The modulation of Smac/DIABLO on mitochondrial apoptosis induced by LPS in Crassostrea gigas. Issue 84 (January 2019)

• Record Type: Journal Article
• Authors: Lv, Zhao; Song, Xiaorui; Xu, Jiachao; Jia, Zhihao; Yang, Bin; Jia, Yunke; Qiu, Limei; Wang, Lingling; Song, Linsheng
• Is Part Of: Fish & shellfish immunology. Issue 84(2019)
• Page Start: 587
• Page End: 598
• Publication Date: 2019-01
• Subjects: Mitochondrial pathway -- Smac/DIABLO -- LPS -- Primary cultured hemocytes -- C. gigas; Fishes -- Immunology -- Periodicals; Shellfish -- Immunology -- Periodicals; Poissons -- Immunologie -- Périodiques; Crustacés -- Immunologie -- Périodiques; 571.9617
• DOI: 10.1016/j.fsi.2018.10.035
• Languages: English
• ISSNs: 1050-4648

Abstract

Abstract: The mitochondrial pathway of apoptosis is well studied as the major mechanism of physiological cell death in vertebrates. In the present study, a second mitochondria-derived activator of caspases (Smac)/direct inhibitor of apoptosis-binding protein (IAP) with low pI protein (DIABLO) (designated as Cg Smac) was identified from oyster Crassostrea gigas . The open reading frame of Cg Smac was of 966 bp nucleotides encoding a predicted polypeptide of 321 amino acids with a conserved Smac/DIABLO domain containing a potential IAP-binding motif of VMPV. Cg Smac proteins were distributed in hemocytes and co-localized with mitochondria. Western blotting analysis revealed that Cg Smac proteins mainly existed in the dimer form in hemocytes, and the monomeric precursors and mature monomers were also detected. After lipopolysaccharide (LPS) stimulation, the mRNA expression of Cg Smac in hemocytes was significantly up-regulated and peaked at 6 h (12.26-fold, p < 0.05), and the protein level of its dimers was significantly up-regulated at 6 h, 12 h, 24 h, and 48 h, while that of Cg Smac monomers was up-regulated at 6 h, 12 h and down-regulated at 24 h, 48 h. The decrease of mitochondrial membrane potential indicated that the occurrence of early stage of apoptosis in primary cultured hemocytes was induced by LPS, and RNA interference (RNAi) of Cg Smac could not rescue this decrease. The caspase-3 activity in primary cultured hemocytes was significantly suppressed after RNAi of Cg Smac. Correspondingly, the total apoptotic rate of primary cultured hemocytes was also significantly suppressed in ds Cg Smac + LPS group (31.57%) compared to dsEGFP + LPS group (40.27%, p < 0.05), which in turn demonstrated the conserved pro-apoptotic function of Cg Smac. Furthermore, the early apoptotic rate (10.4% vs. 8.5%, p < 0.05) was significantly higher in dsCgSmac + LPS group than that of dsEGFP + LPS group, while the necrosis (7.7% vs. 10.0%, p < 0.05) and late apoptotic rates (13.4% vs. 21.9%, p < 0.05) were lower in ds Cg Smac + LPS group than those of dsEGFP + LPS group. Collectively, Cg Smac could activate mitochondrial apoptosis pathway by promoting caspase-3 activity in oyster hemocytes against exogenous LPS invasion. These results provided new insights on oyster apoptosis and the immune defense mechanisms in invertebrates. Highlights: A new pro-apoptotic molecule Cg Smac was identified in Crassostrea gigas. Cg Smac contained a potential IAP-binding motif of VMPV. Cg Smac proteins mainly existed in the dimer form to exert biological function. Cg Smac promoted mitochondrial apoptosis by elevating intracellular caspase-3 activity.