Fluorescence Turn-On Folding Sensor To Monitor Proteome Stress in Live Cells.
Fluorescence Turn-On Folding Sensor To Monitor Proteome Stress in Live Cells.
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DOI：
10.1021/jacs.5b04366
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发表时间：
2015-09-09
影响因子：
15
通讯作者：
Kelly JW
中科院分区：
文献类型：
--
作者：
Liu Y;Zhang X;Chen W;Tan YL;Kelly JW
由热扰动或相关应激引起的蛋白质组错误折叠和/或聚集，通过消耗伴侣/伴侣蛋白途径和降解途径能力，暂时挑战细胞的细胞蛋白质稳态（蛋白质稳态）网络能力。开发基于蛋白质客户端的探针来实时量化细胞蛋白质稳态网络容量是非常可取的。在此，我们介绍了一种基于从头设计的逆醛缩酶（RA）酶的热不稳定突变体的小分子调节荧光蛋白折叠传感器。由于任何细胞中都不存在 RA 酶活性，因此蛋白质折叠传感器是生物正交的。荧光小分子被设计为当它与折叠的功能性 RA 结合并发生共价反应时发出荧光。因此，在第一个实验范式中，细胞蛋白质稳态网络能力及其动态通过 RA-小分子共轭荧光反映，这与存在的折叠和功能性 RA 的量相关，前提是药理学伴侣作用最小化。在第二个实验场景中，只需将荧光探针添加到细胞培养基中，即可在细胞中预形成 RA-荧光探针缀合物。然后，在脉冲追逐型实验中施加蛋白质组错误折叠应力之前，将未反应的探针洗掉。荧光 RA-荧光探针缀合物的聚集体形成反映了蛋白质稳态网络能力不足。应激的去除导致明显的 RA-荧光探针结合物重折叠，部分是通过热休克反应转录程序增强胞质蛋白稳态网络能力介导的，部分是通过细胞蛋白水解作用的时间依赖性 RA-荧光探针结合物降解介导的。
Proteome misfolding and/or aggregation, caused by a thermal perturbation or a related stress, transiently challenges the cellular protein homeostasis (proteostasis) network capacity of cells by consuming chaperone/chaperonin pathway and degradation pathway capacity. Developing protein client-based probes to quantify the cellular proteostasis network capacity in real time is highly desirable. Herein we introduce a small-molecule-regulated fluorescent protein folding sensor based on a thermo-labile mutant of the de novo designed retroaldolase (RA) enzyme. Since RA enzyme activity is not present in any cell, the protein folding sensor is bioorthogonal. The fluorogenic small molecule was designed to become fluorescent when it binds to and covalently reacts with folded and functional RA. Thus, in the first experimental paradigm, cellular proteostasis network capacity and its dynamics are reflected by RA–small molecule conjugate fluorescence, which correlates with the amount of folded and functional RA present, provided that pharmacologic chaperoning is minimized. In the second experimental scenario, the RA–fluorogenic probe conjugate is pre-formed in a cell by simply adding the fluorogenic probe to the cell culture media. Unreacted probe is then washed away before a proteome misfolding stress is applied in a pulse-chase-type experiment. Insufficient proteostasis network capacity is reflected by aggregate formation of the fluorescent RA–fluorogenic probe conjugate. Removal of the stress results in apparent RA–fluorogenic probe conjugate re-folding, mediated in part by the heat-shock response transcriptional program augmenting cytosolic proteostasis network capacity, and in part by time-dependent RA–fluorogenic probe conjugate degradation by cellular proteolysis.