隐球菌转录因子Frt1功能和调控机理研究

Cryptococcus neoformans is a major yeast pathogen that often infects the human central nervous system (CNS) to cause meningitis, resulting in over 600,000 deaths worldwide annually. The SCF (Skp1, Cullins, F-box proteins) E3 ubiquitin ligases-mediated ubiquitin-proteasome system (UPS) is a principle intracellular mechanism for controlled protein degradation in eukaryotes and has recently emerged as an attractive drug target for human diseases. UPS was found to be essential for fungal virulence, as removal of the F-box protein Fbp1 in the SCF E3 ligase led to fungal virulence lost. The fbp1Δ mutant also showed reduced cell membrane integrity and defect in meiosis. By employing Co-immunoprecipitation and protein stability assays, we identified a novel transcription factor Frt1 that functions as a substrate of Fbp1. Our preliminary study results showed that the frt1Δ mutant and its overexpression strain both showed similar phenotype as the fbp1Δ mutant, indicating that Fbp1 regulates fungal virulence through Frt1. Therefore, it is significant to investigate the Frt1 function to understand how the Fbp1-Frt1 pathway regulates Cryptococcus pathogenesis. In this project, we will analyze the Frt1 gene spatial and temporal expression, protein interaction and downstream gene regulation by using GFP protein fusion technology, co-immunoprecipitation and chromatin immunoprecipitation, respectively. The outcome of this study will be a better understanding of the mechanism of UPS in physiological and biochemical functions, morphological development and fungal pathogenesis in C. neoformans. The knowledge gained from this project will lay a foundation for the molecular mechanism study of the ubiquitin-mediated protein degradation in fungal pathogenesis.

新型隐球菌是自然界普遍存在的能侵染人类中枢神经系统的酵母菌，每年能导致大约60万人死亡。泛素蛋白酶体系统是真核细胞内主要的蛋白降解途径，参与调节诸如细胞周期、细胞增生与分化，以及信号传导等多种细胞生理过程。本研究发现泛素化蛋白降解途径与隐球菌致病性密切相关，泛素连接酶E3关键蛋白Fbp1缺失后，隐球菌细胞膜完整性降低，减数分裂被阻断，致病性丧失；本项目通过免疫共沉淀和蛋白稳定性实验鉴定转录因子Frt1为Fbp1一下游底物；初步分析表明 frt1Δ突变体和fbp1Δ突变体具有相同的表型。通过分析转录因子Frt1的时空表达、蛋白互作以及下游基因调控，揭示泛素蛋白降解系统在隐球菌生理生化、形态发育及致病过程中的作用机制，阐明泛素化蛋白降解系统在隐球菌致病过程中的地位，为隐球菌泛素化蛋白降解分子机制和致病机理研究奠定基础。