Post-diapause synthesis of ArHsp40-2, a type 2 J-domain protein from Artemia franciscana, is developmentally regulated and induced by stress

Post-diapause cysts of Artemia franciscana undergo a well-defined developmental process whereby internal differentiation leads to rupture of the cyst shell, release of membrane-enclosed nauplii and hatching to yield swimming larvae. The post-diapause development of A. franciscana has been examined at biochemical and molecular levels, yet little is known about molecular chaperone function during this process. In addressing this we recently described ArHsp40, a type 1 J-domain protein in post-diapause A. franciscana cysts and larvae. The current report describes ArHsp40-2, a second J-domain protein from A. franciscana. ArHsp40-2 is a type 2 J-domain protein, lacking a zinc binding domain but containing other domains characteristic of these proteins. Notably, ArHsp40-2 possesses a double barrel β-domain structure in its substrate binding region, as does ArHsp40. qPCR revealed a relatively low amount of ArHsp40-2 mRNA in 0 h cysts which increased significantly until the E1 stage, most likely as a result of enhanced transcription, after which it declined. An antibody specific to ArHsp40-2 was produced and used to show that like its mRNA, ArHsp40-2 accumulated until the E1 stage and then decreased to amounts lower than those in 0 h cysts. The synthesis of ArHsp40-2 was induced by heat shock indicating that ArHsp40-2 is involved in stress resistance in cysts and nauplii. Accumulation in cysts during early post-diapause development followed by its sharp decline suggests a role in protein disaggregation/refolding, a function of Hsp40s from other organisms, where ArHsp40-2 assists in the rescue of proteins sequestered during diapause by p26, an abundant small heat shock protein (sHsp) in A. franciscana cysts.弗兰西斯卡卤虫（Artemia franciscana）的滞育后囊胞会经历一套特征明确的发育进程：内部分化会触发囊胞外壳破裂，释放出被膜包裹的无节幼体，最终孵化得到可游动的幼虫。目前学界已从生化与分子层面解析了弗兰西斯卡卤虫的滞育后发育过程，但对该进程中分子伴侣的功能却知之甚少。为探究这一科学问题，我们团队近期报道了ArHsp40——一种存在于滞育后弗兰西斯卡卤虫囊胞与幼体中的1型J结构域蛋白（J-domain protein）。本研究报道了第二种来自弗兰西斯卡卤虫的J结构域蛋白ArHsp40-2。ArHsp40-2属于2型J结构域蛋白，不具备锌结合结构域，但包含这类蛋白特有的其他结构域。值得注意的是，与ArHsp40类似，ArHsp40-2的底物结合区域拥有双桶状β结构域结构。实时荧光定量PCR（quantitative real-time polymerase chain reaction, qPCR）结果显示，0小时囊胞中的ArHsp40-2 mRNA含量相对较低，随后其表达量显著上升直至E1阶段，这一变化极有可能由转录增强所介导；在此之后，其mRNA含量开始下降。我们制备了针对ArHsp40-2的特异性抗体，实验结果表明，与mRNA的表达模式类似，ArHsp40-2的蛋白积累量直至E1阶段达到峰值，随后逐渐下降至低于0小时囊胞中的水平。热激处理可诱导ArHsp40-2的合成，这提示ArHsp40-2参与了囊胞与无节幼体的应激抗性调控。ArHsp40-2在滞育后发育早期的囊胞中积累，随后迅速下降，这表明其可能参与蛋白质解聚/重折叠过程——这也是其他物种中热休克蛋白40（heat shock protein 40, Hsp40）的经典功能。在此过程中，ArHsp40-2可协助解救由p26所隔离的蛋白质；p26是弗兰西斯卡卤虫囊胞中一种含量丰富的小型热休克蛋白（small heat shock protein, sHsp）。