Bioluminescence resonance energy transfer detection system for the monitoring of protein-protein interactions, post-translational modifications and cell signalling.

这项对研究工具和仪器赠款计划的应用是为了购买一种适用于高灵敏度和中等吞吐量生物发光共振能量转移(BRET)测量的多模式平板读取器。对自然产生的Bret的操纵使传感器的设计能够实时检测各种细胞事件。主要申请人的实验室开创了Bret的生物用途，并继续开发基于Bret的传感器(迄今已设计和验证了70多种不同的传感器/化验)，探测蛋白质-蛋白质相互作用，以及蛋白质构象变化、运输和信号传递。申请的BRET兼容板读取器将用于许多NSERC支持的(Discovery Grants)项目，其中包括使用BRET。我们在当前的应用中提出了四个这样的项目，这说明了该仪器可能的广泛应用。项目1旨在开发和使用基于BRET的新方法来监测G蛋白偶联受体(GPCR)信号传递的时空传播。GPCR效应器的移位和活性将以亚细胞分辨率实时监测。在项目2中，基于Bret的分析将通过监测细胞周期调节的亚细胞蛋白质在不同细胞器之间的运输来研究有丝分裂过程中细胞表面蛋白质组变化的控制机制。在项目3中，将使用基于Bret的分子内传感器来评估控制T细胞受体相关的LCK激酶活性的结构决定因素，该传感器在与CD3结合时监测该激酶的构象重排。项目4旨在表征SUMO化在控制与癌细胞衰老和迁移相关的蛋白质-蛋白质相互作用中的作用。为此，将开发一种基于Bret的检测方法，以监测小分子对Vimentin和UBC9 SUMO化状态的影响，以及它们对蛋白质-蛋白质相互作用和活性的影响。学生、博士后研究员和高素质人员将参与每个项目，使他们能够学习如何设计、执行和解释BRET结果，并获得对他们所解决的生物学问题的见解。由于Bret原理类似于荧光共振能量转移(FRET)和其他基于邻近的分析方法，如蛋白质互补、蛋白质连接和邻近依赖的生物素鉴定(BioID)，学员将接触到这些创新和备受追捧的方法。鉴于现有仪器已经达到最大容量，NSERC支持的其他项目需要购买新的BRET读取器。新一代仪器的灵敏度和吞吐量更高，将使这些项目得以有效实现。

This application to the Research Tools and Instruments Grants program is for the acquisition of a multimodal plate reader adapted for high sensitivity and medium throughput bioluminescence resonance energy transfer (BRET) measurements. Manipulation of the naturally occurring BRET has enabled the design of sensors detecting various cellular events in real time. The laboratory of the principal applicant pioneered the biological use of BRET and continues to develop BRET-based sensors (over 70 distinct sensors/assays have been designed and validated so far), probing protein-protein interactions, as well as protein conformational changes, trafficking and signaling. The BRET-compatible plate reader requested will be used in many NSERC-supported (Discovery Grants) projects that include the use of BRET. We present four of these projects in the current application, which illustrates the wide spectrum of possible applications of the instrument. Project 1 aims at developing and using new BRET-based approaches to monitor the spatio-temporal propagation of signal transmission by G protein-coupled receptors (GPCR). The translocation and activity of GPCR effectors will be monitored at subcellular resolution in real time. In project 2, BRET-based assays will be used to investigate the mechanisms controlling the changes in the cell surface proteome during mitosis by monitoring the cell cycle-regulated subcellular trafficking of proteins between various organelles. Structural determinants controlling the activity of the T cell receptor-associated Lck kinase will be assessed, in project 3, with the use of intramolecular BRET-based sensors monitoring the conformational rearrangement of the kinase upon binding to CD3. Project 4 aims at characterizing the role of SUMOylation in controlling protein-protein interactions associated with the senescence and migration of cancer cells. For this purpose, a BRET-based assay will be developed to monitor the effects of small molecules on the SUMOylation state of vimentin and UBC9, and their impact on protein-protein interactions and activity. Students, post-doctoral fellows and highly qualified personnel will be involved in each project, allowing them to learn how to design, perform and interpret BRET results in addition to gaining insights into the biological questions that they address. Because the BRET principles are similar to those of fluorescence resonance energy transfer (FRET) and other proximity-based assays, such as protein-complementation, protein-ligation and proximity-dependent biotin identification (BioID), the trainees will be exposed to these innovative and highly sought after approaches. Given that the existing instrument is already used at its maximal capacity, the additional NSERC supported projects necessitate the acquisition of a new BRET reader. The greater sensitivity and throughput of the new generation instruments will allow the efficient realization of these projects.