Department of Chemical Engineering and Biotechnology, University of Cambridge, Pembroke Street, Cambridge CB2 3RA (U.K.).
Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW (U.K.).
Chemphyschem. 2011 Feb 25;12(3):673-680. doi: 10.1002/cphc.201000996. Epub 2011 Feb 9.
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 a-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.
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通过已建立的生物物理技术在体外进行研究，但直接观察体内发生的聚集过程的性质和动力学要困难得多。然而，我们在这里描述了一种荧光共振能量转移传感器，该传感器利用我们的观察结果，即淀粉样体可以作为荧光蛋白变体的能量受体，从而可以通过活系统来量化淀粉样蛋白的聚集动力学。观察到的寿命缩短可归因于荧光能量转移到与生长中的淀粉样物质相关的固有能量状态。事实上，对于与帕金森病相关的聚集蛋白α-突触核蛋白，我们已经使用该传感器来跟踪在体外和体内发生的自缔合反应的动力学，并揭示随之而来的聚集物质的性质。实验在体外、培养的细胞中和活体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。在活体动物中测量致病性淀粉样物质的出现和发展并将此类数据与体外观察到的类似过程相关联的能力为研究错误折叠疾病家族的病理学提供了一种强大的新工具。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，以及它们对定义与其相关的疾病的分子生理学的意义。
