Functions of the ubiquitin -specific processing protease family of deubiquitinating enzymes: Mechanisms of catalysts and substrate recognition

This dissertation discusses the catalytic mechanisms, substrate recognition, and functional regulation of HAUSP and USP14, two representative members of the UBP (ubiquitin-specific processing protease) family of deubiquitinating enzymes. Structural and biochemical studies of these two proteins not only reveal the conserved structural features and catalytic mechanisms of the UBP family of deubiquitinating enzymes, but also provide insights into the mechanisms of substrate recognition by HAUSP, and the functional regulation of USP14 through proteasome association.;The crystal structures of the catalytic core domain of HAUSP in isolation and in complex with ubiquitin aldehyde revealed a novel three-domain architecture comprised of Fingers, Palm, and Thumb. The ubiquitin moiety is specifically coordinated by the Fingers, with its C terminus placed in the active site between the Palm and Thumb. These structures also revealed that the active site of the free HAUSP is in an unproductive conformation. Binding by ubiquitin aldehyde induces a large conformational change in the active site that aligns the catalytic residues for catalysis.;The HAUSP N-terminal domain adopts a nine-stranded antiparallel beta-sandwich structure, closely resembling the TRAF domain architecture. HAUSP interacts with multiple protein substrates, including p53, EBNA1, and Mdm2. HAUSP recognizes all these proteins through specific protein-peptide interactions between its N-terminal domain and the peptide sequences in the protein substrates.;The crystal structures of the catalytic core domain of USP14 in isolation and in complex with ubiquitin aldehyde revealed that the catalytic core domain of USP14 also adopts the Fingers-Palm-Thumb architecture, as first seen in HAUSP structure. The recognition between USP14 and ubiquitin also closely resembles that between HAUSP and ubiquitin. These findings suggest that the three-domain architecture and the mechanisms of ubiquitin recognition and catalysis seen in HAUSP and USP14 are conserved themes among the UBPs. In the free USP14 structure, the catalytic cleft is blocked by two loops right above the active site. Ubiquitin binding relieves the steric hindrance from these two loops, allowing the access of ubiquitin C terminus to the active site. USP14 binds to the 19S regulatory particle of the proteasome via its N-terminal ubiquitin-like domain, and proteasome binding greatly enhances its deubiquitinating activity.