Ubiquitin-Family Modifications of Topoisomerase I in Camptothecin-Treated Human Breast Cancer Cells
Ubiquitin-Family Modifications of Topoisomerase I in Camptothecin-Treated Human Breast Cancer Cells
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DOI：
10.1021/bi802179t
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发表时间：
2009-04-14
期刊：
影响因子：
2.9
通讯作者：
Snapka, Robert M.
中科院分区：
文献类型：
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作者：
Kanagasabai, Ragu;Liu, Shujun;Snapka, Robert M.
喜树碱通过稳定拓扑异构酶I-DNA链传递中间体杀死哺乳动物细胞，所述中间体在DNA复制叉碰撞中转化为致死性双链DNA断裂。哺乳动物细胞中喜树碱稳定的拓扑异构酶I-DNA切割中间体被泛素家族蛋白独特地修饰。这些泛素家族修饰的结构、组成和功能知之甚少。我们已经使用毛细管液相色谱-纳米喷雾串联质谱分析的内源性泛素家族修饰的拓扑异构酶I纯化从喜树碱稳定的拓扑异构酶I-DNA裂解复合物在人乳腺癌细胞。SUMO-2和SUMO-3共有的肽是丰富的，并且鉴定了SUMO-2特有的肽。在这些复合物中还鉴定了泛素。在人拓扑异构酶I-DNA切割复合物中未检测到SUMO-1肽。与纯化的SUMO旁系同源物的相同实验表明，SUMO-I被很好地消化，我们的协议和片段很容易通过LC-MS/MS分析。与纯化的SUMO旁系同源物的加标实验确定，我们可以检测到少至0.5 SUMO-1每个拓扑异构酶I分子的残基。这些结果表明，SUMO-1低于该检测水平，SUMO-2或SUMO-2和SUMO-3的混合物占优势。SUMO-1加帽似乎不太可能限制喜树碱稳定的拓扑异构酶I-DNA切割复合物上形成的SUMO-2/3链的生长。
Camptothecins kill mammalian cells by stabilizing topoisomerase I-DNA strand passing intermediates that are converted to lethal double strand DNA breaks in DNA replication fork collisions. Camptothecin-stabilized topoisomerase I-DNA cleavage intermediates in mammalian cells are uniquely modified by ubiquitin-family proteins. The structure, composition, and function of these ubiquitin-family modifications are poorly understood. We have used capillary liquid chromatography-nanospray tandem mass spectrometry to analyze the endogenous ubiquitin-family modifications of topoisomerase I purified from camptothecin-stabilized topoisomerase I-DNA cleavage complexes in human breast cancer cells. Peptides shared by SUMO-2 and SUMO-3 were abundant, and a peptide unique to SUMO-2 was identified. Ubiquitin was also identified in these complexes. No SUMO-1 peptide was detected in human topoisomerase I-DNA cleavage complexes. Identical experiments with purified SUMO paralogues showed that SUMO-I was well digested by our protocol and that fragments were easily analyzed by LC-MS/MS. Spiking experiments with purified SUMO paralogues determined that we could detect as little as 0.5 SUMO-1 residue per topoisomerase I molecule. These results indicate that SUMO-1 is below this detection level and that SUMO-2 or a mixture of SUMO-2 and SUMO-3 predominates. SUMO-1 capping seems unlikely to be limiting the growth of SUMO-2/3 chains formed on camptothecin-stabilized topoisomerase I-DNA cleavage complexes.