Department of Biology, York Biomedical Research Institute, University of York, York, United Kingdom.
Front Cell Infect Microbiol. 2022 May 6;12:894213. doi: 10.3389/fcimb.2022.894213. eCollection 2022.
Proximity labelling is a powerful and rapidly developing technology for exploring the interaction space and molecular environment of a protein of interest at the nanometre scale. In proximity labelling, a promiscuous biotinylating enzyme is genetically fused to the protein of interest, initiation of labelling then results in the biotinylating enzyme generating reactive biotin which covalently 'tags' nearby molecules. Importantly, this labelling takes place whilst the protein of interest continues to perform its normal functions in the cell. Due to its unique advantageous characteristics, proximity labelling is driving discoveries in an ever increasing range of organisms. Here, we highlight the applications of proximity labelling to the study of kinetoplastids, a group of eukaryotic protozoa that includes trypanosomes and which can cause serious disease in humans and livestock. We first provide a general overview of the proximity labelling experimental workflow including key labelling enzymes used, proper experimental design with appropriate controls and robust statistical analysis to maximise the amount of reliable spatial information that is generated. We discuss studies employing proximity labelling in kinetoplastid parasites to illustrate how these key principles of experimental design are applied. Finally, we highlight emerging trends in the development of proximity labelling methodology.
邻近标记是一种强大且快速发展的技术，可用于探索纳米尺度下目标蛋白质的相互作用空间和分子环境。在邻近标记中，一种杂乱的生物素酰化酶与目标蛋白质发生基因融合，标记的起始导致生物素酰化酶产生反应性生物素，生物素通过共价键“标记”附近的分子。重要的是，这种标记发生在目标蛋白质在细胞中继续执行其正常功能的同时。由于其独特的优势特征，邻近标记正在推动越来越多的生物体的发现。在这里，我们重点介绍邻近标记在研究动基体生物中的应用，动基体生物是一组包括锥虫在内的真核原生动物，它们可导致人类和牲畜的严重疾病。我们首先提供了邻近标记实验工作流程的概述，包括使用的关键标记酶、适当的实验设计和适当的对照以及稳健的统计分析，以最大限度地生成可靠的空间信息。我们讨论了在动基体寄生虫中使用邻近标记的研究，以说明这些实验设计原则如何得到应用。最后，我们强调了邻近标记方法学发展中的新兴趋势。
