小球藻病毒编码的泛素连接酶E3复合物组分cvANK2和cvSkp1功能研究

The constant synthesis and proteolysis of cellular proteins is a very important homeostatic procedure for eukaryotic cells. The degradation of useless proteins is mainly processed by ubiquitin-proteasome system. Ubiquitin-protein ligase E3 is an enzyme superfamily of the most complex, high specificity in the system, while Skp1-Cullin1-F-Box (SCF) complex categorized into E3 superfamily is the enzyme group that has been mostly studied and has the largest member body. Many animal and plant viruses encode F-Box protein to interact with host proteins, mainly the SCF complex, for benefiting their replication. Genome sequencing indicates that chlorella viruses are the only viruses that encode both F-Boxes, named ankyrin repeats (ANKs), and Skp1. Every of the chlorella viruses that the genomes have been sequenced encodes 1-5 ANKs, and a sole Skp1. Previous results suggest that the viral ANK and /or Skp1 possibly assemble into a mosaic type of SCF complex with the cellular SCF subunits. In the present proposal a series of experimental protocols has been carefully and rationally designed in order to demonstrate the possibility of the interactions among the viral ANK/Skp1 and cellular SCF complex subunits by yeast two hybrid, to reconstruct the hypothesized mosaic-type SCF complex by expressing multiple subunits in mammalian cells, to isolate cellular SCF complex post infection with the antibodies raised against viral ANK and Skp1, and to determine the identity of each of the subunits with LC-MS/MS analysis and the protein database search, and finally to perform in vitro polyubiquitination using the reconstructed SCF complex.

蛋白质的不断合成与降解是真核细胞维持正常细胞功能的重要手段。细胞内蛋白质的降解主要由泛素-蛋白酶体系统来完成。泛素连接酶E3是这一系统中最复杂，最具特异性的酶类家族，而SCF（Skp1-Cul1-F-Box）复合体则是其中最大，研究最多的一组。基因组测序显示小球藻病毒是目前唯一一组同时编码F-Box和Skp1蛋白的病毒，不同的小球藻病毒株都编码1-5个F-Box蛋白ANK和1个Skp1蛋白。已有的实验结果显示病毒的ANK和/或Skp1有可能与宿主SCF组分聚合成病毒-细胞混合型SCF复合物。本项目设计了下列实验方法试图加以证实并提出一个新型SCF作用模式：通过酵母双杂交来证明病毒ANK和Skp1与宿主SCF复合物相互作用的可能性；利用哺乳动物细胞异源表达进行病毒-细胞混合型SCF复合物重建； 用病毒蛋白抗体从被感染的细胞中分离SCF并经质谱分析其组分，及体外泛素化底物蛋白来检测其功能。