Kim Eui Tae, Kim Kyeong Kyu, Matunis Mike J, Ahn Jin-Hyun
Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Cheoncheondong Jangangu, Suwon, Gyeonggido, Republic of Korea.
Biochem Biophys Res Commun. 2009 Oct 9;388(1):41-5. doi: 10.1016/j.bbrc.2009.07.103. Epub 2009 Jul 25.
Identifying new targets for SUMO and understanding the function of protein SUMOylation are largely limited by low level of SUMOylation. It was found recently that Ubc9, the SUMO E2 conjugating enzyme, is covalently modified by SUMO at a lysine 14 in the N-terminal alpha helix, and that SUMO-modified Ubc9 has enhanced conjugation activity for certain target proteins containing a SUMO-interacting motif (SIM). Here, we show that, compared to intact Ubc9, the SUMO-Ubc9 fusion protein has higher conjugating activity for SIM-containing targets such as Sp100 and human cytomegalovirus IE2. Assays using an IE2 SIM mutant revealed the requirement of SIM for the enhanced IE2 SUMOylation by SUMO-Ubc9. In pull-down assays with cell extracts, the SUMO-Ubc9 fusion protein bound to more diverse cellular proteins and interacted with some SIM-containing proteins with higher affinities than Ubc9. Therefore, the devised SUMO-Ubc9 fusion will be useful for identifying SIM-containing SUMO targets and producing SUMO-modified proteins.
SUMO新靶点的识别以及对蛋白质SUMO化功能的理解在很大程度上受到SUMO化水平较低的限制。最近发现，SUMO E2缀合酶Ubc9在N端α螺旋的赖氨酸14处被SUMO共价修饰，并且SUMO修饰的Ubc9对某些含有SUMO相互作用基序（SIM）的靶蛋白具有增强的缀合活性。在这里，我们表明，与完整的Ubc9相比，SUMO-Ubc9融合蛋白对含有SIM的靶标（如Sp100和人巨细胞病毒IE2）具有更高的缀合活性。使用IE2 SIM突变体的分析揭示了SIM对于SUMO-Ubc9增强IE2 SUMO化的必要性。在细胞提取物的下拉分析中，SUMO-Ubc9融合蛋白与更多种类的细胞蛋白结合，并且与一些含有SIM的蛋白相互作用，其亲和力高于Ubc9。因此，设计的SUMO-Ubc9融合蛋白将有助于识别含有SIM的SUMO靶标并产生SUMO修饰的蛋白。
