Groettrup M, Ruppert T, Kuehn L, Seeger M, Standera S, Koszinowski U, Kloetzel P M
Institute for Biochemistry, Medical Faculty (Charité), Humboldt University Berlin, Federal Republic of Germany.
J Biol Chem. 1995 Oct 6;270(40):23808-15. doi: 10.1074/jbc.270.40.23808.
Antigenic peptides presented on major histocompatibility complex (MHC) class I molecules to cytotoxic T cells are generated in the cytosol by the 20 S proteasome. Upon stimulation of antigen presenting cells with interferon-gamma, two constitutive subunits of the 20 S proteasome are replaced by the MHC-encoded subunits low molecular mass polypeptide (LMP) 2 and LMP 7. In addition the expression of the two subunits of the 11 S regulator of the 20 S proteasome (PA28) are increased. As the function of LMP2 and LMP7 in antigen presentation is still controversial, we tested whether these subunits might operate by modifying proteasome activation through the 11 S regulator. We strongly overexpressed the two LMP subunits separately or together by transfection in murine fibroblasts. Isolated 20 S proteasomes from LMP transfectants were applied in digests of a 25-mer peptide in the presence or absence of a purified preparation of 11 S regulator from rabbit erythrocytes. Analysis of the cleavage products by high performance liquid chromatography and electrospray mass spectroscopy revealed marked differences in the peptide product profile in dependence on the LMP2 and LMP7 content. While the 11 S regulator did not preferentially activate LMP2 or 7 containing proteasomes, the binding of the 11 S regulator to any of the proteasome preparations markedly changed both the quality and quantity of peptides produced. These results suggest that the 11 S regulator increases the spectrum of peptides which can be generated in antigen presenting cells.
主要组织相容性复合体（MHC）I类分子呈递给细胞毒性T细胞的抗原肽是由20S蛋白酶体在胞质溶胶中产生的。在用γ干扰素刺激抗原呈递细胞后，20S蛋白酶体的两个组成亚基被MHC编码的低分子量多肽（LMP）2和LMP7亚基取代。此外，20S蛋白酶体11S调节因子（PA28）的两个亚基的表达也增加。由于LMP2和LMP7在抗原呈递中的功能仍存在争议，我们测试了这些亚基是否可能通过11S调节因子来修饰蛋白酶体的激活。我们通过转染在小鼠成纤维细胞中分别或共同强烈过表达这两个LMP亚基。在存在或不存在从兔红细胞中纯化的11S调节因子制剂的情况下，将从LMP转染体中分离的20S蛋白酶体应用于25聚体肽的消化。通过高效液相色谱和电喷雾质谱对裂解产物进行分析，结果显示肽产物谱根据LMP2和LMP7的含量有显著差异。虽然11S调节因子不会优先激活含有LMP2或7的蛋白酶体，但11S调节因子与任何一种蛋白酶体制剂的结合都显著改变了产生的肽的质量和数量。这些结果表明，11S调节因子增加了抗原呈递细胞中可产生的肽的种类。
