邻近标记技术在弓形虫蛋白质组学研究中的应用进展

Application Advances of Proximity Labeling Technology in Toxoplasma gondii Proteomics Research

弓形虫编码数量众多的分泌蛋白,储存在微线体、棒状体和致密颗粒等虫体特有的细胞器中,于虫体入侵宿主细胞前后分泌到不同部位,并在虫体的感染、寄生和致病过程中发挥重要作用,对这些分泌蛋白的鉴定和功能研究是弓形虫致病机制研究中的热点和难点。传统的蛋白质组学技术在弓形虫亚细胞器的分离和组分鉴定上存在样品纯化困难和蛋白质组学信息覆盖率低等不足,且该技术在检测瞬时的或较弱的蛋白质相互作用时灵敏性较低。近年来,快速发展的邻近标记技术(PL)克服了传统技术的局限性。PL是将具有特定催化活性的工具酶与诱饵蛋白进行融合,进而添加生物素或生物素苯酚对诱饵蛋白邻近的靶蛋白进行生物素酰化标记的一门技术。该技术整合了酶促反应效率高的优点,可将特定空间内的蛋白进行高效标记,并可检测较弱的蛋白质相互作用,近年来在生物学研究中被广泛使用。本文对基于生物素连接酶(BirA)和抗坏血酸过氧化物酶(APEX)催化的PL在弓形虫蛋白质组学研究中的应用进行阐述,介绍2种标记方法在弓形虫分泌蛋白的挖掘和蛋白质相互作用研究中的贡献,为弓形虫致病机制的研究和弓形虫病的防控提供参考。

Toxoplasma gondii(T.gondii)encodes numerous secretory proteins stored in organelles such as micronemes,rhoptries,and dense granules,which are secreted to different locations before and after the parasite invades host cells and play crucial roles in the infection,parasitism,and pathogenesis processes.The identification and functional studies of these secretory proteins are hotspots and challenges in the research of T.gondii pathogenesis.Traditional proteomic techniques face difficulties in sample purification and low coverage of proteomic information for T.gondii subcellular organelles,and they exhibit lower sensitivity in detecting transient or weak protein interactions.In recent years,the rapidly advancing proximity labeling technology(PL)has overcome these limitations.PL involves fusing bait proteins with specific catalytic enzymes to subsequently add biotin or biotin phenol to label nearby target proteins for biotinylation.This technology integrates the advantages of highly efficient enzyme-catalyzed reactions,enabling efficient labeling of proteins within specific spatial proximity,and can detect weak protein interactions.It has been widely used in biological research in recent years.This paper elaborates on the application of PL catalyzed by biotin ligase(BirA)and ascorbate acid peroxidase(APEX)in T.gondii proteomics research,introduces the contributions of these two labeling methods in the exploration of T.gondii secretory proteins and protein-protein interaction studies,and provides reference for the study of T.gondii pathogenesis and the prevention and control of toxoplasmosis.