Implications of ß-subunit exchanges and of interactions with the activator PA28 for the cleavage preferences of the 20S proteasome: A quantitative analysis based on kinetic experiments

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The structural and kinetic features accounting for the highly selective processing of protein substrates by eukaryotic 20S proteasomes are still poorly understood. The research proposal aims at a comprehensive kinetic modelling of the 20S proteasome. This enables one to test by computer simulations how various putative cleavage mechanisms will affect the pattern of cleavage fragments derived from a given protein substrate. Comparing the model predictions with experimentally determined patterns of proteol ytic fragments the most probable cleavage mechanism will eventually be figured out. To this end an appropriate data base will be established digesting various oligomeric substrates under strictly controlled external conditions (proteasome preparation, pre-treatment of substrates, presence of activating agents, time points etc.) by the constitutive and 'immuno' 20S proteasome and recording the time-dependent formation of the major cleavage products. It is planned to apply the kinetic model as a predictor of major proteasomal cleavage products to scan arbitrary protein sequences for the presence of potential antigenic epitopes.