Topological Energetics and the Cellular Quality Control of Integral Membrane Proteins

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。保持这一点 因此，蛋白质稳态(蛋白质稳态)的平衡对细胞健康至关重要。此外，一个失误， 细胞蛋白平衡与多种遗传性疾病的分子基础有关。不过， 细胞缓冲蛋白质平衡中适应性摆动的方式以及突变对这一过程的影响 人们对此仍然知之甚少。对于完整的膜蛋白来说尤其如此，它占了 四分之一的人类蛋白质组和大多数当前的药物靶点。新出现的证据表明 在细胞中生产折叠的、有功能的和适当定位的膜蛋白通常是低效的和 对突变的影响敏感。新生细胞膜蛋白与分子伴侣的相互作用 而蜂窝质量控制(QC)网络的其他组件似乎在效率中发挥着核心作用 膜蛋白的生物合成和运输。然而，共平移折叠的结构性质 中间体及其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 新生的膜蛋白。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非天然拓扑异构体的形成驱动QC介导的 内质网中新生蛋白质的滞留。以G蛋白偶联受体视紫红质为模型系统，我们 已经使用了一种新的蛋白质工程方法来证明细胞QC的活动是敏感的 到拓扑能量学。此外，我们提供了初步证据表明，致病的错误折叠 视紫红质与视网膜色素变性有关，它可以由非天然的拓扑物稳定产生。 使用这种方法，我们将探索拓扑能量图景和 蜂窝QC网络的活动。为了深入了解这些发现的普遍性和进化 在折叠和功能之间权衡，我们将使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对蛋白质的抑制作用。选举结果将揭示出 视紫红质的构象平衡已经进化为亚稳定的或最大化的生物合成效率。 对结果的计算分析也将提供对结构缺陷的本质的洞察，这些缺陷给出了 上升到蛋白质代谢的扰动。最后，我们提供了初步的证据，证明了合作翻译的约束 折叠在完整的膜蛋白的天然结构集合中施加了接触顺序偏差。去探索 在这种模式下，已知结构的多孔膜蛋白的计算分析与 螺旋相互作用的实验测量将被用来确定自然的， 序列局部接触影响共翻译折叠。总而言之，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。

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.