Short-lived green fluorescent proteins for quantifying ubiquitin/proteasome-dependent proteolysis in living cells
Short-lived green fluorescent proteins for quantifying ubiquitin/proteasome-dependent proteolysis in living cells
复制标题
DOI：
10.1038/75406
复制
发表时间：
2000-05-01
影响因子：
46.9
通讯作者：
Masucci, MG
中科院分区：
文献类型：
--
作者：
Dantuma, NP;Lindsten, K;Masucci, MG
泛素/蛋白酶体依赖的蛋白水解途径是一个有吸引力的治疗目标，因为它的关键参与细胞周期进程和抗原呈递。然而，由于系统的复杂性和缺乏易于检测的真实底物，阻碍了对途径的剖析和调节剂的开发。我们已经开发了一个方便的报告系统，通过生产N-末端规则和泛素融合降解（UFD）靶向的绿色荧光蛋白，允许量化的泛素/蛋白酶体依赖的蛋白水解活细胞。这些报告基因的积累可作为蛋白酶体抑制剂处理细胞中G2/M期阻滞和细胞凋亡的早期预测因子。报告积累和切割的荧光底物的比较表明，限速胰凝乳蛋白酶样活性的蛋白酶体可以基本上削减没有显着影响泛素依赖的蛋白水解。这些报告提供了一个新的强大的工具，阐明泛素/蛋白酶体途径和高通量筛选的化合物，选择性地修改体内蛋白水解。
The ubiquitin/proteasome-dependent proteolytic pathway is an attractive target for therapeutics because of its critical involvement in cell cycle progression and antigen presentation. However, dissection of the pathway and development of modulators are hampered by the complexity of the system and the lack of easily detectable authentic substrates. We have developed a convenient reporter system by producing N-end rule and ubiquitin fusion degradation (UFD)-targeted green fluorescent proteins that allow quantification of ubiquitin/proteasome-dependent proteolysis in living cells. Accumulation of these reporters serves as an early predictor of G2/M arrest and apoptosis in cells treated with proteasome inhibitors. Comparison of reporter accumulation and cleavage of fluorogenic substrates demonstrates that the rate-limiting chymotrypsin-like activity of the proteasome can be substantially curtailed without significant effect on ubiquitin-dependent proteolysis. These reporters provide a new powerful tool for elucidation of the ubiquitin/proteasome pathway and for high throughput screening of compounds that selectively modify proteolysis in vivo.