Allosteric regulation of the proteasomes catalytic sites by the proteasome activator PA28gamma/REGgamma

Proteasome Activator 28{gamma} (PA28{gamma}) is a member of the 11S family of proteasomal regulators that is constitutively expressed in the nucleus and is implicated in certain cancers, lupus, rheumatoid arthritis, and Poly-glutamine neurodegenerative diseases. However, how PA28{gamma} functions in protein degradation remains unclear. Though PA28{gamma}s mechanism has been investigated for some time, many alternative hypotheses have not been tested: e.g. 1) substrate selection, 2) allosteric upregulation of the Trypsin-like catalytic site, 3) allosteric inhibition of the Chymotrypsin- and Caspase-like catalytic sites, 4) conversion of the Chymotrypsin- or Caspase-like sites to new Trypsin-like catalytic sites, and 5) gate-opening in combination with these. The purpose of this study was to conclusively determine how PA28{gamma} regulates proteasome function. Here, we rigorously and definitively show that PA28{gamma} uses an allosteric mechanism to upregulate the proteolytic activity of the 20S proteasomes Trypsin-like catalytic site. Using a constitutively open channel proteasome, we were able to dissociate gating affects from catalytic affects demonstrating that the PA28{gamma}-increases the affinity (Km) and Vmax for Trypsin-like peptide substrates. Mutagenesis of PA28{gamma} also reveals that it does not select for (i.e. filter) peptide substrates, and does not change the specificity of the other active sites to trypsin-like. Further, using Cryo-EM we were able to visualize the C7 symmetric PA28{gamma}-20S proteasome complex at 4.4[A] validating its expected 11S-like quaternary structure and proteasome binding mode. The results of this study provide unambiguous evidence that PA28{gamma} functions by allosterically upregulating the T-L like site in the 20S proteasome. Significance StatementThis study rigorously demonstrates that PA28g allosterically activates the b-2 proteolytic site of the 20S proteasome directly without affecting 20S gating. Further, we generated the first human 11S-human 20S proteasome cryo-EM structure of the PA28g-20S complex showing that, despite its different affects on 20S activity, it has a similar quaternary structure as the other 11S family members. The significance of these findings is paramount as the b-2 site is responsible for post-basic cleavage and suggests that PA28g is specialized to degrade positively charged DNA binding proteins. Further, b-2 upregulation via PA28g could provide a protective effect against poly-glutamine expanded proteins, like Huntingtin. This work provides a framework for PA28g drug development to treat PA28g addicted cancers and Huntingtons Disease.