Parasporin-1, a novel cytotoxic protein from Bacillus thuringiensis, induces Ca2+ influx and a sustained elevation of the cytoplasmic Ca2+ concentration in toxin-sensitive cells
Parasporin-1, a novel cytotoxic protein from Bacillus thuringiensis, induces Ca2+ influx and a sustained elevation of the cytoplasmic Ca2+ concentration in toxin-sensitive cells
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DOI：
10.1074/jbc.m611382200
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发表时间：
2007-03-09
影响因子：
4.8
通讯作者：
Mizuki, Eiich
中科院分区：
文献类型：
--
作者：
Katayama, Hideki;Kusaka, Yoshitomo;Mizuki, Eiich
副孢子蛋白-1（Parasporin-1）是苏云金芽孢杆菌（Bacillus thuringiensis）的一种新型非杀虫包涵体蛋白，对特定的哺乳动物细胞具有细胞毒性。在这项研究中，我们研究了parasporin-1对toxinsensitive细胞系的影响，以阐明parasporin-1的细胞毒机制。Parasporin-1不是膜孔形成毒素，如通过测量乳酸脱氢酶释放、碘化丙啶渗透和Parasporin-1处理的细胞中的膜电位所证明的。Parasporin-1降低了对Parasporin-1敏感的HeLa细胞的蛋白质和DNA合成水平。在用这种毒素处理的细胞中观察到的最早变化是细胞内游离Ca 2+浓度的快速升高;在副孢蛋白-1处理后1-3分钟观察到细胞内Ca 2+水平的增加。使用四种不同的细胞系，我们发现细胞对parasporin-1的敏感性程度与细胞内Ca 2+浓度增加的大小呈正相关。毒素诱导的细胞内Ca ~（2+）浓度升高在低Ca ~（2+）缓冲液中明显降低，而在无Ca ~（2+）缓冲液中未观察到。相应地，在低Ca 2+缓冲液中副孢蛋白-1的细胞毒性降低，并通过向细胞外培养基中加入Ca 2+而恢复。抑制三聚体G蛋白信号传导的苏拉明抑制了Ca 21内流和parasporin-1的细胞毒性。在parasporin-1处理的HeLa细胞中，观察到caspase-3前体和poly（ADPribose）聚合酶的降解。此外，合成半胱天冬酶抑制剂阻断parasporin-1的细胞毒活性。这些结果表明，parasporin-1激活这些细胞中的细胞凋亡信号作为Ca 2+水平增加的结果，并且Ca 2+内流是parasporin-1毒性的基础途径中的第一步。
Parasporin-1 is a novel non-insecticidal inclusion protein from Bacillus thuringiensis that is cytotoxic to specific mammalian cells. In this study, we investigated the effects of parasporin-1 on toxinsensitive cell lines to elucidate the cytotoxic mechanism of parasporin-1. Parasporin-1 is not a membrane pore-forming toxin as evidenced by measurements of lactate dehydrogenase release, propidium iodide penetration, and membrane potential in parasporin-l-treated cells. Parasporin-1 decreased the level of cellular protein and DNA synthesis in parasporin-l-sensitive HeLa cells. The earliest change observed in cells treated with this toxin was a rapid elevation of the intracellular free-Ca2+ concentration; increases in the intracellular Ca2+ levels were observed 1-3 min following parasporin-1 treatment. Using four different cell lines, we found that the degree of cellular sensitivity to parasporin-1 was positively correlated with the size of the increase in the intracellular Ca2+ concentration. The toxin-induced elevation of the intracellular Ca2+ concentration was markedly decreased in low-Ca2+ buffer and was not observed in Ca2+-free buffer. Accordingly, the cytotoxicity of parasporin-1 decreased in the low-Ca2+ buffer and was restored by the addition of Ca2+ to the extracellular medium. Suramin, which inhibits trimeric G-protein signaling, suppressed both the Ca 21 influx and the cytotoxicity of parasporin-1. In parasporin-1-treated HeLa cells, degradation of pro-caspase-3 and poly(ADPribose) polymerase was observed. Furthermore, synthetic caspase inhibitors blocked the cytotoxic activity of parasporin-1. These results indicate that parasporin-1 activates apoptotic signaling in these cells as a result of the increased Ca2+ level and that the Ca2+ influx is the first step in the pathway that underlies parasporin-1 toxicity.