A Chemical and Enzymatic Approach to Study Site-Specific Sumoylation.
A Chemical and Enzymatic Approach to Study Site-Specific Sumoylation.
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DOI：
10.1371/journal.pone.0143810
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Zhou H
中科院分区：
文献类型：
--
作者：
Albuquerque CP;Yeung E;Ma S;Fu T;Corbett KD;Zhou H
多种细胞通路通过泛素家族蛋白的蛋白修饰来调节。SUMO是一种小泛素样修饰剂，通过E1、E2和E3酶催化的级联反应共价附着在目标蛋白上的赖氨酸上。了解SUMO的多种调节作用的主要障碍是缺乏合适的方法来识别蛋白质SUMO化位点。在这里，我们开发了一种基于质谱（MS）的方法，结合化学和酶修饰来鉴定sumoylation位点。我们应用该方法在体外分析了E1酶的自聚合化，并将其与以Smt3-I96R为底物的gg -残基法进行了比较。我们进一步研究了smt3-I96R突变在体内的影响，并对酿酒酵母的蛋白质聚合位点进行了蛋白质组范围的分析。为了验证这些发现，我们在体内确认了Aos1和Uba2的几个sumomylation位点。总之，这些结果表明，我们的化学和酶的方法鉴定蛋白质的summoylation位点提供了一个有用的工具和方法的组合允许蛋白质的summoylation位点的详细分析。
A variety of cellular pathways are regulated by protein modifications with ubiquitin-family proteins. SUMO, the Small Ubiquitin-like MOdifier, is covalently attached to lysine on target proteins via a cascade reaction catalyzed by E1, E2, and E3 enzymes. A major barrier to understanding the diverse regulatory roles of SUMO has been a lack of suitable methods to identify protein sumoylation sites. Here we developed a mass-spectrometry (MS) based approach combining chemical and enzymatic modifications to identify sumoylation sites. We applied this method to analyze the auto-sumoylation of the E1 enzyme in vitro and compared it to the GG-remnant method using Smt3-I96R as a substrate. We further examined the effect of smt3-I96R mutation in vivo and performed a proteome-wide analysis of protein sumoylation sites in Saccharomyces cerevisiae. To validate these findings, we confirmed several sumoylation sites of Aos1 and Uba2 in vivo. Together, these results demonstrate that our chemical and enzymatic method for identifying protein sumoylation sites provides a useful tool and that a combination of methods allows a detailed analysis of protein sumoylation sites.