Characterization of a new apoptosis-inducing protein from the cabbage butterfly

菜粉蝶中一种新的细胞凋亡诱导蛋白的表征

• 批准号: 13660344
• 负责人: WATANABE Masahiko
• 金额: $ 2.24万
• 依托单位: National Cancer Center
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13660344/
• 关键词: cabbage butterfly; pierisin; ADP-rivosylation; DNA adduct; bacterial toxin; ADP-リボシル化

Pierisin-1 is a 98-kDa cytotoxic protein found in the cabbage butterfly and shows sequence similarity with ADP-ribosylating toxins such as cholera toxin. To identify a substrate protein for ADP-ribosyltransferase activity of pierisin-1, a mixture of[adenylate-^<32>P]-NAD, HeLa cell extract and pierisin-1 was incubated and analyzed by SDS/PAGE and autoradiography. The labeled material was protease resistant but DNase sensitive. This result suggested that DNA might be the substrate for ADP-ribosylation by pierisin. When a variety of oligonucleotides having different sequences were used for acceptors from the efficiency strongly correlated with the proportion of guanine base in each oligonucleotide. For structural determination of the reaction product by pierisin-1, 2'-deoxyguanosine was used as an acceptor molecule. The UV spectrum and ESI-MS suggested the presence of ADP-ribosylated dG. Several NMR analyses revealed the structure as α and β forms of N^2-(ADP-ribos-1-yl)-2'-deoxyguanosine. Finally, we independently synthesized chemically and confirmed the structural identity with the enzyme reaction product. When high-molecular-weight dsDNA was used as acceptor molecule, the same position of guanine base was ADP-ribosylated, as confirmed by structural analyses including ^1H-MNR. The ^<32>P-postlaveling method for detection of ADP-ribosylated dG indicated that ADP-ribosylation of N-2 of guanine base also occurred in HeLa cells treated with pierisin-1. From the above results, it is concluded that pierisin-1 transfers an ADP-ribosyl moiety from NAD to N-2 of guanine base of cellular DNA, and this causes apoptosis induction of the cells.

Pierisin-1 是一种在菜粉蝶中发现的 98 kDa 细胞毒性蛋白，显示出与 ADP-核糖基化毒素（例如霍乱毒素）相似的序列。培养^ 32 P]-NAD、HeLa细胞提取物和pierisin-1，并通过SDS/PAGE和放射自显影进行分析。该结果表明，当使用具有不同序列的多种寡核苷酸作为受体时，DNA可能是与每种寡核苷酸中鸟嘌呤碱基比例相关的ADP-核糖基化的强烈底物。为了通过 Pierisin-1 确定反应产物的结构，使用 2'-脱氧鸟苷作为受体分子，UV 光谱和 ESI-MS 表明存在。 ADP-核糖基化 dG。几次 NMR 分析揭示了 N^2-(ADP-ribos-1-yl)-2'-deoxyguanosine 的 α 和 β 形式的结构。最后，我们独立进行了化学合成并证实了该酶的结构一致性。当使用高分子量双链DNA作为受体分子时，鸟嘌呤碱基的相同位置被ADP核糖基化，这通过结构分析证实。 ^ 1 H-MNR。用于检测ADP-核糖基化dG的^ 32 P-postlaveling方法表明，用Pierisin-1处理的HeLa细胞中也发生了鸟嘌呤碱基的ADP-核糖基化。结论是，pierisin-1将ADP-核糖基部分从NAD转移到细胞DNA鸟嘌呤碱基的N-2，这导致细胞凋亡诱导细胞。