Allosteric regulation of the proteasome’s catalytic sites by the proteasome activator PA28γ /REGγ

Abstract

Proteasome Activator 28γ (PA28γ) 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 PA28γ functions in protein degradation remains unclear. Though PA28γs 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 PA28γ regulates proteasome function. Here, we rigorously and definitively show that PA28γ 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 PA28γ-increases the affinity (Km) and Vmax for Trypsin-like peptide substrates. Mutagenesis of PA28γ 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 PA28γ-20S proteasome complex at 4.4A validating it's expected 11S-like quaternary structure and proteasome binding mode. The results of this study provide unambiguous evidence that PA28γ functions by allosterically upregulating the T-L like site in the 20S proteasome.