【作者】 张颖；

【导师】 李长润；

【作者基本信息】 安徽大学 ， 生物学， 2018， 硕士

【摘要】 白色念珠菌是一种最普遍的致病性真菌。本文研究了三种与白色念珠菌生长相关蛋白,分别是支架蛋白Iqg1,Septin蛋白Cdc3,以及腺苷酸环化酶Cyr1。详细研究了 Iqg1在白色念珠菌形态发生中的作用,Cdc3的磷酸化对白色念珠菌菌丝生长的影响。并对Cyr1的不同功能区段进行了蛋白表达、纯化和结晶实验。白色念珠菌Iqg1是一种支架蛋白,在很多生物学过程中发挥重要作用。本文主要研究了 Iqg1与Sec3、Hof1之间的相互作用关系。通过离体蛋白结合试验,我们发现Iqg1的Gap-C功能区段与Sec3的N端有直接的相互作用,在Iqg1敲除菌株中,Sec3-GFP的极性分布完全消失,表明Iqg1直接影响Sec3在细胞内的分布,即Iqg1与Sec3之间功能上也相互影响,这些结果进一步验证了离体蛋白结合试验的结果。同样地,在离体蛋白结合试验中,我们也检测到Iqg1与Hof1之间的相互作用。通过对Hof1-GFP的Time-lapse microscopy观察,我们发现在野生型菌株中Hof1-GFP的极性分布在30分钟后完全消失,而在Iqg1敲除菌株中Hof1-GFP的极性分布在120分钟后依然不消失,表明Iqg1直接影响Hof1的极性分布与蛋白质稳定性。该结果也进一步验证了上述离体蛋白结合试验的结果。Septin环的架构转变是白色念珠菌细胞分裂的关键。Cdc3是Septin环中的重要一员,所以在白色念珠菌细胞分裂等生物学过程中发挥重要作用。本实验室的前期研究发现Cdc3有被磷酸化的现象。为了探讨Cdc3磷酸化的生理意义,我们构建了 Cdc3的磷酸化氨基酸位点的突变体。结果表明,把Cdc3的磷酸化氨基酸位点突变成模拟磷酸化的氨基酸后,跟野生型菌株相比,突变体菌株在菌丝生长条件下形成的菌丝比较短而粗,表明Cdc3的磷酸化可能在白色念珠菌的菌丝生长过程中发挥重要作用。环腺苷酸-蛋白激酶A信号通路(cAMP-PKApathway)是白色念珠菌调节菌丝生长的重要信号通路,腺苷酸环化酶在该信号通路中起着信号受体的关键性作用。为了更好地研究其功能,本实验对Cyr1四个主要功能区段进行了离体表达和纯化,并发现Cyr1的NRA和PP2C功能区与菌丝生长相关蛋白的Pho81的ANK功能区发生直接相互作用。更多还原

【Abstract】 Candida albicans is one of the most common pathogenic fungi.In this study,the function of the scaffold protein Iqg1,the Septin protein Cdc3,and the adenylate cyclase Cyrl was examined.Iqgl is a scaffold protein homologous to human IQGAPs which play important roles in a lot of biological processes.It has been reported that the interaction of IQGAP1 with the exocyst complex(directly through exocyst component Sec3)is required for tumor cell invasion.Here we showed that the GAPC domain of Iqg1 directly associated with the N-terminal of Sec3 by in vitro binding assay.Furthermore,the polarized localization of Sec3-GFP was completely abolished in the iqg1 Δ deletion mutant,indicating a genetic interaction between Iqgl and Sec3.These results suggest that Iqg1 associates with Sec3 both physically and genetically.Simiarly,a physical interaction between Iqg1 and Hof1 was detected in the in vitro binding assay.Time-lapse microscopy revealed that Hof1-GFP remained at the bud neck much longer in the iqg1△deletion mutant than that in the wild type cells,indicating that Iqg1 might affect the stabiligy of Hof1 in Candida albicans.These results suggest that Iqg1 associates with Hofl both physically and genetically.The architectural change of the Septin ring is the key to the division of Candida albicans.Cdc3 is an important member of the Septin ring.The previous work in the lab has shown that Cdc3 was phosphorylated in Candida albicans.To find out the physiological significance of Cdc3 phosphorylation,the phosphorylated serine or threorine residues identified by mass spectrometry were mutated to either non-phosphorylatable alannine(A)or phosphomimic aspartic acid(D).Results showed that the hyphae of the phosphomimic mutant of Cdc3 was much shorter than that of wild type Cdc3 cells,indicating that the phosphorylation of Cdc3 might be involved in the hyphal development in Candida albicans.更多还原