with molecular chaperones remain poorly understood. Nevertheless, the formation of these interactions implies that conformational defects are common among nascent membrane proteins. Based on the physicochemical mechanisms of cotranslational membrane protein folding, we hypothesize that the formation of non-native topomers during biosynthesis drives the QC-mediated retention of nascent proteins in the ER. Using the G-protein coupled receptor rhodopsin as a model system, we have employed a novel protein engineering approach to demonstrate that the activity of cellular QC is sensitive to the topological energetics. Moreover, we provide preliminary evidence that the pathogenic misfolding of rhodopsin, which is associated with retinitis pigmentosa, can arise from the stabilization of a non-native topomer. Using this approach, we will probe the nature of the interface between the topological energy landscape and the activity of the cellular QC network. To gain insights into the generality of these findings and the evolutionary trade-offs between folding and function, we will employ a novel adaptation of deep mutational scanning to survey the proteostatic effects of every possible point mutation in rhodopsin. The results will reveal whether the conformational equilibria of rhodopsin has evolved to be metastable or to maximize the efficiency of biosynthesis. Computational analysis of the results will also provide insights into the nature of the structural defects that give rise to proteostatic perturbations. Finally, we provide preliminary evidence that the constraints of cotranslational folding impose a contact order bias in the native structural ensembles of integral membrane proteins. To explore this paradigm, computational analyses of polytopic membrane proteins of known structure in conjunction with experimental measurements of helical interactions will be employed to determine the extent to which native, sequence-local contacts influence co-translational folding. Together, these results will provide fundamental insights into the mechanisms of membrane protein folding in the cell and the molecular basis of disease.

摘要 几乎所有的生物过程都需要细胞蛋白质的适当产生和降解。保持这种 因此，蛋白质稳态（蛋白质稳态）的平衡对于细胞适应性是必不可少的。此外， 细胞蛋白质稳态与多种遗传疾病的分子基础有关。然而，尽管如此， 细胞缓冲蛋白质稳态适应性波动的方式以及突变对这一过程的影响 仍然知之甚少。这对于膜整合蛋白来说尤其如此，它们是膜整合蛋白的一个重要组成部分。 人类蛋白质组的四分之一和目前大多数药物靶点。新出现的证据表明， 在细胞中产生折叠的、功能性的和适当定位的膜蛋白通常是低效的， 对突变的影响很敏感新生膜蛋白与分子伴侣的相互作用 和蜂窝质量控制（QC）网络的其他组件似乎在效率方面发挥着核心作用， 膜蛋白的合成和运输。然而，共翻译折叠的结构特性 中间体以及它们与分子伴侣相互作用的性质仍然知之甚少。 然而，这些相互作用的形成意味着构象缺陷是常见的， 新生膜蛋白基于共翻译膜蛋白的物理化学机制， 折叠，我们假设，在生物合成过程中形成的非天然拓扑异构体驱动QC介导的 新生蛋白质保留在ER中。以G蛋白偶联受体视紫红质为模型系统， 已经采用了一种新的蛋白质工程方法来证明细胞QC的活性是敏感的， 到拓扑能量学。此外，我们提供的初步证据表明，致病性错误折叠的 与色素性视网膜炎相关的视紫红质可能由非天然拓扑异构体的稳定化产生。 使用这种方法，我们将探索拓扑能量景观和 蜂窝QC网络的活动。为了深入了解这些发现的一般性和进化的 在折叠和功能之间的权衡，我们将采用一种新的适应深度突变扫描来调查 视紫红质中每一个可能的点突变的蛋白抑制作用。结果将揭示是否 视紫红质的构象平衡已经进化为亚稳态或最大化生物合成效率。 计算分析的结果也将提供洞察的性质，结构缺陷，使 上升到蛋白质稳定扰动。最后，我们提供了初步的证据，共翻译的限制， 折叠在完整膜蛋白的天然结构集合中施加接触顺序偏差。探讨 这种范例，已知结构的多位膜蛋白的计算分析， 将采用螺旋相互作用的实验测量来确定天然的， 序列-局部接触影响共翻译折叠。总之，这些结果将提供基本的 深入了解细胞膜蛋白折叠的机制和疾病的分子基础。