Abstract：
Iron homeostasis is vital for the host\'s defense against pathogenic invasion and the ferritinophagy is a crucial mechanism in maintaining intracellular iron homeostasis by facilitating the degradation and recycling of stored iron. The nuclear receptor coactivator 4 (NCOA4) serves as a ferritinophagy receptor, facilitating the binding and delivery of ferritin to the autophagosome and lysosome. However, NCOA4 of the sea cucumber Apostichopus japonicus (AjNCOA4) has not been reported until now. In this study, we identified and characterized AjNCOA4 in A. japonicus. This gene encodes a polypeptide containing 597 amino acids with an open reading frame of 1794 bp. The inferred amino acid sequence of AjNCOA4 comprises an ARA70 domain. Furthermore, a multiple sequence alignment demonstrated varying degrees of sequence homology between AjNCOA4 from A. japonicus and other NCOA4 orthologs. The phylogenetic tree of NCOA4 correlates with the established timeline of metazoan evolution. Expression analysis revealed that AjNCOA4 is expressed in all tested tissues, including the body wall, muscle, intestine, respiratory tree, and coelomocytes. Following challenge with Vibrio splendidus, the coelomocytes exhibited a significant increase in AjNCOA4 mRNA levels, peaking at 24 h. We successfully obtained recombinant AjNCOA4 protein through prokaryotic expression and prepared a specific polyclonal antibody. Immunofluorescence and co-immunoprecipitation experiments demonstrated an interaction between AjNCOA4 and AjFerritin in coelomocytes. RNA interference-mediated knockdown of AjNCOA4 expression resulted in elevated iron ion levels in coelomocytes. Bacterial stimulation enhanced ferritinophagy in coelomocytes, while knockdown of AjNCOA4 reduced the occurrence of ferritinophagy. These findings suggest that AjNCOA4 modulates ferritinophagy induced by V. splendidus in coelomocytes of A. japonicus.
摘要：
铁稳态对于宿主防御病原体入侵至关重要，铁吞噬是通过促进储存铁的降解和再循环来维持细胞内铁稳态的重要机制。核受体共激活因子4（NCOA4）作为铁细胞吞噬受体，促进铁蛋白与自噬体和溶酶体的结合和递送。然而,海参刺参（AjNCOA4）的NCOA4至今尚未见报道。在这项研究中,我们鉴定并表征了刺槐中的AjNCOA4。该基因编码包含597个氨基酸的多肽，开放阅读框为1794bp。推断的AjNCOA4的氨基酸序列包含ARA70结构域。此外，多重序列比对显示了来自A.japonicus的AjNCOA4和其他NCOA4直向同源物之间不同程度的序列同源性。NCOA4的系统发育树与已建立的后生动物进化时间表相关。表达分析显示，AjNCOA4在所有受试组织中均有表达，包括身体的墙壁,肌肉，肠,呼吸树,和腔体细胞。在挑战脾弧菌之后，腔体细胞的AjNCOA4mRNA水平显着增加，在24h达到峰值。我们通过原核表达成功获得了重组AjNCOA4蛋白，并制备了特异性多克隆抗体。免疫荧光和免疫共沉淀实验表明，在腔体细胞中，AjNCOA4和AjFerritin之间存在相互作用。RNA干扰介导的AjNCOA4表达敲低导致腔体细胞中铁离子水平升高。细菌刺激增强了腔体细胞中的铁蛋白自噬，而敲除AjNCOA4减少了铁细胞吞噬的发生。这些发现表明，AjNCOA4调节日本血吸虫的腔体细胞中脾弧菌诱导的铁蛋白吞噬。
