STRUCTURE/FUNCTION OF A PROTEASOME INHIBITOR

P131是一种抑制合成肽和大分子蛋白质降解的蛋白质 20S蛋白酶体的蛋白质底物(Ma等人，Biochim。生物群落。行为 1119(1992)，303-311)。我们已经克隆了人类基因并对其进行了测序 第131页。推导出的P131的一级结构为271个氨基酸，具有 分子量为29,792，与十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法测定的分子量一致 从牛血细胞中提纯的蛋白质。P131不同于任何 先前报道的蛋白质，并具有富含Pro的羧基末端的一半 (26%的脯氨酸残留量)。重组P131已在大肠杆菌中表达， 纯化至均一，并发现显示出类似的抑制特性 与牛P131的同源性比较。凝胶测定的60 kDa二聚体天然P131 过滤，通过直接与20s结合来抑制蛋白酶体的活性 蛋白酶体的摩尔比为2：1。一个蛋白酶体-P131复合体已经被 甘油密度梯度离心法分离。我们已经建造了 缺失突变体，表明p131富含脯氨酸的羧基末端 负责抑制功能。圆二色谱揭示了 富含脯氨酸的区域具有随机卷曲构象，这表明两者 P131序列和扩展的随机卷曲结构是重要的 为了抑制。这导致了小肽片段的合成 P131，并且我们已经证明了抑制活性定位于 富含Pro的羧基末端内的一小块区域。此外，我们 已有研究表明，P131能抑制与PA28结合的蛋白酶体。 蛋白酶体激活剂被认为在免疫反应中起作用。P131做到了 然而，不抑制与PA700复合的蛋白酶体，PA700是一种多亚单位 参与泛素依赖的蛋白质降解的调节蛋白。 这些数据表明，监管机构之间存在着一个等级关系 蛋白酶体功能的调控。

P131 is a protein that inhibits degradation of synthetic peptides and large protein substrates by the 20S proteasome (Ma et al., Biochim. Biophys. Acta 1119 (1992), 303-311). We have cloned and sequenced the human gene for P131. The deduced primary structure of P131, 271 amino acids, has a molecular weight of 29,792 in accord with that estimated by SDS-PAGE for the protein purified from bovine blood cells. P131 is not similar to any previously reported protein and has a proline-rich carboxyterminal half (26% proline residues). Recombinant P131 has been expressed in E. coli, purified to homogeneity, and found to display inhibitory properties similar to those of bovine P131. Native P131, a 60 kDa dimer as determined by gel filtration, inhibits proteasome activity by directly binding to the 20S proteasome in a 2:1 molar ratio. A proteasome-P131 complex has been isolated by glycerol density gradient centrifugation. We have constructed deletion mutants which show that the proline-rich carboxyterminus of p131 is responsible for inhibitory function. Circular-dichroism revealed that the proline-rich region has a random coil conformation suggesting that both the sequence of P131 and an extended random coil structure are important for inhibition. This lead to the synthesis of small peptide fragments of p131, and we have demonstrated that the inhibitory activity is localized to a small region within the proline-rich carboxyterminus. In addition, we have shown that P131 can inhibit the proteasome complexed to PA28, a proteasome activator believed to function in the immune response. P131 did not, however, inhibit the proteasome complexed to PA700, a multisubunit regulatory protein involved in ubiquitin-dependent protein degradation. These data indicate that there is a hierarchy among regulators for the control of proteasome function.