Characterization of zebrafish caspase-3 and induction of apoptosis through ceramide generation in fish fathead minnow tailbud cells and zebrafish embryo
Characterization of zebrafish caspase-3 and induction of apoptosis through ceramide generation in fish fathead minnow tailbud cells and zebrafish embryo
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DOI：
10.1042/0264-6021:3600039
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发表时间：
2001-11-15
影响因子：
4.1
通讯作者：
Yamashita, M
中科院分区：
文献类型：
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作者：
Yabu, T;Kishi, S;Yamashita, M
Caspase-3 是从斑马鱼胚胎中克隆出来的，对其特性进行了表征，以确定 Caspase 在斑马鱼胚胎发生和细胞凋亡中的生物学意义，斑马鱼是脊椎动物发育生物学和遗传学中的模型生物。预测的氨基酸序列共有282个氨基酸残基，由结构域前体和大小亚基组成。系统发育分析表明，克隆的斑马鱼 caspase 是 caspase-3 亚家族的一员，其长度约为 1000 bp。与非洲爪蟾、鸡和哺乳动物的 caspase-3 具有 60% 的同一性。此外，重组斑马鱼caspase可水解乙酰基-Asp-Glu-Val-Asp-4-甲基-香豆酰-7-ainide，并表现出与哺乳动物caspase-3亚家族相似的底物特异性。因此，该 caspase 被命名为斑马鱼 caspase-3。斑马鱼 caspase-3 的过度表达诱导鱼黑头鲦鱼尾芽细胞和斑马鱼胚胎细胞凋亡并增加神经酰胺水平。使用半胱天冬酶抑制剂（苄氧基羰基-天冬氨酸-谷氨酸-缬氨酸-天冬氨酸-氟甲基酮）处理可抑制神经酰胺生成和细胞凋亡诱导。此外，斑马鱼caspase-3 mRNA作为母体因子存在于早期胚胎直至1000细胞阶段，然后在原肠胚阶段后通过合子表达在全身表达。这些发现表明，分离的 caspase-3 在诱导神经酰胺生成以及鱼细胞和斑马鱼胚胎细胞凋亡中发挥着重要作用，并表明 caspase-3 在发育过程中充当促凋亡信号的调节剂。
Caspase-3 was cloned from zebrafish embryos and its properties were characterized to identify the biological implications of caspase in embryogenesis and apoptosis in zebrafish, which is a model organism in vertebrate developmental biology and genetics. The predicted amino acid sequence, totalling 282 amino acid residues, consisted of the prodomain and large and small subunits. Phylogenetic analysis showed that the cloned zebrafish caspase was a member of the caspase-3 subfamily with approx. 60% identity with caspase-3 from Xenopus, chicken and mammals. In addition, recombinant zebrafish caspase hydrolysed acetyl-Asp-Glu-Val-Asp-4-methyl-coumaryl-7-ainide, and exhibited similar substrate specificity to the mammalian caspase-3 subfamily. Therefore this caspase was designated zebrafish caspase-3. Overexpression of zebrafish caspase-3 induced apoptosis and increased ceramide levels in fish fathead minnow tailbud cells and zebrafish embryos. Both ceramide generation and apoptosis induction were inhibited by treatment with a caspase inhibitor, benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone. Moreover, zebrafish caspase-3 mRNA was present in early embryos up to the 1000-cell stage as a maternal factor, and was then expressed throughout the body after the gastrula stage by zygotic expression. These findings indicate that the isolated caspase-3 plays an important role in the induction of ceramide generation as well as apoptosis in fish cells and the zebrafish embryo, and suggest that caspase-3 functions as a modulator of the pro-apoptotic signal in development.