前蛋白转化酶枯草溶菌素9参与Caspase-3活化的生物信息学分析

Bioinformatics Analysis of Proprotein Convertase Cubtilisin/Kexin 9 Involved in Caspase-3 Activation

目的研究前蛋白转化酶枯草溶菌素9促凋亡的作用是否与活化凋亡通路Caspase-3相关。方法用80mg/L氧化型低密度脂蛋白孵育人脐静脉内皮细胞24h,同时设空白对照组、转染试剂组、阴性对照组和80nmol/L前蛋白转化酶枯草溶菌素9siRNA+80mg/L氧化型低密度脂蛋白组,流式细胞术测定细胞凋亡率,免疫印迹法和酶联免疫吸附法分别检测Caspase-3的表达及活性。然后通过生物信息学方法对前蛋白转化酶枯草溶菌素9与Caspase-8和Caspase-9进行比较,找出它们之间的序列相似程度,以及是否存在共同的功能结构域。结果前蛋白转化酶枯草溶菌素9siRNA能抑制氧化型低密度脂蛋白诱导的Caspase-3蛋白表达和活性增加。生物信息学分析表明,前蛋白转化酶枯草溶菌素9、Caspase-8、Caspase-9三者的序列同源性高达22.14%,三者在C端的二级结构十分接近,特别是前蛋白转化酶枯草溶菌素9和Caspase-9的螺旋、片层、转角、无规则卷曲含量十分接近,三级结构分析发现前蛋白转化酶枯草溶菌素9和Caspase-9的外观轮廓相近,且在腹部中央都有一个裂口,显示前蛋白转化酶枯草溶菌素9和Caspase-9可能具有相同的酶切活性。结论前蛋白转化酶枯草溶菌素9具有与Caspase-9相似的功能结构域,可能具有激活Caspase-3的功能。

Aim To determine whether the proapoptotic effect of proprotein-convertase cubtilisin/kexin 9（PCSK9）is related to its effect on Caspase-3 activation by bioinformatics analysis.Methods HUVEC-12 were incubated with 80 mg/L oxidized low density lipoprotein（ox-LDL）for 24 h,and groups of blank control,transfection reagent control,negative control,and 80 nmol/L PCSK9 siRNA＋80 mg/L ox-LDL were established.Apoptosis rate was measured by flow cytometry.Western blotting and ELISA were used to measure Caspase-3 protein expression and activity,respectively.Bioinformatics methods were used to compare PCSK9,Caspase-8 and Caspase-9,in order to find the similarity of sequence and whether a common conservative motif existed.Results An inhibitory effect of PCSK9 siRNA on both expression and activity of Caspase-3 was observed.The sequence similarity of PCSK9,Caspase-8 and Caspase-9 was 22.14%,there was a similar second structure in C-terminal of the three proteins,the content of helix,strand,turn and coil was nearly the same in PCSK9 and Caspase-9,the profile of PCSK9 and Caspase-9 was similar,and there was common cleft in their abdomen,this cleft may be related to their similar enzyme activity.Conclusion There are similar motif in PCSK9 and Caspase-9.The proapoptotic capacity of PCSK9 is related to its effect on Caspase-3 activation.