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

基本信息

批准号：
10437748
负责人：
Jonathan Patrick Schlebach
金额：
$ 30.43万
依托单位：
TRUSTEES OF INDIANA UNIVERSITY
依托单位国家：
美国
项目类别：
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/10437748
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

项目摘要

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.

抽象的几乎所有生物过程都需要细胞蛋白质的适当产生和降解。维持这个因此，蛋白质稳态（蛋白质稳态）的平衡对于细胞健康至关重要。此外，失误细胞蛋白质稳态与多种遗传疾病的分子基础有关。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此过程的影响仍然知之甚少。对于整合膜蛋白来说尤其如此，它解释了四分之一的人类蛋白质组和大多数当前药物靶点。新出现的证据表明细胞中折叠的、有功能的、正确定位的膜蛋白的生产通常效率低下，而且对突变的影响敏感。新生膜蛋白与分子伴侣的相互作用和细胞质量控制 (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生物合成和运输。然而，共平移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在新生膜蛋白。基于共翻译膜蛋白的理化机制折叠，我们假设生物合成过程中非天然拓扑异构体的形成驱动了 QC 介导的新生蛋白质保留在内质网中。使用 G 蛋白偶联受体视紫红质作为模型系统，我们采用了一种新颖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致病性错误折叠与色素性视网膜炎相关的视紫红质可以由非天然拓扑异构体的稳定化产生。使用这种方法，我们将探讨拓扑能量景观和能量场之间界面的本质。细胞 QC 网络的活动。为了深入了解这些发现的普遍性和进化折叠和功能之间的权衡，我们将采用深度突变扫描的新颖改编来调查视紫红质中每种可能的点突变的蛋白质抑制作用。结果将揭示是否视紫红质的构象平衡已进化为亚稳态或最大化生物合成效率。对结果的计算分析还将深入了解结构缺陷的性质，从而提供引起蛋白质稳态扰动。最后，我们提供了初步证据表明共翻译的限制折叠在完整膜蛋白的天然结构整体中施加了接触顺序偏差。探索在这种范式中，对已知结构的多面体膜蛋白进行计算分析将采用螺旋相互作用的实验测量来确定天然的程度，序列局部接触影响共翻译折叠。总之，这些结果将为深入了解细胞中膜蛋白折叠的机制和疾病的分子基础。

项目成果

期刊论文数量（3）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Modeling membrane geometries implicitly in Rosetta.

在 Rosetta 中隐式模拟膜几何形状。

DOI：
发表时间：
2024-03
期刊：
影响因子：
0
作者：
Woods, Hope;Leman, Julia Koehler;Meiler, Jens
通讯作者：
Meiler, Jens

Divergent Folding-Mediated Epistasis Among Unstable Membrane Protein Variants.

不稳定膜蛋白变体中不同折叠介导的上位性。

DOI：
发表时间：
2024-02-01
期刊：
影响因子：
0
作者：
Chamness, Laura M;Kuntz, Charles P;McKee, Andrew G;Penn, Wesley D;Hemmerich, Christopher M;Rusch, Douglas B;Woods, Hope;Dyotima;Meiler, Jens;Schlebach, Jonathan P
通讯作者：
Schlebach, Jonathan P

Towards generalizable predictions for G protein-coupled receptor variant expression.

对 G 蛋白偶联受体变异表达的普遍预测。

DOI：
发表时间：
2022-07-19
期刊：
影响因子：
3.4
作者：
Kuntz, Charles P;Woods, Hope;McKee, Andrew G;Zelt, Nathan B;Mendenhall, Jeffrey L;Meiler, Jens;Schlebach, Jonathan P
通讯作者：
Schlebach, Jonathan P