Investigating Interaction Partners Of Yeast UBPS and Human USP7 Deubiquitinases

Ubiquitin-specific proteases (USPs; Ubps) are the most abundant family of deubiquitinating enzymes. Their involvement in various cellular processes, which are implicated in development and diseases such as cancer, have made them an important subject of investigation. Protein function correlates with its three-dimensional fold; therefore, structural studies can help identify evolutionary conserved features, catalytic mechanisms and provide clues to specificity and interactions. We examined the expression of soluble Saccharomyces cerevisiae deubiquitinating enzymes in E. coli for structural studies. Following extensive optimizations Ubp1, the Ubp6 catalytic domain and the Ubp12 N-terminal domain crystallized. The Ubp6 crystals led to the determination of its three-dimensional structure and revealed that the catalytic triad residues are arranged in an active conformation. We showed that the Ubp6 catalytic domain superimposes well with that of USP14, its human homologue. This suggests a similar regulatory mechanism for yeast Ubp6 as compared to human USP14. Further, we investigated the mode of protein interaction of yeast Ubp15. Both S. cerevisiae Ubp15 and its human homologue, USP7, harbor a highly conserved DWGF motif in their N-terminal domains. The DWGF motif in USP7-NTD efficiently binds substrates or interacting proteins containing (E/P/A)XXS sequences. We showed that similar to USP7-NTD, the DWGF motif in Ubp15-NTD mediates interaction with (E/P/A)XXS motifs. To establish latency, human herpesviruses (HHVs) have evolved various mechanisms for host immune evasion. Proteins expressed by some members of the HHV family interfere with the USP7-p53-Hdm2 pathway. They competitively bind to USP7, block USP7-substrate interaction and suppress p53 mediated apoptosis. We identified an EGPS motif in both vIRF1 protein (expressed by Kaposi's sarcoma-associated herpesvirus; KSHV) and pp71 protein (expressed by human cytomegalovirus; HCMV). This motif is identical to the sequence reported in EBNA1 (of Epstein-Barr virus) and ORF45 (of KSHV) responsible for mediating their interaction with USP7-NTD. We demonstrated that both vIRF1 and pp71 interact with USP7-NTD in vitro and characterized the interaction using functional studies. The crystal structures of the USP7-NTD:vIRF1 and USP7-NTD:pp71 peptide complexes revealed identical modes of binding as that of the EBNA1 EGPS peptide to USP7-NTD. Our results support new roles for vIRF1 and pp71 through deregulation of the deubiquitinating enzyme USP7, which destabilize p53 and inhibit cellular antiviral responses.