Strutural And Functional Studies On Deubiquitinases Usp25 And Usp28

Ubiquitination is a reversible protein post-translational modification. The E1(ubiquitin activating enzyme), E2(ubiquitin conjugating enzyme) and E3(ubiquitin ligase) enzymatic cascade catalyzes protein ubiquitination, whereas deubiquitinases(DUBs) promote the removal of ubiquitin. To date, approximately 100 DUBs, which are classified into six subfamilies according to their different structural and functional features, have been identified in human genome. DUBs play important roles in maintaining the ubiquitination-deubiquitination balances of proteins, and contribute to the tight regulations of multiple cellular processes including cell metabolism, cell proliferation and differentiation. USP domain-containing DUBs constitute the largest deubiquitinase subfamily, which include more than 50 DUB members. According to the recently published articles, quite a few of USPs have been confirmed to play essential roles in cancer-related pathways, and the aberrant expressions and functional disorders of these USPs attribute to the developing of cancers. Therefore, the USP-related structural biology studies are quite important, and surely have distinguishable scientific significances. Besides, the extending discoveries of structure-based inhibitors may provide new therapy for cancers.Both of the two research targets of this thesis, Usp25 and Usp28, belong to the USP subfamily of deubiquitinases. They have been identified to play important roles in multiple cellular processes. Usp25 has been demonstrated to be involved in biological processes including myogenesis, endoplasmic reticulum-associated protein degradation and immune system responses. And Usp28 is confirmed to play essential roles in cancer-related pathways. Usp25 and Usp28 share a high sequence homology. They both contain an N-terminal ubiquitin-binding region(UBR) with ubiquitin-associated domain(UBA) and ubiquitin-interacting motif(s)(UIM) included and a conserved USP catalytic domain. The UBR regions of Usp25 and Usp28 modulate the enzymatic activities of these two enzymes by participating in the ubiquitin substrate recognition processes. Moreover, it is worth of noting that besides the ubiquitin binding domains a typical small ubiquitin-like modifier(SUMO)-interacting motif(SIM) is also identified in the UBR regions of Usp25 and Usp28. This finding indicates that SUMO molecules might also be involved in the enzymatic activity regulations of Usp25 and Usp28.In this thesis, solution NMR techniques and biochemical approaches were applied to investigate how the UBRs of Usp25 and Usp28 regulate their enzymatic activities. We then found that the UBRs of Usp25 and Usp28 could specifically bind to both the ubiquitin substrates and the SUMO2 protein. The ubiquitin-binding abilities of UBRs are required for the full activity displays of Usp25 and Usp28. Meanwhile, SUMO2 impairs the catalytic activities of Usp25 and Usp28 by competitively preventing their interactions with ubiquitin substrates. Finally, based on our aboved-mentioned findings, the working models to depict the regulatory roles of UBRs in the functional displays of Usp25 and Usp28 are proposed.