Leu Jiann-Horng, Kuo Yu-Chen, Kou Guang-Hsiung, Lo Chu-Fang
Institute of Zoology, National Taiwan University, Taipei 10617, Taiwan, ROC.
Dev Comp Immunol. 2008;32(2):121-33. doi: 10.1016/j.dci.2007.05.005. Epub 2007 Jun 14.
The inhibitor of apoptosis proteins (IAPs) play important roles in both apoptosis and innate immunity. Here, we report the first cloning and characterization of a novel IAP family member, PmIAP, from Penaeus monodon. The full-length PmIAP cDNA is 4769bp, with an ORF encoding a protein of 698 amino acids. The PmIAP protein contains three BIR domains and a C-terminal RING domain, and its mRNA was expressed in all analyzed tissues. In insect cells, PmIAP, together with Spodoptera frugiperda IAP, AcMNPV P35, and WSSV449 (or ORF390, an anti-apoptosis protein encoded by white spot syndrome virus), could all block the apoptosis induced by Drosophila Reaper protein (Rpr), whereas only P35 and WSSV449 could block the apoptosis induced by actinomycin D. Co-immunoprecipitation showed that PmIAP physically interacted with Rpr, and in an immunofluorescent analysis the two proteins produced co-localized punctate signals in the cytoplasm. Deletion analysis revealed that both the BIR2 and BIR3 domains of PmIAP could independently bind to and inhibit Rpr, whereas the BIR1 domain could not. These results strongly suggest that PmIAP blocks Rpr's pro-apoptotic activity through mechanisms that are evolutionarily conserved across crustaceans, insects, and mammals.
凋亡抑制蛋白（IAPs）在细胞凋亡和固有免疫中均发挥着重要作用。在此，我们报道了首个从斑节对虾中克隆并鉴定的新型IAP家族成员PmIAP。PmIAP cDNA全长4769bp，其开放阅读框编码一个含698个氨基酸的蛋白质。PmIAP蛋白包含三个杆状病毒IAP重复序列（BIR）结构域和一个C端的类泛素连接酶（RING）结构域，其mRNA在所有分析的组织中均有表达。在昆虫细胞中，PmIAP与草地贪夜蛾IAP、苜蓿银纹夜蛾核型多角体病毒P35以及白斑综合征病毒449（或ORF390，由白斑综合征病毒编码的一种抗凋亡蛋白）均可阻断果蝇促凋亡蛋白（Rpr）诱导的细胞凋亡，而只有P35和WSSV449可阻断放线菌素D诱导的细胞凋亡。免疫共沉淀显示PmIAP与Rpr存在物理相互作用，免疫荧光分析表明这两种蛋白在细胞质中产生共定位的点状信号。缺失分析表明，PmIAP的BIR2和BIR3结构域均可独立结合并抑制Rpr，而BIR1结构域则不能。这些结果有力地表明，PmIAP通过在甲壳类动物、昆虫和哺乳动物中进化保守的机制来阻断Rpr的促凋亡活性。
