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

真核20S蛋白酶体高度选择性加工蛋白质底物的结构和动力学特征仍然知之甚少。本研究计划旨在建立20S蛋白酶体的综合动力学模型。这使人们能够通过计算机模拟来测试各种假定的切割机制如何影响来自给定蛋白质底物的切割片段的模式。将模型预测与实验确定的蛋白裂解片段模式进行比较，最终将找出最可能的裂解机制。为此，将建立一个合适的数据库，在严格控制的外部条件下（蛋白酶体制备、底物预处理、活化剂的存在、时间点等），由本型和“免疫”20S蛋白酶体消化各种低聚底物，并记录主要裂解产物的时间依赖性形成。计划应用动力学模型作为主要蛋白酶体切割产物的预测因子来扫描任意蛋白质序列以寻找潜在抗原表位的存在。

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.