Insight into the Biochemical Interaction of USP7 C-terminal Domain

Ubiquitination is a post-translational modification, involving the covalent attachment of ubiquitin, important for the regulation of cellular pathways. Deubiquitinases reverse this process through the hydrolysis of ubiquitin moieties from substrates. The deubiquitinase, Ubiquitin Specific Protease 7 (USP7), regulates many important nuclear processes. The USP7 N-terminus (USP7-NTD) is a TRAF domain which functions predominantly in substrate-binding while the USP7 C-terminus (USP7- CTD) was predicted to have 4-5 ubiquitin like domains (Ubl1-5). The aims of this thesis are biochemical and structural analyses of the C-terminus of human USP7. Various Ubl domains of human USP7-CTD were cloned, expressed, purified and crystallized. Subsequently, the biochemical interaction of an ICP0 peptide with these Ubl domains was investigated using in vitro GST-pulldown experiments and fluorescence polarization assays where the ICP0 peptide showed tightest interaction with Ubl123. Co-crystals of Ubl123 and ICP0 peptide need further refinement prior to structure determination.