Ma Li, Aslanian Aaron, Sun Huaiyu, Jin Mingji, Shi Yu, Yates John R, Hunter Tony
From the ‡Molecular and Cell Biology Laboratory, Salk Institute for Biological Studies, La Jolla, California 92037, USA;
From the ‡Molecular and Cell Biology Laboratory, Salk Institute for Biological Studies, La Jolla, California 92037, USA; §Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California 92037, USA.
Mol Cell Proteomics. 2014 Jul;13(7):1659-75. doi: 10.1074/mcp.M113.035626. Epub 2014 May 5.
Post-translational modification by SUMO is a highly conserved pathway in eukaryotes that plays very important regulatory roles in many cellular processes. Deregulation of the SUMO pathway contributes to the development and progression of many diseases including cancer. Therefore, identifying additional SUMO substrates and studying how their cellular and biological functions are regulated by sumoylation should provide new insights. Our studies showed that sumoylation activity was significant in Xenopus egg extracts, and that a high level of sumoylation was associated with sperm chromatin when SUMO was incubated with Xenopus egg extracts. By isolating SUMO-conjugated substrates using His-tagged SUMO1 or SUMO2 proteins under denaturing conditions, we identified 346 proteins by mass spectrometry analysis that were not present in control pull-downs. Among them, 167 proteins were identified from interphase egg extracts, 86 proteins from mitotic phase egg extracts, and 93 proteins from both. Thirty-three proteins were pulled down by SUMO1, 85 proteins by SUMO2, and 228 proteins by both. We validated the sumoylation of five candidates, CKB, ATXN10, BTF3, HABP4, and BZW1, by co-transfecting them along with SUMO in HEK293T cells. Gene ontology analysis showed that SUMO substrates identified in this study were involved in diverse biological processes. Additionally, SUMO substrates identified from different cell cycle stages or pulled down by different SUMO homologs were enriched for distinct cellular components and functional categories. Our results comprehensively profile the sumoylation occurring in the Xenopus egg extract system.
小泛素样修饰蛋白（SUMO）介导的翻译后修饰是真核生物中一条高度保守的途径，在许多细胞过程中发挥着非常重要的调节作用。SUMO途径失调会导致包括癌症在内的许多疾病的发生和发展。因此，识别更多的SUMO底物并研究它们的细胞和生物学功能是如何通过SUMO化进行调节的，应该会提供新的见解。我们的研究表明，SUMO化活性在非洲爪蟾卵提取物中很显著，并且当SUMO与非洲爪蟾卵提取物一起孵育时，高水平的SUMO化与精子染色质相关。通过在变性条件下使用His标签的SUMO1或SUMO2蛋白分离SUMO缀合的底物，我们通过质谱分析鉴定了346种在对照下拉实验中不存在的蛋白质。其中，167种蛋白质从间期卵提取物中鉴定出来，86种蛋白质从有丝分裂期卵提取物中鉴定出来，93种蛋白质在两者中都有。33种蛋白质被SUMO1下拉，85种蛋白质被SUMO2下拉，228种蛋白质被两者都下拉。我们通过在HEK293T细胞中与SUMO共转染来验证五个候选蛋白CKB、ATXN10、BTF3、HABP4和BZW1的SUMO化。基因本体分析表明，本研究中鉴定的SUMO底物参与了多种生物学过程。此外，从不同细胞周期阶段鉴定的或被不同SUMO同源物下拉的SUMO底物在不同的细胞成分和功能类别中富集。我们的结果全面描绘了非洲爪蟾卵提取物系统中发生的SUMO化情况。
