A FRET sensor for non-invasive imaging of amyloid formation in vivo.
A FRET sensor for non-invasive imaging of amyloid formation in vivo.
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DOI：
10.1002/cphc.201000996
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发表时间：
2011-02-25
期刊：
影响因子：
--
通讯作者：
Kaminski CF
中科院分区：
文献类型：
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作者：
Kaminski Schierle GS;Bertoncini CW;Chan FTS;van der Goot AT;Schwedler S;Skepper J;Schlachter S;van Ham T;Esposito A;Kumita JR;Nollen EAA;Dobson CM;Kaminski CF
淀粉样多肽的错误折叠和聚集是许多神经退行性疾病的根源。虽然蛋白质聚集可以很容易地在体外研究，通过建立生物物理技术，直接观察的性质和动力学的聚集过程发生在体内是更具挑战性的。然而，我们在这里描述的Förster共振能量转移传感器，允许淀粉样蛋白的聚集动力学在生活系统中进行量化，利用我们的观察，淀粉样蛋白组件可以作为能量受体的荧光蛋白的变体。所观察到的寿命减少可归因于荧光能量转移到与生长的淀粉样物质相关的固有能量状态。事实上，对于α-突触核蛋白，一种其聚集与帕金森病有关的蛋白质，我们已经使用这种传感器来跟踪体外和体内发生的自缔合反应的动力学，并揭示随后聚集物质的性质。实验在体外、培养的细胞和活的秀丽隐杆线虫中进行。对于后者，读数与毒性表型的出现直接相关。测量活体动物中致病性淀粉样蛋白种类的出现和发展的能力以及将这些数据与体外观察到的类似过程相关的能力为错误折叠疾病家族的病理学研究提供了强大的新工具。我们的研究证实了聚集反应发生的分子环境的重要性，突出了体外和体内发生的过程之间的相似性和差异，以及它们对定义与它们相关的疾病的分子生理学的意义。
Misfolding and aggregation of amyloidogenic polypeptides lie at the root of many neurodegenerative diseases. Whilst protein aggregation can be readily studied in vitro by established biophysical techniques, direct observation of the nature and kinetics of aggregation processes taking place in vivo is much more challenging. We describe here, however, a Förster resonance energy transfer sensor that permits the aggregation kinetics of amyloidogenic proteins to be quantified in living systems by exploiting our observation that amyloid assemblies can act as energy acceptors for variants of fluorescent proteins. The observed lifetime reduction can be attributed to fluorescence energy transfer to intrinsic energy states associated with the growing amyloid species. Indeed, for α-synuclein, a protein whose aggregation is linked to Parkinson’s disease, we have used this sensor to follow the kinetics of the self-association reactions taking place in vitro and in vivo and to reveal the nature of the ensuing aggregated species. Experiments were conducted in vitro, in cells in culture and in living Caenorhabditis elegans. For the latter the readout correlates directly with the appearance of a toxic phenotype. The ability to measure the appearance and development of pathogenic amyloid species in a living animal and the ability to relate such data to similar processes observed in vitro provides a powerful new tool in the study of the pathology of the family of misfolding disorders. Our study confirms the importance of the molecular environment in which aggregation reactions take place, highlighting similarities as well as differences between the processes occurring in vitro and in vivo, and their significance for defining the molecular physiology of the diseases with which they are associated.