Detection of protein ubiquitination.
Detection of protein ubiquitination.
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DOI：
10.3791/1293
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发表时间：
2009-08-19
期刊：
影响因子：
--
通讯作者：
Zhang, Zhuohua
中科院分区：
文献类型：
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作者：
Choo, Yeun Su;Zhang, Zhuohua
泛素化，即多肽泛素与靶蛋白的共价连接，是由一组三种酶进行的关键翻译后修饰。它们包括泛素激活酶E1、泛素缀合酶E2和泛素连接酶E3。与E1和E2不同，E3泛素连接酶显示底物特异性。另一方面，许多去泛素化酶在加工多聚泛素化蛋白质中起作用。泛素化可导致蛋白质稳定性、细胞定位和生物活性的改变。参与泛素化/去泛素化途径的基因突变或改变的泛素系统功能与许多不同的人类疾病相关，例如各种类型的癌症、神经变性和代谢紊乱。检测靶蛋白的泛素化改变或正常，可以更好地了解这些疾病的发病机制。在这里，我们描述的协议，以检测蛋白质泛素化在体外培养的细胞在体内和试管。这些方案也可用于检测其他泛素样小分子修饰，如sumolyation和neddylation。
Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.