Dipartimento di Scienze Biomediche, UniVersità di Modena e Reggio Emilia, Via G Campi 287, I-41125 Modena, Italy, Italy.
Chem Res Toxicol. 2009 Dec;22(12):2009-16. doi: 10.1021/tx900297g.
We have analyzed the proteome of MCF-7 cells exposed to palytoxin (PlTX), to characterize protein components involved in the death response induced by the toxin. The protein profiles of cell lysates were obtained by two-dimensional (2D) electrophoresis, and we found that four components were increased by PlTX treatment. By tryptic digestion of protein spots in the gels and LC-ESI-MS/MS analysis of resulting peptides, those four components were found to include three isoforms of the heat shock protein (hsp) 27 differing with regard to their phosphrylation state, as well as DJ-1/PARK7. The effects exerted by PlTX on hsp 27 and DJ-1 proteins were further quantified by immunoblotting analyses of proteins separated by monodimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and using antibodies recognizing total hsp 27, the hsp 27 forms phosphorylated in Ser(82), and DJ-1 protein. Dose-response and time-course experiments yielded results that only partially confirmed those found by protein staining after 2D electrophoresis. These findings were further checked by immunoblotting of proteins after fractionation by 2D electrophoresis, and we found that only some forms of those comigrating in a single band upon monodimensional SDS-PAGE were actually increased in extracts from PlTX-treated cells. We obtained evidence that the three hsp 27 isoforms whose relative abundance was increased in MCF-7 cells exposed to PlTX comprised two proteins phosphorylated in Ser(82), whereas the third form most likely contains a phosphorylated amino acid residue other than Ser(82). Moreover, we could show that PlTX treatment determined the accumulation of an oxidized isoform of DJ-1 in MCF-7 cells. We conclude that the toxicity pathway of PlTX in MCF-7 cells involves post-translational modifications of hsp 27 and DJ-1 stress response proteins, comprising a shift in the equilibria among hsp 27 isoforms toward those phosphorylated in Ser(82), as well as the oxidation of DJ-1.
我们分析了 MCF-7 细胞暴露于海兔毒素（PlTX）后的蛋白质组，以鉴定与毒素诱导的死亡反应相关的蛋白质成分。通过二维（2D）电泳获得细胞裂解物的蛋白质图谱，我们发现 PlTX 处理后有四个成分增加。通过凝胶中蛋白质斑点的胰蛋白酶消化和衍生肽的 LC-ESI-MS/MS 分析，发现这四个成分包括热休克蛋白（hsp）27 的三种同工型，其磷酸化状态不同，以及 DJ-1/PARK7。通过单维十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分离的蛋白质的免疫印迹分析和识别总 hsp 27、Ser(82)磷酸化的 hsp 27 形式和 DJ-1 蛋白的抗体进一步定量 PlTX 对 hsp 27 和 DJ-1 蛋白的作用。剂量反应和时间过程实验的结果仅部分证实了 2D 电泳后蛋白质染色发现的结果。通过 2D 电泳分离蛋白质后的免疫印迹检查进一步检查了这些发现，我们发现仅在 PlTX 处理细胞的提取物中，一些在单维 SDS-PAGE 上共迁移的同种型实际上增加。我们有证据表明，在暴露于 PlTX 的 MCF-7 细胞中相对丰度增加的三种 hsp 27 同工型包括两个在 Ser(82)磷酸化的蛋白质，而第三种形式很可能含有 Ser(82)以外的磷酸化氨基酸残基。此外，我们可以证明 PlTX 处理导致 MCF-7 细胞中 DJ-1 氧化同工型的积累。我们的结论是，PlTX 在 MCF-7 细胞中的毒性途径涉及 hsp 27 和 DJ-1 应激反应蛋白的翻译后修饰，包括 hsp 27 同工型之间的平衡向 Ser(82)磷酸化的同工型转移，以及 DJ-1 的氧化。
