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Cooperative Effect of KCl and Polyethylene Glycol on Liquid-Liquid Phase Separation of Polyproline-Arginine
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摘要: 由脯氨酸（Proline，P）和精氨酸（Arginine，R）构成的双重复肽是人体内C9ORF72基因异常扩增产生的富含毒性的多肽，其异常扩增及液-液相分离（liquid-liquid phase separation，LLPS）行为是神经退行性疾病发病的重要原因，因此研究多肽的调控行为可以为治疗相关疾病提供帮助. 主要利用光学显微成像以及紫外分光光度计技术，研究35个脯氨酸-精氨酸重复多肽（PR35）的LLPS现象及其调控，并对机制进行分析. 发现PR35的LLPS的发生依赖溶液中氯化钾的浓度，当氯化钾浓度低于2 700 mmol/L时，LLPS不会出现，而其浓度高于2 700 mmol/L时，LLPS会发生. 同时在此临界浓度（2 700 mmol/L）溶液中，加入不同质量分数的分子拥挤剂聚乙二醇（PEG1000）后，发现PEG对LLPS具有促进作用，并且随着PEG质量分数升高，促进作用增强. 同时通过紫外分光光度计对吸光度进行了分析，得到的结论与显微镜的观察结果一致.Abstract: The double complex peptide composed of proline (Proline, P) and arginine (Arginine, R) is a toxicity-rich polypeptide produced by the abnormal amplification of C9ORF72 gene in the human body. The abnormal amplification and liquid-liquid phase separation (LLPS) behavior of the peptide are important causes of neurodegenerative diseases, so to study the regulatory behavior of the peptide is helpful to the therapeutic treatment of related diseases. In the paper, optical microscopic imaging and ultraviolet spectrophotometric technique were used to study the LLPS phenomenon and the regulation of related 35 proline-arginine repeat peptides (PR35), and the regulation mechanism was analyzed. It was found that the occurrence of LLPS in PR35 depended on the concentration of potassium chloride in the solution. LLPS did not take place when the concentration of KCl was lower than 2 700 mmol/L, but LLPS took place when the concentration was higher than 2 700 mmol/L. In this critical concentration (2 700 mmol/L) of the KCl solution, after adding different mass fractions of the molecular crowding agent polyethylene glycol (PEG1000), it was found that PEG had a promoting effect on LLPS, and the promoting effect increased with the increase of mass fraction of PEG concentration. At the same time, the conclusion of ultraviolet spectrophotometric absorbance analysis was consistent with the results of microscopic survey.
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Key words:
- PR35 /
- optical microscope /
- liquid-liquid phase separation /
- polyethylene glycol
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图 1 不同KCl溶液中PR35发生LLPS的相图及紫外吸光度变化散点图（PR35最终浓度始终为100 μmol/L，比例标尺为2 μm）
（a）50 mmol/L KCl，（b）1 200 mmol/L KCl，（c）2 700 mmol/L KCl，（d）3 000 mmol/L KCl，（e） 3 200 mmol/L KCl，（f）3 600 mmol/L KCl，（g） PR35在不同KCl溶液中的相行为变化范围图，（h）PR35在不同KCl浓度下吸光度变化的散点图
Figure 1. LLPS phase images and UV absorbance variation scattering diagrams of PR35 in different KCl solutions (final concentration of PR35 always 100 μmol/L，Bar=2 μm)
(a) 50 mmol/L KCl, (b) 1 200 mmol/L KCl, (c) 2 700 mmol/L KCl, (d) 3 000 mmol/L KCl, (e) 3 200 mmol/L KCl, (f) 3 600 mmol/L KCl, (g) variation range diagrams of phase behavior of PR35 in different KCl solutions, (h) scattering diagrams of absorbance variation of PR35 at different concentrations of KCl
图 2 不同质量分数的PEG1000分别在KCl溶液低浓度及高浓度下PR35的显微成像变化图及PEG1000的作用示意图（蓝色为PR35，红色为PEG1000，比例标尺为2 μm）
（a） 50 mmol/L KCl+ 0% PEG1000，（b） 50 mmol/L KCl+ 10% PEG1000，（c）~（h） 2 700 mmol/L KCl+（分别为0%，3%，7%，10%，13%，15%）PEG1000
Figure 2. Microscopic imaging variations of PEG1000 with different concentrations of PR35 at low and high concentrations of KCl solutions and schematic diagram of the effect of PEG1000 (PR35 in blue and PEG1000 in red, Bar=2 μm)
(a) 50 mmol/L KCl+ 0% PEG1000, (b) 50 mmol/L KCl+ 10% PEG1000, (c)~(h) 2 700 mmol/L KCl+ (0%, 3%, 7%, 10%, 13%, 15%, respectively) PEG1000
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