Study Of Structure And Molecular Mechanism Of Ubiquitin-like Family Proteins

Post-translational modification is an important mechanism that regulates a wide variety of cellular events such as protein degradation, singal transduction, gene expression, cell cycle, and DNA repair. Post-translational modifications include phosphorylation, acetylation, methylation, glycosylation, ubiquitylation and so on. Ubiquitin proteins family includes more than ten proteins. So far, many studies have been focused on ubiquitin and SUMO (small ubiquitin-like proteins). Our studies can be devided into two parts:one is about the study of solution structure of SUMO of Trypanosoma brucei and its interaction with hUbc9; the other is about the structural study of Pup and its interaction with Mpa (mycobacterium proteasomal ATPase).Trypanosoma brucei is the most ancient unicellular eukaryotic organism that causes sleeping sickness in human and nagana diseases in animals. In this study, we use NMR method to solve the solution structure of SUMO of Trypanosoma brucei (Tb-SUMO). This is the first structure of SUMO determined in protist. Tb-SUMO has the typical ubiquitin-fold containing five 13 stands and two a halix. Compared with other SUMO structures, Tb-SUMO has an extended region with a length of 32 amino acids at the N-terminus. We also study the interaction between Tb-SUMO and human Ubc9 (the E2 enzyme has not been identified in Trypanosoma brucei so far). On SUMO, the surface is composed byβsheet and some loops, which is highly complementary in its electrostatic potentials and hydrophobicity to the positively charged surface of Ubc9. The result shows that the structure and binding surface of Tb-SUMO is conserved.In eukaryotic cells, ubiquitin is attached to substracts as a tag to bring proteins into proteasome for degradation. Proteasome in mycobacterial has been found for many years. Ubiquitin protein is not known in prokaryotae, until Pup is reported to be coupled to proteins, rendering them as substrates for proteasome-mediated degradation in mycobacterium in 2008. Pup is a small protein encoded by 64 amino acids. Like eukaryotic ubiquitin proteins, there are two glycines in the C-terminus of Pup. Sequence alignment shows that Pup and ubiquitin proteins share very low similarity. Diverse structure predictions combined with CD and NMR spectroscopic studies all demonstrate that Pup is an intrinsically disordered protein. Moreover, 1H-15N NOE (nuclear Overhauser effect) data and CSI (chemical shift index) analyses indicate that there is a residual secondary structure at the C-terminus of Pup. In M. tuberculosis, Mpa is the regulatory cap ATPase of the proteasome that interacts with Pup and brings the substrates to the proteasome for degradation. SPR (surface plasmon resonance) and NMR perturbation studies imply that the C-terminus of Pup, ranging from residue 30 to 59, binds to Mpa probably through a hydrophobic interface. In addition, phylogenetic analysis clearly shows that the Pup family belongs to a unique and divergent evolutionary branch, suggesting that it is the most ancient and deeply branched family among ubiquitinlike proteins. This might explain the structural distinction between Pup and other ubiquitin-like superfamily proteins.