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.

抽象的 几乎所有的生物过程都需要适当的细胞蛋白产生和降解。维护这一点 因此，蛋白质稳态（蛋白质稳定）的平衡对于细胞适应性至关重要。此外，有一段 细胞蛋白抑制剂已与多种遗传疾病的分子基础联系起来。尽管如此， 细胞缓冲蛋白抑制剂中的自适应波和突变对此过程的影响的方式 仍然很了解。关于整体膜蛋白的尤其如此，这说明了 人类蛋白质组的四分之一和大多数当前药物靶标。新兴证据表明 细胞中折叠，功能和正确局部局部膜蛋白的产生通常效率低下，并且 对突变的影响敏感。新生膜蛋白与分子伴侣的相互作用 细胞质量控制（QC）网络的其他组件似乎在效率中起着核心作用 膜蛋白的生物合成和运输。但是，共同折叠的结构特性 中间体以及它们与分子伴侣的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在 新生的膜蛋白。基于共透明膜蛋白的理化机理 折叠，我们假设生物合成期间非母层的形成驱动QC介导的 在ER中保留新生蛋白。使用G蛋白偶联受体视紫红蛋白作为模型系统，我们 已经采用了一种新型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，我们提供了初步证据表明 与色素性视网膜炎有关的视紫红质可能是由于非本地拓平的稳定而引起的。 使用这种方法，我们将探测拓扑能量景观与 细胞QC网络的活性。了解这些发现的普遍性和进化论 折叠和功能之间的权衡，我们将采用深层突变扫描的新颖调整进行调查 视紫红质中每个可能点突变的蛋白抑制作用。结果将揭示是否是否 视紫红蛋白的构象平衡已经演变为可稳定或最大化生物合成的效率。 结果的计算分析还将提供有关结构缺陷的性质的见解 上升到蛋白抑制性扰动。最后，我们提供了初步证据，表明共转倾的约束 折叠在整体膜蛋白的天然结构集合中施加了接触顺序偏置。探索 这种范式，对已知结构的多重膜蛋白的计算分析以及 将使用螺旋相互作用的实验测量来确定天然的程度， 序列 - 本地触点会影响共转折叠。这些结果将共同提供基本 洞悉细胞中膜蛋白折叠的机制和疾病的分子基础。