Molecular mechanisms used by chaperones to reduce the toxicity of aberrant protein oligomers
Molecular mechanisms used by chaperones to reduce the toxicity of aberrant protein oligomers
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DOI：
10.1073/pnas.1117799109
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发表时间：
2012-07-31
影响因子：
11.1
通讯作者：
Chiti, Fabrizio
中科院分区：
文献类型：
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作者：
Mannini, Benedetta;Cascella, Roberta;Chiti, Fabrizio
伴侣蛋白是蛋白质稳态网络的主要调节剂，并且已知促进蛋白质折叠、抑制蛋白质聚集、以及促进细胞内错误折叠聚集体的解聚和清除。我们已经测试了五种分子伴侣对错误折叠的寡聚体的毒性的影响，所述错误折叠的寡聚体是从细胞外添加到培养细胞中的三种不同蛋白质预先形成的。所有的伴侣被发现降低低聚物的毒性显着，即使在非常低的伴侣/蛋白质的摩尔比，只要它们被添加到细胞外，而不是被过度表达在胞质溶胶。红外光谱和定点标记实验，使用芘排除了离散的低聚物内的结构重组。相反，共聚焦显微镜，SDS-PAGE，和内在荧光测量表明紧密结合的寡聚体和分子伴侣之间。此外，原子力显微镜成像表明，更大的组件的低聚物形成的伴侣的存在下。这表明，伴侣结合到低聚物，并促进其组装成更大的物种，随之而来的反应性表面的屏蔽和减少其扩散流动性。总的来说，数据表明伴侣蛋白有效地中和细胞外错误折叠的寡聚体的一般能力，并揭示蛋白质寡聚体进一步组装成更大的种类可以是中和这种细胞外种类的有效策略。
Chaperones are the primary regulators of the proteostasis network and are known to facilitate protein folding, inhibit protein aggregation, and promote disaggregation and clearance of misfolded aggregates inside cells. We have tested the effects of five chaperones on the toxicity of misfolded oligomers preformed from three different proteins added extracellularly to cultured cells. All the chaperones were found to decrease oligomer toxicity significantly, even at very low chaperone/protein molar ratios, provided that they were added extracellularly rather than being overexpressed in the cytosol. Infrared spectroscopy and site-directed labeling experiments using pyrene ruled out structural reorganizations within the discrete oligomers. Rather, confocal microscopy, SDS-PAGE, and intrinsic fluorescence measurements indicated tight binding between oligomers and chaperones. Moreover, atomic force microscopy imaging indicated that larger assemblies of oligomers are formed in the presence of the chaperones. This suggests that the chaperones bind to the oligomers and promote their assembly into larger species, with consequent shielding of the reactive surfaces and a decrease in their diffusional mobility. Overall, the data indicate a generic ability of chaperones to neutralize extracellular misfolded oligomers efficiently and reveal that further assembly of protein oligomers into larger species can be an effective strategy to neutralize such extracellular species.