Regulation of the ubiquitin-proteasome-system during oxidative stress

许多环境毒物、医疗药物和滥用药物通过氧自由基和相关活性氧发挥毒性作用。尽管对脂质过氧化进行了大量的研究，但对蛋白质氧化和负责这些氧化蛋白质降解的蛋白水解酶的可能调节知之甚少。我们的目的是测试蛋白酶体系统的调节和氧化蛋白与泛素化系统的相互作用。虽然蛋白酶体系统降解氧化的胞质蛋白似乎已经确定，但泛素系统识别的机制尚不清楚。多个研究小组发现20S蛋白酶体在体外系统中能够识别氧化蛋白并优先降解氧化蛋白。另一方面，在活细胞中，降解细胞内蛋白质的主要蛋白酶是26S蛋白酶体。由于这种蛋白酶体形式能够识别和降解泛素化蛋白，因此了解泛素化系统对氧化蛋白的识别信号是很有趣的。因此，氧化蛋白和模型底物将被用作泛素化分析的底物，并通过使用氧化标记来研究选择性识别。结果表明，在氧化应激过程中，除了泛素化酶与底物的可能相互作用外，蛋白酶体本身也发生了调节。在氧化应激期间和氧化应激后，将研究11S和19S等主要调节因子与20S核心蛋白酶体的结合以及蛋白酶体的其他调节机制。因此，本提案将重点放在哺乳动物细胞的蛋白酶体和泛素化系统的功能和调控上，似乎符合“20S/26S泛素-蛋白酶体系统的结构、功能和调控”项目的标准。

Many environmental toxicants, medical drugs, and abuse substances exert their toxic effects via oxygen free radicals and related active oxygen species. Despite extensive work on lipid peroxidation little is known about protein oxidation and the possible regulation of proteolytic enzymes, responsible for degradation of these oxidized proteins. Our objective is to test the regulation of the proteasomal system and the interaction of oxidized proteins with the ubiquitination system.Although it seems to be established, that the proteasomal system degrades oxidized cytosolic proteins, the mechanism of recognition by the ubiquitin system is still not known. It was found by several groups that the 20S proteasome can recognize oxidized proteins and degrade them preferentially in in vitro systems. On the other hand in living cells the major protease degrading intracellular proteins is the 26S proteasome. Since this proteasomal form is able to recognize and degrade polyubiquitinated proteins it is interesting to know the recognition signals of oxidized proteins by the ubiquitination system. Therefore, oxidized proteins and model substrates will be used as substrates for ubiquitination assays and by using of oxidation markers the selective recognition is investigated. It was demonstrated, that besides the possible interactions of the ubiquitinating enzymes with the substrate a modulation of the proteasome itself occurs during oxidative stress. The binding of the major regulators, like the 11S and the 19S particles to the 20S core proteasome will be investigated during and after oxidative stress together with other regulatory mechanisms of the proteasome. Therefore, the proposal is focused on the function and regulation of the proteasomal and the ubiquitination system of mammalian cells and seems to fulfill the criteria of the program "Structure, function and regulation of the 20S/26S ubiquitin-proteasome system".