BIOCHEMICAL CHARACTERIZATION OF 26S PROTEASOMES IN FARM ANIMALS TO IMPROVE KNOWLEDGE ABOUT ANTIGEN PROCESSING

Aim of the present work was to perform a biochemical characterization of 26S proteasomes in farm animals through a comparative study in eight different animal species. 26S proteasome is a 2,5 MDa ATP-dependent macromolecular protease that degrades ubiquitinated and some non ubiquitinated proteins in eukaryotic cells (1). Furthermore proteasomes are involved in antigen presentation since they produce the peptides (epitopes) that are presented on cellular surface in association with MHC class-I molecules (2). Firstly, we performed a partial purification, through differential centrifugation, of 26S constitutive proteasome (from muscle) and 26S immunoproteasome (from spleen) in eight different animal species (dog, goat, swine, cat, rabbit, cattle, horse and sheep). Then, we assessed the three proteasomal peptidase activities using specific fluorogenic peptides: 100 µM Suc-LLVY-amc (for the chymotrypsin-like activity), 100 µM Z-YVAD-amc (for the caspase-like activity), 100 µM Bz-VGR-amc (for the trypsin-like activity). Finally, we also performed western blot analysis using primary antibodies against the non-catalytic subunits α3, α4, α5 and α6. The results of the spectrofluorimetric measurements show that overall proteasomal enzymatic activity is conserved in the animal species examined despite some species-specific differences. Most importantly the ratios between different proteasomal peptidase activities are maintained, both for constitutive and immuno 26S proteasomes. Furthermore, a substantial conservation of the proteasomal active sites in animals of veterinary interest is demonstrated by a similar sensitivity of the β5 subunit (responsible of the chymotrypsin-like activity) to three of the most specific inhibitors currently available. Finally, western blot analysis of four different non-catalytic α subunits confirm that also the non-catalytic proteasomal structure is conserved in the species examined. Our results are consistent with a generally conserved overall structure and enzymatic activities. Nevertheless, to clarify more in detail how the proteasome system works in farm animals, we plan to test the sensitivity of the other proteasomal active sites to specific inhibitors and to study in vitro degradation of model protein substrates. REFERENCES: 1. Glickman M.H. and Ciechanover A., “The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction”. Physiol Rev. 2002 apr;82(2):373-428. 2. Goldberg A.L., Cascio P., Saric T. and Rock K.L., “The importance of the proteasome and subsequent proteolytic steps in the generation of antigenic peptides”. Mol Immunol 2002 oct;39(3-4):147-64.