MICROINJECTION STUDIES ON PROTEIN DEGRADATION

本提案的长期目标是通过以下方式阐明机制： 这些细胞选择性地降解它们的细胞内蛋白质， 发现蛋白质的特征，促进他们的catalysis。 两种细胞 可获得这样的系统，其中可以消除脯氨酸或脯氨酸， 内肽酶或泛素（Ub）缀合。 单个放射性标记 将蛋白质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。 这个过程已经揭示了 泛素和牛血清白蛋白在缺乏 脯氨酸内肽酶和氧化血红蛋白在TS 85中稳定 含有不稳定的Ub激活酶的小鼠细胞。 的半衰期 还将测量一系列温度敏感的T4溶菌酶， 测试热力学稳定性与代谢相关的假设 稳定 令人惊讶的是，T4溶菌酶在HeLa中降解非常迅速 细胞，我们将确定是否表面半胱氨酸或精氨酸精氨酸 对是它寿命短的原因 此外，特定的蛋白质 将瞄准原子核，以发现它们的降解率 都受到定位错误的影响 最后，将泛素注入 ts85小鼠细胞或HeLa细胞和Ub代谢的几个方面将被 测量，包括Ub在自噬和释放注射的 蛋白质，以及Ub缀合物在之前和之后的亚细胞位置。 热休克后。

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.