Chau Edward, Kim Jin Ryoun
Departments of Chemical and Biomolecular Engineering, New York University, 6 MetroTech Center, Brooklyn, NY 11201, USA.
Biochem Eng J. 2022 Jan;178. doi: 10.1016/j.bej.2021.108292. Epub 2021 Nov 29.
The aggregation of α-synuclein (αS) into oligomers and fibrils is implicated in the pathology of Parkinson's Disease (PD). While a molecular probe for rapid and comprehensive evaluation of αS aggregation states is critical for a better understanding of PD pathology, identification of therapeutic candidates, and the development of early diagnostic strategies, no such probe has yet to be developed. A structurally flexible αS variant, PG65, was previously developed as a target binding-driven, conformation-switching molecular probe for rapid αS oligomer detection. Though informative, detection using PG65 provides no comprehensive assessment of the αS aggregation states. In the present study, we report engineering of a molecular probe, PG65-MIMO (a PG65 variant with Multiple-Inputs and Multiple-Outputs), that rapidly (within 2 hr) produces comprehensive information on αS aggregation states. PG65-MIMO generates distinct fluorescence responses to the three major αS conformers (monomers, oligomers, and fibrils). PG65-MIMO also displays unique fluorescent signals for αS oligomers, depending on the tris(2-carboxyethyl)phosphine (TCEP) concentration. Our results suggest that the TCEP dependent signaling of PG65-MIMO may be associated with its conformational states. Overall, our study illustrates engineering of an αS variant to create a molecular probe for handling multiple inputs and multiple outputs, addressing the technological gap in αS detection.
α-突触核蛋白（αS）聚集成寡聚体和原纤维与帕金森病（PD）的病理过程有关。虽然用于快速全面评估αS聚集状态的分子探针对于更好地理解PD病理、鉴定治疗候选物以及开发早期诊断策略至关重要，但尚未开发出这样的探针。一种结构灵活的αS变体PG65，此前被开发为一种基于靶标结合驱动的构象转换分子探针，用于快速检测αS寡聚体。尽管具有信息价值，但使用PG65进行的检测无法全面评估αS的聚集状态。在本研究中，我们报告了一种分子探针PG65-MIMO（一种具有多输入和多输出的PG65变体）的工程设计，该探针能在2小时内快速产生关于αS聚集状态的全面信息。PG65-MIMO对三种主要的αS构象体（单体、寡聚体和原纤维）产生不同的荧光响应。根据三（2-羧乙基）膦（TCEP）的浓度，PG65-MIMO对αS寡聚体也显示出独特的荧光信号。我们的结果表明，PG65-MIMO的TCEP依赖性信号传导可能与其构象状态有关。总体而言，我们的研究展示了通过工程设计αS变体来创建一种用于处理多输入和多输出的分子探针，解决了αS检测中的技术差距。
