Structural Characteristic Of Pup From Mycobacterium Tuberculosis And Cellular Function Of Trypanosoma Brucei SUMO And Nedd8system

With the identification of ubiquitin-like modification system in prokaryotes and archaea and an increasing body of research on eukaryotic ubiquitin-like proteins, it’s well.known that ubiquitin-like proteins are ubiquitiously distributed in organisms and are essential for many cellular processes. In this thesis, we investigated the structure-functional relationship of Pup from Mycobacterium tuberculosis (Mtb), and the cellular function of SUMO and Nedd8system in Trypanosoma brucei (T. brucei). Our results are presented in three chapters of the thesis.One work in this thesis focuses on structure and function of Pup from Mycobacterium tuberculosis. Although different ubiquitin-like proteins from eukaryotes display extremely low sequence similarity, their3D (three-dimensional) structures exhibit highly conserved typical ubiquitin-like folds. Interestingly, our studies reveal that Pup not only shares low sequence similarity, but also presents a totally distinguished structure compared with other ubiquitin-like superfamily proteins. Diverse structure predictions combined with CD and NMR spectroscopic studies all demonstrate that Pup is an intrinsically disordered protein. Moreover, SPR and NMR perturbation studies imply that the C-terminus of Pup binds to Mpa probably through a hydrophobic interface, which indicated the Pup-mediated protein degradation pathway.Major work in this thesis focuses on the identification and functional analyses of SUMO and Nedd8systems in T. brucei, a kind of eukaryotic unicellular parasite. So far, ubiquitin-like modification system has not been reported in T. brucei yet. We first identified the SUMOylation and Neddylation pathway in T. brucei. Furthermore, function analyses of SUMO and Nedd8system reveal their distinct roles in T. brucei. SUMO system is essential for sister-chromatin segregation in mitosis, and plays key roles in maintaining genome integrity in T. brucei. In addition, we identified and purified the enzymes E1, E2involved in SUMO modification, and reconstructed SUMOylation assay in vitro for further research. Wherease, we indicate that Nedd8system is important for ubiquitination, DNA replication licensing and flagellum attachment. It’s the first time to report the role of Nedd8system in regulating flagellum attachment in T. brucei. Further researches on trypanosomatid SUMO and Nedd8system will reveal the detailed mechanism, by which SUMO and Nedd8regulate the cellular processes.