Hamajima Rina, Iwamoto Asako, Tomizaki Moe, Suganuma Ikue, Kitaguchi Koji, Kobayashi Michihiro, Yamada Hayato, Ikeda Motoko
Laboratory of Sericulture and Entomoresources, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan.
Laboratory of Sericulture and Entomoresources, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan.
Insect Biochem Mol Biol. 2016 Dec;79:97-107. doi: 10.1016/j.ibmb.2016.10.012. Epub 2016 Oct 29.
Recent advances in genome-wide surveys have revealed a number of lepidopteran insect homologs of mammalian and Drosophila genes that are responsible for apoptosis regulation. However, the underlying molecular mechanisms for apoptosis regulation in lepidopteran insect cells remain poorly understood. In the present study, we demonstrated that the transfection of Bombyx mori BM-N cells with dsRNA against the B. mori cellular iap1 gene (cbm-iap1) induces severe apoptosis that is accompanied by an increase of caspase-3-like protease activity. In these apoptotic cells, the cleaved form of the endogenous initiator caspase Dronc (Bm-Dronc) was detected, indicating that cBm-IAP1 protein depletion by RNAi silencing resulted in the activation of Bm-Dronc. In transient expression assays in BM-N cells, cBm-IAP1 suppressed the apoptosis triggered by Bm-Dronc overexpression and depressed the elevation of caspase-3-like protease activity, but also increased the cleaved form of Bm-Dronc protein. cBm-IAP1 also suppressed the caspase-3-like protease activity stimulated by Bm-caspase-1 overexpression. Co-immunoprecipitation experiments demonstrated that cBm-IAP1 strongly interacts with Bm-Dronc, but only has weak affinity for Bm-caspase-1. Transient expression analyses showed that truncated cBm-IAP1 proteins defective in the BIR1, BIR2 or RING domain were unable to suppress Bm-Dronc-induced apoptosis. In addition, BM-N cells expressing truncated cBm-IAP1 proteins underwent apoptosis, suggesting that intact cBm-IAP1, which has anti-apoptotic activity, was replaced or displaced by the overexpressed truncated cBm-IAP1 proteins, which are incapable of interfering with the apoptotic caspase cascade. Taken together, the present results demonstrate that cBm-IAP1 is a vital negative regulator of apoptosis in BM-N cells and functions by preventing the activation and/or activity of Bm-Dronc and Bm-caspase-1.
全基因组调查的最新进展揭示了许多鳞翅目昆虫中与哺乳动物和果蝇基因同源的、负责细胞凋亡调控的基因。然而，鳞翅目昆虫细胞中细胞凋亡调控的潜在分子机制仍知之甚少。在本研究中，我们证明用针对家蚕细胞凋亡抑制蛋白1基因（cbm-iap1）的双链RNA转染家蚕BM-N细胞会诱导严重的细胞凋亡，并伴随着类半胱天冬酶-3蛋白酶活性的增加。在这些凋亡细胞中，检测到内源性起始半胱天冬酶Dronc（Bm-Dronc）的裂解形式，这表明RNAi沉默导致的cBm-IAP1蛋白缺失会导致Bm-Dronc的激活。在BM-N细胞的瞬时表达试验中，cBm-IAP1抑制了Bm-Dronc过表达引发的细胞凋亡，并降低了类半胱天冬酶-3蛋白酶活性的升高，但也增加了Bm-Dronc蛋白的裂解形式。cBm-IAP1还抑制了Bm-半胱天冬酶-1过表达刺激的类半胱天冬酶-3蛋白酶活性。免疫共沉淀实验表明，cBm-IAP1与Bm-Dronc强烈相互作用，但对Bm-半胱天冬酶-1只有弱亲和力。瞬时表达分析表明，在杆状病毒IAP重复序列1（BIR1）、杆状病毒IAP重复序列2（BIR2）或指环结构域（RING）有缺陷的截短cBm-IAP1蛋白无法抑制Bm-Dronc诱导的细胞凋亡。此外，表达截短cBm-IAP1蛋白的BM-N细胞发生了凋亡，这表明具有抗凋亡活性的完整cBm-IAP1被过表达的、无法干扰凋亡半胱天冬酶级联反应的截短cBm-IAP1蛋白所取代或替代。综上所述，目前的结果表明，cBm-IAP1是BM-N细胞中细胞凋亡的重要负调节因子，其功能是通过阻止Bm-Dronc和Bm-半胱天冬酶-1的激活和/或活性来实现的。
