泛素-蛋白酶体是真核生物主要的蛋白降解系统，UBL-UBA穿梭蛋白是该系统的重要成分，其能够识别、结合泛素化蛋白然后将其转运到蛋白酶体进行降解。已知弓形虫存在泛素-蛋白酶体系统，但未见UBL-UBA穿梭蛋白在泛素蛋白降解中的功能相关研究报道。前期发现弓形虫能够表达UBL-UBA穿梭蛋白DDI1，其基因缺失虫株对小鼠的致病性显著减弱；分析发现弓形虫还存在与DDI1有类似功能结构域的穿梭蛋白RAD23和DSK2，推测该三种蛋白在泛素化蛋白降解过程中具有重要作用。本项目拟通过基因编辑、免疫共沉淀、质谱分析以及蛋白质组学等技术，确认此三种蛋白在弓形虫中的表达和定位，解析三者的功能和相互关系，确定各穿梭蛋白的转运底物；通过比较各穿梭蛋白和底物蛋白的基因调控虫株在分裂周期、DNA损伤修复能力等方面的变化，深度揭示穿梭蛋白转运泛素化蛋白及其影响弓形虫生命活动的机制。

Eukaryotic cells are equipped to degrade proteins with the ubiquitin-proteasome system, UBL-UBA shuttle proteins are important components of the ubiquitin-proteasome system (UPS). Ubiquitinated proteins are recognized and combined by the shuttle proteins, which then drive the ubiquitinated proteins to the proteasome for degradation. The Toxoplasma gondii genome encodes all of the machinery required for UPS, however, there are no studies about the function of shuttle proteins. DDI1 has been identified in T. gondii in our current study and the virulence of ddi1 knockout strain in mice is attenuated significantly. The other two proteins of this family are also identified in T. gondii, so we speculated this family of proteins play important roles in T. gondii. This project intends to confirm the expression and localization of these three shuttle proteins in T. gondii, analyze the functions and relations of the three proteins and determine the substrates of the shuttle proteins through gene editing, co-immunoprecipitation, mass spectrometry and proteomics technology. By observing the differences of the cell cycle and DNA damage repair in gene editing strains, we intend to reveal the mechanisms of shuttle proteins transporting ubiquitinated proteins and affecting the life activities of T. gondii.