School of Agriculture, Ludong University, Yantai, 264025, China.
School of Agriculture, Ludong University, Yantai, 264025, China.
Int J Biol Macromol. 2024 Aug;275(Pt 1):133645. doi: 10.1016/j.ijbiomac.2024.133645. Epub 2024 Jul 3.
Fas-associated protein with death domain (FADD) was initially identified as a crucial adaptor protein in the apoptotic pathway mediated by death receptor (DR). Subsequently, many studies have confirmed that FADD plays a vital role in innate immunity and inflammatory responses in animals. However, the function of this pleiotropic molecule in mollusk species has not been well explored. In this study, we successfully verified the gene sequence of FADD in the Zhikong scallop (Chlamys farreri) and designated it as CfFADD. The CfFADD protein contains a conserved death effector and death domains. Phylogenetic analysis showed that CfFADD is a novel addition to the molluscan FADD family with a close evolutionary relationship with molluscan FADD subfamily proteins. CfFADD mRNA expression in various scallop tissues was significantly induced by challenge with pathogen-associated molecular patterns (lipopolysaccharide, peptidoglycan, and poly(I:C)), suggesting its role in innate immunity in scallops. Co-immunoprecipitation showed that CfFADD interacted with the scallop DR (tumor necrosis factor receptor) and a signaling molecule involved in the Toll-like receptor pathway (interleukin-1 receptor-associated kinase), confirming that CfFADD may be involved in DR-mediated apoptosis and innate immune signaling pathways. Further studies showed that CfFADD interacted with CfCaspase-8 and activated caspase-3. HEK293T cells exhibited distinct apoptotic features after transfection with a CfFADD-expression plasmid, suggesting a functional DR-FADD-caspase apoptotic pathway in scallops. Overexpression of CfFADD led to a significant dose-dependent activation of interferon β and nuclear factor-κB reporter genes, demonstrating the key role of CfFADD in innate immunity. In summary, our research has confirmed the critical roles of CfFADD in innate immunity and apoptosis and provides valuable information for developing comparative immunology theories.
Fas 相关死亡结构域蛋白（FADD）最初被鉴定为死亡受体（DR）介导的凋亡途径中的关键衔接蛋白。随后，许多研究证实 FADD 在动物的固有免疫和炎症反应中发挥着重要作用。然而，这种多功能分子在软体动物物种中的功能尚未得到充分探索。在这项研究中，我们成功地验证了栉孔扇贝（Chlamys farreri）中 FADD 的基因序列，并将其命名为 CfFADD。CfFADD 蛋白包含保守的死亡效应结构域和死亡结构域。系统进化分析表明，CfFADD 是软体动物 FADD 家族的一个新成员，与软体动物 FADD 亚家族蛋白具有密切的进化关系。CfFADD 在栉孔扇贝各种组织中的 mRNA 表达在受到病原体相关分子模式（脂多糖、肽聚糖和聚 I:C）的刺激后显著诱导，表明其在扇贝固有免疫中的作用。共免疫沉淀表明 CfFADD 与扇贝 DR（肿瘤坏死因子受体）和参与 Toll 样受体途径的信号分子（白细胞介素 1 受体相关激酶）相互作用，证实 CfFADD 可能参与 DR 介导的凋亡和固有免疫信号通路。进一步的研究表明，CfFADD 与 CfCaspase-8 相互作用并激活 caspase-3。转染 CfFADD 表达质粒的 HEK293T 细胞表现出明显的凋亡特征，表明扇贝中存在功能性 DR-FADD-caspase 凋亡途径。CfFADD 的过表达导致干扰素 β 和核因子-κB 报告基因的显著剂量依赖性激活，证明 CfFADD 在固有免疫中的关键作用。总之，我们的研究证实了 CfFADD 在固有免疫和凋亡中的关键作用，并为比较免疫学理论的发展提供了有价值的信息。
