Enhanced rate of degradation of basic proteins by 26S immunoproteasomes.

Immunoproteasomes are alternative forms of proteasomes specialized in the generation of MHC class I antigenic peptides and important for efficient cytokine production. We have identified a new biochemical property of 26S immunoproteasomes, namely the ability to hydrolyze basic proteins at greatly increased rates compared to constitutive proteasomes. This enhanced degradative capacity is specific for basic polypeptides, since substrates with a lower content in lysine and arginine residues are hydrolyzed at comparable rates by constitutive and immunoproteasomes. Crucially, selective inhibition of the immunoproteasome tryptic subunit ß2i strongly reduces degradation of basic proteins. Therefore, our data demonstrate the rate limiting function of the proteasomal trypsin-like activity in controlling turnover rates of basic protein substrates and suggest new biological roles for immunoproteasomes in maintaining cellular homeostasis by rapidly removing a potentially harmful excess of free histones that can build up under different pathophysiological conditions.

Comparative study of the biochemical properties of proteasomes in domestic animals.

Information on the biochemical properties of proteasomes is lacking or, at best, only fragmentary for most species of veterinary interest. Moreover, direct comparison of the limited data available on the enzymatic features of proteasomes in domestic animals is rendered difficult due to the heterogeneity of the experimental settings used. This represents a clear drawback in veterinary research, given the crucial involvement of proteasomes in control of several physiological and pathological processes. We performed the first comparative analysis of key biochemical properties of proteasomes obtained from 8 different domestic mammals. Specifically, we investigated the three main peptidase activities of constitutive and immunoproteasomes in parallel and systematically checked the sensitivity of the chymotryptic site to three of the most potent and selective inhibitors available. Overall, there was substantial similarity in the enzymatic features of proteasomes among the species examined, although some interesting species-specific features were observed.

PA28αß 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 full-length proteins by 20S, 26S, and PA28αß-20S immunoproteasomes and analyzed the spectrum of peptides released. Notably, PA28αß-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, binding of PA28αß to 20S greatly reduces the size of proteasomal products and favors the release of specific, more hydrophilic, longer peptides. Hence, PA28αß 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.

Pivotal Advance: Protein synthesis modulates responsiveness of differentiating and malignant plasma cells to proteasome inhibitors.

A previously unsuspected, considerable proportion of newly synthesized polypeptides are hydrolyzed rapidly by proteasomes, possibly competing with endogenous substrates and altering proteostasis. In view of the anti-cancer effects of PIs, we set out to achieve a quantitative assessment of proteasome workload in cells hallmarked by different PI sensitivity, namely, a panel of MM cells, and in a dynamic model of plasma cell differentiation, a process that confers exquisite PI sensitivity. Our results suggest that protein synthesis is a key determinant of proteasomal proteolytic burden and PI sensitivity. In different MM cells and in differentiating plasma cells, the average proteolytic work accomplished per proteasome ranges over different orders of magnitude, an unexpected degree of variability, with increased workload invariably associated to increased PI sensitivity. The unfavorable load-versus-capacity balance found in highly PI-sensitive MM lines is accounted for by a decreased total number of immunoproteasomes/cell coupled to enhanced generation of RDPs. Moreover, indicative of cause-effect relationships, attenuating general protein synthesis by the otherwise toxic agent CHX reduces PI sensitivity in activated B and in MM cells. Our data support the view that in plasma cells protein synthesis contributes to determine PI sensitivity by saturating the proteasomal degradative capacity. Quantitating protein synthesis and proteasome workload may thus prove crucial to design novel negative proteostasis regulators against cancer.