PA28αβ Reduces Size and Increases Hydrophilicity of 20S Immunoproteasome Peptide Products

The specific roles that immunoproteasome variants play in MHC class I antigen presentation are unknown at present. To investigate the biochemical properties of different immunoproteasome forms and unveil the molecular mechanisms of PA28 activity, we performed in vitro degradation of loosely folded, nonubiquitinated full-length proteins (casein and IGF-1) by 20S, 26S, and PA28αβ-20S immunoproteasomes and analyzed qualitatively and quantitatively the spectrum of peptides released by HPLC and tandem mass spectrometry. Notably, PA28αβ-20S immunoproteasomes hydrolyze proteins at the same low rates as 20S alone, which is in line with PA28, neither stimulating nor preventing entry of unfolded polypeptides into the core particle. Most importantly, our study demonstrates that binding of PA28αβ to 20S greatly reduces the size of proteasomal products and favors the release of specific, more hydrophilic, longer peptides. In accordance with our findings, PA28αβ may either allosterically modify proteasome active sites or act as a selective ‘‘smart’’ sieve that controls the efflux of products from the 20S proteolytic chamber.