MICROINJECTION STUDIES ON PROTEIN DEGRADATION

这项建议的长期目标是通过以下方式阐明这些机制 哪些细胞选择性地降解它们的细胞内蛋白并 发现促进分解代谢的蛋白质的特性。两个单元格 有一些系统可以消除这两种情况中的任何一种 内肽酶或泛素(Ub)结合。个人，放射性标记 蛋白质将被注射到每个细胞系中，以确定哪些蛋白质 是这些途径的底物。这一过程已经揭示了 泛素和牛血清白蛋白AR在缺乏细胞中稳定 脯氨酸内肽酶和氧化血红蛋白在TS85中稳定 含有一种不稳定的Ub激活酶的小鼠细胞。人类的半衰期 一系列对温度敏感的T4溶菌酶也将被检测到 检验热力学稳定性与新陈代谢相关的假设 稳定性。令人惊讶的是，T4溶菌酶在HeLa中降解得非常快 细胞，我们将确定表面半胱氨酸或精氨酸-精氨酸 Pair对其短暂的寿命负有责任。此外，特定的蛋白质 将针对原子核，以发现它们的降解率 都受到本地化错误的影响。最后，泛素将被注射到 TS85小鼠细胞或HeLa细胞和Ub代谢的几个方面 测量，包括Ub在自噬和注射的释放中的作用 蛋白质，以及Ub结合物的亚细胞定位。 在热震之后。

The long term objective of this proposal os to elucidate the mechanisms by which cells selectively degrade their intracellular proteins and to discover the features of protein that promote their catabolism. Two cell systems are available in which it is possible to eliminate either proline endopeptidase or ubiquitin (Ub) conjugation. Individual, radiolabeled proteins will be injected into each cell line to determine which proteins are substrates for these pathways. This procedure has already revealed that ubiquitin and bovine serum albumin ar stabilized in cells lacking proline endopeptidase and that oxidized hemoglobin is stabilized in ts85 mouse cells which contain a labile Ub-activating enzyme. The half-lives of a series of temperature-sensitive T4 lysozymes will also be measured to test the hypothesis that thermodynamic stability correlates with metabolic stability. Surprisingly, T4 lysozyme is degraded very rapidly in HeLa cells, and we will determine whether surface cysteines or arginine-arginine pairs are responsible for its short life. In addition, specific proteins will be targeted to the nucleus to discover whether their degradation rates are affected by mislocalization. Finally, ubiquitin will be injected into ts85 mouse cells or HeLa cells and several aspects of Ub metabolism will be measured, including Ub's role in autophagy and release of injected proteins, as well as the subcellular location of Ub conjugates before and after heat-shock.