A universal strategy for proteomic studies of SUMO and other ubiquitin-like modifiers
A universal strategy for proteomic studies of SUMO and other ubiquitin-like modifiers
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DOI：
10.1074/mcp.m400149-mcp200
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发表时间：
2005-01-01
影响因子：
7
通讯作者：
Wilson, VG
中科院分区：
文献类型：
--
作者：
Rosas-Acosta, G;Russell, WK;Wilson, VG
小泛素样修饰物的结合是影响蛋白质功能的重要机制。目前，大多数这些修饰物的底物已知数量有限，从而限制了我们对它们在细胞生理学中的作用和相关性的了解。在这里，我们报告了泛素样修饰物蛋白质组学研究的通用策略的发展。这一策略包括在严格负调控启动子的控制下建立稳定的表达双标记修饰物的细胞系，诱导标记修饰物的表达和与细胞蛋白的结合，串联亲和纯化标记修饰物共价修饰的蛋白质池，以及用LC和MS鉴定修饰的蛋白质。将该方法应用于SUMO-1和SUMO-3的蛋白质组学分析，我们确定SUMO-1和SUMO-3是半衰期超过20h的稳定蛋白质，证明MG-132和热休克处理都能极大地刺激相扑-1和相扑-3的相扑修饰。证明了相扑-1或相扑-3对某些已知相扑底物的优先使用，并确定了122种假定相扑底物，其中只有27种似乎同时被相扑-1和相扑-3修饰。相扑-1和相扑-3修饰的蛋白质子集的这种有限重叠支持相扑类似物很可能在功能上是不同的。鉴定的三种新的相扑底物，即多嘧啶结合蛋白相关剪接因子PSF，结构微管成分α-微管蛋白和与GTP结合的核蛋白RAN，被确认为真正的相扑底物。将这一通用策略应用于鉴定由其他泛素样修饰物修饰的细胞底物池，将极大地增加我们对不同泛素样偶联物系统在细胞中的生物学作用的了解。
Post-translational modification by the conjugation of small ubiquitin-like modifiers is an essential mechanism to affect protein function. Currently, only a limited number of substrates are known for most of these modifiers, thus limiting our knowledge of their role and relevance for cellular physiology. Here, we report the development of a universal strategy for proteomic studies of ubiquitin-like modifiers. This strategy involves the development of stable transfected cell lines expressing a double-tagged modifier under the control of a tightly negatively regulated promoter, the induction of the expression and conjugation of the tagged modifier to cellular proteins, the tandem affinity purification of the pool of proteins covalently modified by the tagged modifier, and the identification of the modified proteins by LC and MS. By applying this methodology to the proteomic analysis of SUMO-1 and SUMO-3, we determined that SUMO-1 and SUMO-3 are stable proteins exhibiting half-lives of over 20 h, demonstrated that sumoylation with both SUMO-1 and SUMO-3 is greatly stimulated by MG-132 and heat shock treatment, demonstrated the preferential usage of either SUMO-1 or SUMO-3 for some known SUMO substrates, and identified 122 putative SUMO substrates of which only 27 appeared to be modified by both SUMO-1 and SUMO-3. This limited overlapping in the subset of proteins modified by SUMO-1 and SUMO-3 supports that the SUMO paralogues are likely to be functionally distinct. Three of the novel putative SUMO substrates identified, namely the polypyrimidine tract-binding protein-associated splicing factor PSF, the structural microtubular component alpha-tubulin, and the GTP- binding nuclear protein Ran, were confirmed as authentic SUMO substrates. The application of this universal strategy to the identification of the pool of cellular substrates modified by other ubiquitin-like modifiers will dramatically increase our knowledge of the biological role of the different ubiquitin-like conjugations systems in the cell.