Conformational analysis of misfolded protein aggregation by FRET and live-cell imaging techniques.
Conformational analysis of misfolded protein aggregation by FRET and live-cell imaging techniques.
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通过FRET和活细胞成像技术对错误折叠蛋白聚集的构象分析。
DOI：
10.3390/ijms16036076
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发表时间：
2015-03-16
影响因子：
5.6
通讯作者：
Kinjo M
中科院分区：
文献类型：
--
作者：
Kitamura A;Nagata K;Kinjo M
细胞内稳态由几种类型的蛋白质机制维持，包括分子伴侣和蛋白水解系统。蛋白质组的失调破坏细胞、组织和整个生物体的稳态，并且被假设为引起神经退行性疾病，包括肌萎缩侧索硬化症（ALS）和亨廷顿病（HD）。神经退行性疾病的一个标志是在神经元中形成泛素阳性包涵体，这表明错误折叠的蛋白质的聚集过程在疾病进展期间发生变化。因此，高通量测定可溶性寡聚体在聚集过程中，以及隔离的蛋白质在包涵体中的构象，是必不可少的阐明生理调节机制和药物发现在这一领域。为了阐明聚集倾向蛋白的相互作用，积累和构象，原位光谱成像技术，如Förster/荧光共振能量转移（FRET），荧光相关光谱（FCS）和双分子荧光互补（BiFC）已被采用。在这里，我们总结了最近的报告，其中这些技术被应用于分析的聚集倾向的蛋白质（特别是他们的二聚化，相互作用和构象变化），并描述了几个荧光指标用于实时观察的生理状态相关的蛋白质稳态。
Cellular homeostasis is maintained by several types of protein machinery, including molecular chaperones and proteolysis systems. Dysregulation of the proteome disrupts homeostasis in cells, tissues, and the organism as a whole, and has been hypothesized to cause neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and Huntington’s disease (HD). A hallmark of neurodegenerative disorders is formation of ubiquitin-positive inclusion bodies in neurons, suggesting that the aggregation process of misfolded proteins changes during disease progression. Hence, high-throughput determination of soluble oligomers during the aggregation process, as well as the conformation of sequestered proteins in inclusion bodies, is essential for elucidation of physiological regulation mechanism and drug discovery in this field. To elucidate the interaction, accumulation, and conformation of aggregation-prone proteins, in situ spectroscopic imaging techniques, such as Förster/fluorescence resonance energy transfer (FRET), fluorescence correlation spectroscopy (FCS), and bimolecular fluorescence complementation (BiFC) have been employed. Here, we summarize recent reports in which these techniques were applied to the analysis of aggregation-prone proteins (in particular their dimerization, interactions, and conformational changes), and describe several fluorescent indicators used for real-time observation of physiological states related to proteostasis.
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