Li Tianwei, Santockyte Rasa, Shen Rong-Fong, Tekle Ephrem, Wang Guanghui, Yang David C H, Chock P Boon
Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Arch Biochem Biophys. 2006 Sep 1;453(1):70-4. doi: 10.1016/j.abb.2006.03.002. Epub 2006 Mar 20.
Ubiquitin-like modifiers (UBLs) contain ubiquitin homology domains and can covalently modify target proteins in a manner similar to ubiquitylation. In this study, we revealed a general proteomic approach to elucidate the enzymatic pathways and identify target proteins for three UBLs: SUMO-2, SUMO-3, and NEDD8. Expression plasmids containing the cDNAs of Myc/6xHis doubly-tagged processed or non-conjugatable forms of these UBLs were constructed. The constructed vectors were then used to transfect HEK 293 Tet-On cells, and stable cell lines expressing these UBLs and their mutants were established. The epitope-tagged proteins were purified by immunoprecipitation under native conditions or by affinity chromatography on nickel resin under denaturing conditions. Purified proteins were analyzed using liquid chromatography coupled with mass spectrometry (LC-MS/MS). Most of the E1-like activating enzymes, E2-like conjugating enzymes and the majorities of the known target as well as some previously unreported proteins for SUMO-2, SUMO-3, and NEDD8 pathways were identified.
类泛素修饰因子（UBLs）含有泛素同源结构域，能够以类似于泛素化的方式共价修饰靶蛋白。在本研究中，我们揭示了一种通用的蛋白质组学方法，用于阐明三种UBLs（SUMO-2、SUMO-3和NEDD8）的酶促途径并鉴定其靶蛋白。构建了含有Myc/6xHis双标签的这些UBLs加工形式或不可缀合形式的cDNA的表达质粒。然后使用构建的载体转染HEK 293 Tet-On细胞，并建立表达这些UBLs及其突变体的稳定细胞系。通过在天然条件下免疫沉淀或在变性条件下用镍树脂进行亲和层析来纯化表位标记的蛋白质。使用液相色谱-质谱联用（LC-MS/MS）分析纯化的蛋白质。鉴定出了大多数E1样激活酶、E2样缀合酶以及SUMO-2、SUMO-3和NEDD8途径的大多数已知靶蛋白以及一些先前未报道的蛋白质。
