Analysis of oxygen/glucose-deprivation-induced changes in SUMO3 conjugation using SILAC-based quantitative proteomics.
Analysis of oxygen/glucose-deprivation-induced changes in SUMO3 conjugation using SILAC-based quantitative proteomics.
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DOI：
10.1021/pr200834f
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发表时间：
2012-02-03
影响因子：
4.4
通讯作者：
Paschen W
中科院分区：
文献类型：
--
作者：
Yang W;Thompson JW;Wang Z;Wang L;Sheng H;Foster MW;Moseley MA;Paschen W
短暂性脑缺血显著激活小泛素样修饰物（SUMO2/3）缀合。在暴露于6小时的短暂氧/葡萄糖剥夺（OGD）（缺血模型）的细胞中，SUMOylation在再氧合后的0到30分钟内急剧增加。为了阐明瞬时OGD对SUMO靶蛋白结合的影响，我们将表达HA-SUMO3的神经母细胞瘤B35细胞暴露于瞬时OGD中，并使用细胞培养氨基酸稳定同位素标记（SILAC）来量化OGD诱导的特定SUMO化蛋白水平的变化。将对照细胞和ogd处理细胞的裂解液均匀混合，对ha标记的蛋白进行免疫沉淀，并通过1D-SDS-PAGE-LC-MS/MS进行分析。我们鉴定了188个假定的SUMO - 3结合蛋白，包括许多转录因子和共调节因子，以及PIAS2和PIAS4 SUMO连接酶，其中22个增加或减少了bb0±2倍。除SUMO3外，蛋白偶联的SUMO1、SUMO2及泛素水平均升高。重要的是，OGD诱导的蛋白泛素化被SUMO2/3基因沉默完全阻断。总的来说，这些结果表明了ogd调节的SUMO化的几种机制，指出了一些信号通路，这些信号通路可能是基于SUMO的信号传导和缺血应激恢复的目标，并证明了SUMO和泛素偶联通路之间存在严格控制的串串。
Transient cerebral ischemia dramatically activates small ubiquitin-like modifier (SUMO2/3) conjugation. In cells exposed to 6 h of transient oxygen/glucose deprivation (OGD), a model of ischemia, SUMOylation increases profoundly between 0 and 30 min following re-oxygenation. To elucidate the effect of transient OGD on SUMO conjugation of target proteins, we exposed neuroblastoma B35 cells expressing HA-SUMO3 to transient OGD and used stable isotope labeling with amino acids in cell culture (SILAC) to quantify OGD-induced changes in levels of specific SUMOylated proteins. Lysates from control and OGD-treated cells were mixed equally, and HA-tagged proteins were immunoprecipitated and analyzed by 1D-SDS-PAGE-LC-MS/MS. We identified 188 putative SUMO3-conjugated proteins, including numerous transcription factors and coregulators, and PIAS2 and PIAS4 SUMO ligases, of which 22 were increased or decreased >±2-fold. In addition to SUMO3, the levels of protein-conjugated SUMO1 and SUMO2, as well as ubiquitin, were all increased. Importantly, protein ubiquitination induced by OGD was completely blocked by gene silencing of SUMO2/3. Collectively, these results suggest several mechanisms for OGD-modulated SUMOylation, point to a number of signaling pathways that may be targets of SUMO-based signaling and recovery from ischemic stress, as well as demonstrate a tightly controlled crosstalk between the SUMO and ubiquitin conjugation pathways.
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