Analysis of Sumoylation
Analysis of Sumoylation
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DOI：
10.1007/978-1-4939-9055-9_14
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发表时间：
2019-01-01
期刊：
影响因子：
--
通讯作者：
Pichler, Andrea
中科院分区：
文献类型：
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作者：
Breucker, Jan;Pichler, Andrea
SUMO（小泛素相关修饰剂）可逆附着的蛋白质调节在转录调节、核质运输、细胞周期进程、减数分裂和 DNA 修复等多种细胞过程中发挥着重要作用。然而，大多数苏酰化蛋白质在稳态水平下丰度有限，这是由于严格的调控和/或修饰和去修饰的快速周转。因此，体内蛋白质苏酰化的分析非常具有挑战性。尽管如此，还是建立了一种新方法，可以检测脊椎动物细胞和组织内源水平的苏酰化蛋白。该方法涉及通过免疫沉淀富集苏酰化蛋白质，然后进行肽洗脱。在内源性底物苏酰化得到验证后，解决其功能后果是下一个合乎逻辑的步骤。这需要从大量修饰蛋白中受益的 SUMO 位点映射。在这里，我们简要描述了实现许多底物有效体外苏酰化的策略。
Protein regulation by reversible attachment of SUMO (small ubiquitin-related modifier) plays an important role in several cellular processes such as transcriptional regulation, nucleo-cytoplasmic transport, cell-cycle progression, meiosis, and DNA repair. However, most sumoylated proteins are of marginal abundance at steady state levels, which is due to strict regulation and/or rapid turnover of modification and de-modification. Consequently, analysis of protein sumoylation in vivo is very challenging. Nonetheless, a novel method was established that allows detection of sumoylated proteins at endogenous levels from vertebrate cells and tissues. This approach involves the enrichment of sumoylated proteins by immunoprecipitation followed by peptide elution. After endogenous substrate sumoylation is verified, addressing its functional consequences is the next logical step. This requires SUMO site mapping that benefits from larger quantities of modified protein. Here, we shortly describe strategies to achieve efficient in vitro sumoylation of many substrates.