Structural Insights Into20S Proteasome Activation By Human REGγ

The proteasome is a large multisubunit protease found in archaebacteria, bacteria and eukaryotes. The activity of the proteasome is critical for many cellular pathways. The current study of the mechanisim of proteasome activity is focused in two aspects: the ubiquitin ATP-dependent pathway and the ubiquitin ATP-independent pathway.The C2-WW-HECT type ubiquitin ligases Smurfl and Smurf2play a critical role in embryogenesis and RhoA signaling pathways. The intramolecular interaction between C2and HECT domains has been shown to autoinhibit the ligase activity of Smurf2. However, the role of the Smurfl C2domain remains elusive. Therefore, the thesis is mainly focused on the structure of the Smurfl-C2domain, in order to provide an important basis for further functional studies of the Smurfl-C2domain.The REG (or11S activators, PA28) have been shown to regulate specificity of the20S proteasome by binding to the ends of the cylindrical proteasome structure in a ubquitin-and ATP-independent manner. REGy specifically activates proteasomal hydrolysis of peptides after basic residues only. Furthermore, it is unique in targeting degradation of intact protein substrates, such as p21, SRC-3and Smurfl. REGy also plays important roles in cell cycle, Apoptosis, viral infection and tumor development. Therefore, it is critical for the structural study of REGy.The study of the first part in this thesis is the determination of the three-dimensional structure of Smurfl-C2domain to2A resolution. The Smurfl-C2domain possesses a typical anti-parallel β-sandwich fold. Examination of the sulfate binding site reveals two key lysine residues:K28and K85. These two lysine residues within the C2domain are critically important for Smurfl localization at the plasma membrane but do not inhibit the HECT ligase in Smurf2. This work further supports a Ca2+-independent localization mechanism for Smurfl.In the second part of this thesis, the3.0A resolution crystal structure of the human REGy heptamer was determined. The structure of the REGy-linker mutant was determined, in which a homolog-specific insert (residues60-107) in REGy was substituted by an engineered six amino acid linker. Substituting the homolog-specific insert, which contains a putative nuclear localization signal, causes protein translocation from the nucleus to the cytoplasm but does not affect its structure or biochemical activity.In the final part of this thesis, a third mutant structure, REGγ-linker(K188E), was also determined. The K188E mutant of REGyabolishes its specific activation profile and enables proteasome-mediated cleavage after acidic, basic and hydrophobic residues. The K188E mutant crystal structure reveals significant conformational changes in the C-terminal tail and activation loop (residues143-152), which interact with and activate the proteasome. Two residues are shown to be critically important for proteasome activation:G1y150confers essential main chain conformational change to the activation loop, while Glu148mediates activating interactions with the proteasome subunit. Our work provides new understanding of20S activation by REG proteins via a markedly different mechanism than their PA26orthologs from lower eukaryotes.In conclusion, our finding about the C2structure in this thesis is important for further functional studies of Smurfl. The present work on REGy and mutant structures provide a basic understanding of its mechanism in stimulating proteasome activity.