Davydov I V, Patra D, Varshavsky A
Division of Biology, California Institute of Technology, Pasadena 91125, USA.
Arch Biochem Biophys. 1998 Sep 15;357(2):317-25. doi: 10.1006/abbi.1998.0829.
Ubiquitin-dependent degradation of intracellular proteins underlies a multitude of biological processes, including the cell cycle, cell differentiation, and responses to stress. One ubiquitin-dependent proteolytic system is the N-end rule pathway, whose targets include proteins that bear destabilizing N-terminal residues. This pathway, which has been characterized only in somatic cells, is shown here to be present also in germ line cells such as the eggs of the amphibian Xenopus laevis. We demonstrate that the set of destabilizing residues in the N-end rule pathway of Xenopus eggs is similar, if not identical, to that of somatic cells such as mammalian reticulocytes and fibroblasts. It is also shown that the degradation of engineered N-end rule substrates in egg extracts can be strongly and selectively inhibited by dipeptides bearing destabilizing N-terminal residues. This result allowed us to ask whether selective inhibition of the N-end rule pathway in egg extracts influences the apoptosis-like changes that are observed in these extracts. A dipeptide bearing a bulky hydrophobic (type 2) destabilizing N-terminal residue was found to delay the apoptotic changes in egg extracts, whereas dipeptides bearing basic (type 1) destabilizing N-terminal residues had no effect. High activity of the N-end rule pathway in egg extracts provides an alternative to reticulocyte extracts for the in vitro analyses of this pathway.
细胞内蛋白质的泛素依赖性降解是众多生物学过程的基础，包括细胞周期、细胞分化以及应激反应。一种泛素依赖性蛋白水解系统是N端规则途径，其靶标包括带有不稳定N端残基的蛋白质。该途径此前仅在体细胞中得到表征，本文显示其在生殖系细胞中也存在，比如两栖动物非洲爪蟾的卵。我们证明，非洲爪蟾卵的N端规则途径中不稳定残基的集合即便不完全相同，也与诸如哺乳动物网织红细胞和成纤维细胞等体细胞中的相似。研究还表明，带有不稳定N端残基的二肽能够强烈且选择性地抑制卵提取物中工程化N端规则底物的降解。这一结果使我们能够探究，选择性抑制卵提取物中的N端规则途径是否会影响这些提取物中观察到的类似凋亡的变化。结果发现，带有庞大疏水（2型）不稳定N端残基的二肽可延缓卵提取物中的凋亡变化，而带有碱性（1型）不稳定N端残基的二肽则没有效果。卵提取物中N端规则途径的高活性为该途径的体外分析提供了一种替代网织红细胞提取物的方法。
