Yang Zhouning, Zhou Ke, Liu Hao, Wu Andong, Mei Long, Liu Qingzhen
State Key Laboratory of Virology and Modern Virology Research Center, College of Life Sciences, Wuhan University, Wuhan, People's Republic of China.
PLoS One. 2016 Mar 15;11(3):e0151016. doi: 10.1371/journal.pone.0151016. eCollection 2016.
Sf9, a cell line derived from Spodoptera frugiperda, is an ideal model organism for studying insect apoptosis. The first notable study that attempted to identify the apoptotic pathway in Sf9 was performed in 1997 and included the discovery of Sf-caspase-1, an effector caspase of Sf9. However, it was not until 2013 that the first initiator caspase in Sf9, SfDronc, was discovered, and the apoptotic pathway in Sf9 became clearer. In this study, we report another caspase of Sf9, SfDredd. SfDredd is highly similar to insect initiator caspase Dredd homologs. Experimentally, recombinant SfDredd underwent autocleavage and exhibited different efficiencies in cleavage of synthetic caspase substrates. This was attributed to its caspase activity for the predicted active site mutation blocked the above autocleavage and synthetic caspase substrates cleavage activity. SfDredd was capable of not only cleaving Sf-caspase-1 in vitro but also cleaving Sf-caspase-1 and inducing apoptosis when it was co-expressed with Sf-caspase-1 in Sf9 cells. The protein level of SfDredd was increased when Sf9 cells were treated by Actinomycin D, whereas silencing of SfDredd reduced apoptosis and Sf-caspase-1 cleavage induced by Actinomycin D treatment. These results clearly indicate that SfDredd functioned as an apoptotic initiator caspase. Apoptosis induced in Sf9 cells by overexpression of SfDredd alone was not as obvious as that induced by SfDronc alone, and the cleavage sites of Sf-caspase-1 for SfDredd and SfDronc are different. In addition, despite sharing a sequence homology with initiator caspases and possessing weak activity on initiator caspase substrates, SfDredd showed strong activity on effector caspase substrates, making it the only insect caspase reported so far functioning similar to human caspase-2 in this aspect. We believe that the discovery of SfDredd, and its different properties from SfDronc, will improve the understanding of apoptosis pathway in Sf9 cells.
Sf9是一种源自草地贪夜蛾的细胞系，是研究昆虫凋亡的理想模式生物。1997年进行了首次旨在鉴定Sf9细胞凋亡途径的重要研究，其中发现了Sf - caspase - 1，它是Sf9的效应半胱天冬酶。然而，直到2013年才发现Sf9中的首个起始半胱天冬酶SfDronc，Sf9中的凋亡途径才变得更加清晰。在本研究中，我们报告了Sf9的另一种半胱天冬酶SfDredd。SfDredd与昆虫起始半胱天冬酶Dredd同源物高度相似。实验表明，重组SfDredd会进行自我切割，并且在切割合成半胱天冬酶底物时表现出不同的效率。这归因于其半胱天冬酶活性，因为预测的活性位点突变会阻断上述自我切割和合成半胱天冬酶底物的切割活性。SfDredd不仅能够在体外切割Sf - caspase - 1，而且当它与Sf - caspase - 1在Sf9细胞中共表达时，还能切割Sf - caspase - 1并诱导凋亡。当用放线菌素D处理Sf9细胞时，SfDredd的蛋白水平会升高，而沉默SfDredd会减少放线菌素D处理诱导的凋亡和Sf - caspase - 1的切割。这些结果清楚地表明SfDredd起到了凋亡起始半胱天冬酶的作用。单独过表达SfDredd在Sf9细胞中诱导的凋亡不如单独过表达SfDronc诱导的明显，并且SfDredd和SfDronc对Sf - caspase - 1的切割位点不同。此外，尽管SfDredd与起始半胱天冬酶具有序列同源性，并且对起始半胱天冬酶底物具有较弱的活性，但它对效应半胱天冬酶底物表现出很强的活性，这使其成为迄今为止报道的在这方面功能类似于人类半胱天冬酶 - 2的唯一昆虫半胱天冬酶。我们相信，SfDredd的发现及其与SfDronc不同的特性，将增进对Sf9细胞凋亡途径的理解。
