The 20S immunoproteasome and constitutive proteasome bind with the same affinity to PA28αβ and equally degrade FAT10

The 20S immunoproteasome (IP) is an interferon(IFN)-gamma - and tumor necrosis factor (TNF) - inducible variant of the 20S constitutive proteasome (CP) in which all its peptidolytically active subunits beta 1, beta 2, and beta 5 are replaced by their cytokine inducible homologues beta 1i (LMP2), beta 2i (MECL-1), and beta 5i (LMP7). These subunit replacements alter the cleavage specificity of the proteasome and the spectrum of proteasome-generated peptide ligands of MHC class I molecules. In addition to antigen processing, the IP has recently been shown to serve unique functions in the generation of pro-inflammatory T helper cell subtypes and cytokines as well as in the pathogenesis of autoimmune diseases, but the mechanistic involvement of the IP in these processes has remained elusive. In this study we investigated whether the IP differs from the CP in the interaction with two IFN-gamma/TNF inducible factors: the 11S proteasome regulator PA28 alpha beta and the ubiquitin-like modifier FAT10 (ubiquitin D). Using thermophoresis, we determined the affinity of PA28 alpha beta for the CP and IP to be 12.2 nM +/- 2.8 nM and 15.3 nM +/- 2.7 nM, respectively, which is virtually identical. Also the activation of the peptidolytic activities of the IP and CP by PA28 alpha beta did not differ. For FAT10 we determined the degradation kinetics in cycloheximide chase experiments in cells expressing almost exclusively IP or CP as well as in IFN-gamma stimulated and unstimulated cells and found no differences between the degradation rates. Taken together, we conclude that neither differences in the binding strength to, nor activation by PA28 alpha beta, nor a difference in the rate of FAT10-mediated degradation can account for distinct functional capabilities of the IP as compared to the CP.