Untersuchungen zur Rolle der Tripeptidylpeptidase II-vermittelten Proteolyse invivo (In vivo-Role of Tripeptidyl Peptidase II-Mediated Proteolysis in Murine Cells and Mice)

尽管有些人认为蛋白酶体只产生细胞毒性t淋巴细胞（CTL）表位的c端（Craiu等，1997；Beninga等，1998），但我们的工作表明，蛋白酶体在许多表位的两端精确切割。我们打算使用含有至少45个CTL表位的HIV-1-Nef作为底物，用于分离的20S、26S和免疫蛋白酶体的消化，并确定所有降解产物。这将使我们能够澄清蛋白酶体在表位生成中的作用，更好地定义各种类型蛋白酶体的特异性（26S和免疫蛋白酶体的特异性迄今尚未得到系统的研究），并开发预测蛋白酶体切割位点的算法。TPPII是一种比26S蛋白酶体更大的胞质蛋白酶复合物，在蛋白酶体抑制剂适应的细胞中，TPPII的上调允许细胞存活。我们打算研究TPPII是否正常地在蛋白酶体下游的胞浆蛋白水解中起作用，因此是古细菌Tricorn蛋白酶的真核垂坠。此外，我们打算更详细地分析适应细胞关于蛋白酶体功能损伤和TPPII允许生存的精确机制。

Whereas some groups favor that proteasomes only generate the C-termini of cytotoxic T-lymphocyte (CTL) epitopes (Craiu et al., 1997; Beninga et al., 1998), our work has suggested that they cleave precisely at both ends of many epitopes. We intend to use as substrate HIV-1-Nef, which contains at least 45 CTL epitopes, for digestion by isolated 20S, 26S and immunoproteasomes and to determine all degradation products. This will allow us to clarify the role of proteasomes in epitope generation, to better define the specificity of the various types of proteasomes (the specificities of 26S and immunoproteasomes have so far not been investigated systematically) and to develop algorithms for the prediction of proteasomal cleavage sites.Upregulation of TPPII, a cytosolic protease complex larger than the 26S proteasome, in proteasome inhibitor-adapted cells permits cell survival. We intend to investigate whether TPPII normally functions in cytosolic proteolysis down-stream of proteasomes and therefore is the eukaryotic pendant of the archaeal Tricorn protease. Further, we intend to analyze the adapted cells in more detail regarding impairment of proteasome functions and precise mechanisms by which TPPII permits survival.