Visualizing the Multistep Process of Protein Aggregation in Live Cells.
Visualizing the Multistep Process of Protein Aggregation in Live Cells.
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DOI：
10.1021/acs.accounts.1c00648
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发表时间：
2022-02-01
影响因子：
18.3
通讯作者：
Zhang, Xin
中科院分区：
文献类型：
--
作者：
Ye, Songtao;Hsiung, Chia-Heng;Tang, Yuqi;Zhang, Xin
蛋白质聚集是一种生物现象，在这种现象中，经过异常加工或突变的蛋白质会错误折叠并组装成各种不可溶的聚集体。几十年的研究已经描绘了蛋白质的结构、相互作用和活性，这些蛋白质要么是天然折叠的结构，要么是不可溶的聚集体，如淀粉样纤维。然而，在蛋白质折叠的这两个极端状态之间存在着各种中间物种。在这里，我们将这些构象统称为错误折叠的蛋白质低聚物，包括由未折叠或错误折叠的蛋白质形成的可溶性低聚物和淀粉样前体低聚物。虽然已经开发了大量的工具来研究折叠蛋白或淀粉样纤维，但由于缺乏在活细胞中检测和询问这些物种的方法，理解错误折叠的蛋白低聚物的性质和活性的研究一直受到限制。在这篇文章中，我们描述了我们在开发化学方法方面所做的努力，这些方法允许对活细胞中的多步骤蛋白质聚集过程进行表征，特别是错误折叠的蛋白质低聚体。作为这一旅程的开始，我们试图开发一种荧光方法，其中错误折叠的低聚物可以打开连接到目标蛋白(POI)的化学探针的荧光。为此，我们生产了一系列不稳定的HaloTag变体，形成了AgHalo传感器的主要组件，当细胞受到压力时，它会错误折叠和聚集。当AgHalo与溶剂变色的荧光团共价连接时，AgHalo共轭的错误折叠会激活荧光，导致观察到错误折叠的低聚物。在这项工作之后，我们通过AggTag方法将检测范围从AgHalo扩展到任何感兴趣的蛋白质，其中POI基因融合到自我标记的蛋白质标签(HaloTag或SNAP-Tag)。针对基于分子转子的荧光团，我们应用调制的FP发色团核心作为AggTag探针的原型，以实现对活细胞中多种蛋白质的错误折叠的可溶性低聚物的荧光检测。接下来，我们进一步发展了AggTag方法来区分不溶性聚集体和错误折叠的低聚物，使用了两类不同激活这两种构象的荧光发射的探针。为了实现这一目标，我们应用物理有机化学和计算化学发现了一类新的类三极管荧光团，其中电子密度调节器或给体-受体键的π轨道被用来控制荧光团在激发态的转动势垒。这一机制使我们能够合理地设计出对粘度有理想响应的分子旋转型荧光团，从而扩大了AggTag方法的应用范围。综上所述，我们的工作使该领域能够直接监测中间错误折叠的低聚物，并从活细胞中的这种构象中区分出不可溶的聚集体，从而使许多目前尚未回答的蛋白质聚集问题的研究成为可能。未来的方向是开发能够对蛋白质聚集过程进行定量分析的方法。此外，需要新的方法来检测和量化形成无膜细胞器的蛋白质或RNA凝聚体的形成和成熟。
Protein aggregation is a biological phenomenon in which aberrantly processed or mutant proteins misfold and assemble into a variety of insoluble aggregates. Decades of studies have delineated the structure, interaction, and activity of proteins in either their natively folded structures or in insoluble aggregates such as amyloid fibrils. However, a variety of intermediate species exist between these two extreme states in the protein folding landscape. Herein, we collectively term these conformations as misfolded protein oligomers, including soluble oligomers and pre-amyloidal oligomers that are formed by unfolded or misfolded proteins. While extensive tools have been developed to study folded proteins or amyloid fibrils, research to understand the properties and activities of misfolded protein oligomers has been limited by the lack of methods to detect and interrogate these species in live cells. In this Account, we describe our efforts in the development of chemical methods that allow for the characterization of the multi-step protein aggregation process, in particular the misfolded protein oligomers, in living cells. As the start of this journey, we attempted to develop a fluorogenic method, wherein the misfolded oligomers could turn on the fluorescence of chemical probes that are conjugated to the protein-of-interest (POI). To this end, we produced a series of destabilized HaloTag variants, formulating the primary component of the AgHalo sensor, which misfold and aggregate when cells are subjected to stress. When AgHalo is covalently conjugated with a solvatochromic fluorophore, misfolding of the AgHalo conjugation would activate fluorescence, resulting in the observation of misfolded oligomers. Following this work, we extended the scope of detection from AgHalo to any protein-of-interest via the AggTag method, wherein the POIs are genetically fused to self-labeling protein tags (HaloTag or SNAP-tag). Focusing on the molecular rotor-based fluorophores, we applied the modulated FP chromophore core as a prototype for the AggTag probes, to enable the fluorogenic detection of misfolded soluble oligomers of multiple proteins in live cells. Next, we further developed the AggTag method to distinguish insoluble aggregates from misfolded oligomers, using two classes of probes that differently activate fluorescence emission towards these two conformations. To enable this goal, we applied physical organic chemistry and computational chemistry to discover a new category of triode-like fluorophores, wherein the π orbitals of either an electron density regulator or the donor-acceptor linkages are used to control the rotational barriers of fluorophores at the excited states. This mechanism allows us to rationally design molecular rotor-based fluorophores that have desired responses to viscosity, thus extending the application of the AggTag method. In summary, our work allows the field to directly monitor the intermediate misfolded oligomers and differentiate insoluble aggregates from this conformation in live cells, thus enabling the studies of many currently unanswered questions in protein aggregation. Future directions are to develop methods that enable quantitative analyses of the protein aggregation process. Further, new methods are needed to detect and quantify the formation and maturation of protein or RNA condensates that form membraneless organelles.
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影响因子：
15
作者：
Liu Y;Wolstenholme CH;Carter GC;Liu H;Hu H;Grainger LS;Miao K;Fares M;Hoelzel CA;Yennawar HP;Ning G;Du M;Bai L;Li X;Zhang X
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影响因子：
15
作者：
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通讯作者：
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影响因子：
5.6
作者：
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通讯作者：
Kinjo M
影响因子：
19
作者：
Boeynaems S;Alberti S;Fawzi NL;Mittag T;Polymenidou M;Rousseau F;Schymkowitz J;Shorter J;Wolozin B;Van Den Bosch L;Tompa P;Fuxreiter M
通讯作者：
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影响因子：
3.7
作者：
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