...
  • 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Biochemistry >Substrate properties of ubiquitin carboxyl-terminally derived peptide probes for protein ubiquitination
【24h】

Substrate properties of ubiquitin carboxyl-terminally derived peptide probes for protein ubiquitination

机译：泛素羧基末端衍生肽探针对蛋白质泛素化的底物特性

获取原文
获取原文并翻译 | 示例
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

Protein ubiquitination is a widespread protein posttranslational modification in eukaryotes that regulates essentially every aspect of cellular processes. The attachment of ubiquitin to a protein substrate is accomplished through an enzymatic cascade involving the actions of an activating enzyme (E1), a conjugating enzyme (E2), and a ligase (E3). There are more than 600 E3 ligases estimated to exist in the human genome that regulate the targeting specificity of protein ubiquitination. To understand the dynamic role of protein ubiquitination in biological processes, robust tools need to be developed which can be employed to establish the substrate specificity of each of these E3 ligases. In this report, we show that the ubiquitin carboxyl-terminally derived peptide probes can serve as modest ubiquitin surrogates for the ubiquitination pathway. In the E1-catalyzed probe adenylation assay, peptide probe 3 with a RLRGG recognition sequence exhibited the highest activity, with the k(cat)/K-1/2 determined to be 1.1 x 10(4) M-1 s(-1), roughly 470-fold lower than that of ubiquitin. The rate of transfer from the E1 peptide probe thioesters to E2 showed clear sequence dependency, with peptide probe 4 with an LRLRGG recognition sequence showed the fastest rate (t(1/2) = 0.9 min), essentially identical to that of ubiquitin (t(1/2) = 0.8 min) under our assay conditions. Furthermore, peptide probes 4 and 8 also exhibited the selective, parkin-mediated labeling of tubulins in a semipurified tubulin-parkin complex. Finally, these carboxyl-terminally derived peptide probes were shown to label the ubiquitination substrates in fraction II of the rabbit reticulocyte lysate with an efficiency parallel to their substrate properties. The selective use of these ubiquitin carboxyl-terminally derived peptide probes by the ubiquitination pathway suggests that perhaps more potent peptide ubiquitination probes based on the ubiquitin C-terminal scaffold can be developed through additional structural optimization.
机译：蛋白质泛素化是真核生物中广泛的蛋白质翻译后修饰，它基本上调控细胞过程的各个方面。泛素与蛋白质底物的连接是通过涉及激活酶（E1），结合酶（E2）和连接酶（E3）的酶促级联反应完成的。据估计，在人类基因组中存在600多种E3连接酶，它们调节蛋白质泛素化的靶向特异性。为了了解蛋白质泛素化在生物过程中的动态作用，需要开发出可靠的工具，这些工具可用于建立每种E3连接酶的底物特异性。在此报告中，我们表明泛素羧基末端衍生的肽探针可以作为泛素化途径的适度泛素替代物。在E1催化的探针腺苷酸化分析中，具有RLRGG识别序列的肽探针3表现出最高的活性，其中k（cat）/ K-1 / 2被确定为1.1 x 10（4）M-1 s（-1） ），比泛素低约470倍。从E1肽探针硫酯到E2的转移速率显示出明确的序列依赖性，具有LRLRGG识别序列的肽探针4显示最快的速率（t（1/2）= 0.9 min），与泛素（t （1/2）= 0.8分钟）。此外，肽探针4和8还显示出在半纯化的微管蛋白-帕金复合物中的微管蛋白的选择性，帕金介导的标记。最后，这些羧基末端衍生的肽探针显示出标记兔网织红细胞裂解物II中泛素化底物的效率与其底物特性平行。通过泛素化途径对这些泛素羧基末端衍生的肽探针的选择性使用表明，可能可以通过其他结构优化来开发基于泛素C末端支架的更有效的肽泛素化探针。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号