The dynamic alterations of H2AX complex during DNA repair detected by a proteomic approach reveal the critical roles of Ca2+/calmodulin in the ionizing radiation-induced cell cycle arrest
The dynamic alterations of H2AX complex during DNA repair detected by a proteomic approach reveal the critical roles of Ca2+/calmodulin in the ionizing radiation-induced cell cycle arrest
复制标题
DOI：
10.1074/mcp.m500327-mcp200
复制
发表时间：
2006-06-01
影响因子：
7
通讯作者：
Chen, Xian
中科院分区：
文献类型：
--
作者：
Du, Yu-Chun;Gu, Sheng;Chen, Xian
通过使用 DNA 核酸酶消化和集成质谱、稳定同位素标记和亲和纯化的定量“双标记”蛋白质组学方法，我们研究了哺乳动物细胞染色质中组蛋白 H2AX 相关蛋白复合物对电离辐射 (IR) 的响应。在未照射的对照细胞中，钙调蛋白 (CaM) 和转录延伸因子促进染色质转录 (FACT) 与 H2AX 相关。将细胞暴露于 IR 30 分钟后，CaM 和 FACT 复合物解离，而两种 DNA 修复蛋白（聚（ADP-核糖）聚合酶 1 和 DEAH 盒多肽 30 同工型 1）与 H2AX 相互作用。暴露后 2 小时 30 分钟，复合物中不再含有上述蛋白质。在未照射和照射的细胞中，H2B、核磷蛋白/B23和钙网蛋白与H2AX相关。结果表明，H2AX 复合物在诱导 DNA 损伤和 DNA 修复过程中经历动态变化。通过免疫沉淀/蛋白质印迹和哺乳动物双杂交测定验证了每种条件下 H2AX 和 H2B、核磷蛋白/B23、钙网蛋白、聚（ADP-核糖）聚合酶-1 和 CaM 之间的真正相互作用。由于在 H2AX 复合物中发现了多种 Ca2+ 结合蛋白，因此对 Ca2+ 的作用进行了检查。结果表明，Ca2+/CaM 在调节 IR 诱导的细胞周期停滞中发挥重要作用，可能是通过介导染色质结构来实现的。这里提供的数据集表明，对功能性细胞蛋白质-蛋白质相互作用的动力学进行灵敏的分析可以成功地剖析重要的代谢或信号传导途径。
By using DNA nuclease digestion and a quantitative '' dual tagging '' proteomic approach that integrated mass spectrometry, stable isotope labeling, and affinity purification, we studied the histone H2AX-associating protein complex in chromatin in mammalian cells in response to ionizing radiation (IR). In the non-irradiated control cells, calmodulin (CaM) and the transcription elongation factor facilitates chromatin transcription (FACT) were associated with H2AX. Thirty minutes after exposing cells to IR the CaM and FACT complexes dissociated, whereas two DNA repair proteins, poly(ADP-ribose) polymerase-1 and DEAH box polypeptide 30 isoform 1, interacted with H2AX. Two hours and 30 min after exposure, none of the above proteins were in the complex. H2B, nucleophosmin/B23, and calreticulin were associated with H2AX in both non- irradiated and irradiated cells. The results suggest that the H2AX complex undergoes dynamic changes upon induction of DNA damage and during DNA repair. The genuine interactions between H2AX and H2B, nucleophosmin/B23, calreticulin, poly(ADP-ribose) polymerase-1, and CaM under each condition were validated by immunoprecipitation/Western blotting and mammalian two-hybrid assays. Because multiple Ca2+-binding proteins were found in the H2AX complex, the roles of Ca2+ were examined. The results indicate that Ca2+/CaM plays important roles in regulating IR-induced cell cycle arrest, possibly through mediating chromatin structure. The dataset presented here demonstrates that sensitive profiling of the dynamics of functional cellular protein-protein interactions can successfully lead to the dissection of important metabolic or signaling pathways.