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

真核生物20S蛋白酶体对蛋白质底物的高度选择性加工的结构和动力学特征仍然知之甚少。这项研究计划旨在对20多岁的蛋白酶体进行全面的动力学建模。这使得人们能够通过计算机模拟来测试各种假定的切割机制将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质分解片段模式进行比较，最终将找出最可能的切割机制。为此，将建立一个适当的数据库，在严格控制的外部条件下(蛋白酶体制备、底物的前处理、活化剂的存在、时间点等)消化各种寡聚底物。通过结构性和免疫性的20S蛋白酶体，记录主要切割产物的随时间变化的形成。它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，以扫描任意蛋白质序列，以寻找潜在的抗原表位。

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.