Cho W L, Raikhel A S
Department of Parasitology, National Yang-Ming University, 112, Taipei, Taiwan.
Insect Biochem Mol Biol. 2001 Mar 15;31(4-5):283-8. doi: 10.1016/s0965-1748(00)00175-2.
In the female fat body of the mosquito Aedes aegypti, lysosomes play important roles during the cessation of vitellogenesis by degrading the biosynthetic machinery and aiding the remodeling of the fat body cells. A detailed study of a mosquito lysosomal aspartic protease (AaLAP) has shown a unique expression pattern in the vitellogenic fat body: the level of AaLAP mRNA dramatically rises and peaks at 24 h post blood meal (PBM) correlating with the high titer of ecdysteroids; however, there is a 12 h lag before peak levels of AaLAP protein and its enzymatic activity has been observed. These observations suggest that the high titer of 20-hydroxyecdysone (20E) may hinder translation of the AaLAP mRNA. Here, we used an in vitro organ culture to study the effect of 20E on the protein synthesis of AaLAP in the fat body. The increase in the AaLAP protein level in the fat body, dissected at 24 h PBM and incubated for 6 or 12 h, was inhibited by the presence of 10(-5) M 20E in the medium. Incubation in the hormone-free medium did not effect accumulation of the AaLAP protein which proceeded at the levels comparable to the intact insect. Furthermore, the effect of 10(-5) M 20E on the AaLAP accumulation was reversible. These experiments support the hypothesis of the 20E-mediated repression of lysosomal protease mRNAs at the translational level in the regulation of vitellogenic and postvitellogenic events in the mosquito fat body. Analysis of the 5' and 3' -end untranslated regions (UTR) of AaLAP mRNA form secondary structures suggest that they may also contribute to mRNA stability and 20E-mediated translational inhibition.
在埃及伊蚊雌蚊的脂肪体中，溶酶体在卵黄生成停止过程中发挥重要作用，它通过降解生物合成机制并协助脂肪体细胞重塑来实现这一功能。对一种蚊子溶酶体天冬氨酸蛋白酶（AaLAP）的详细研究表明，其在卵黄生成的脂肪体中具有独特的表达模式：AaLAP mRNA水平在吸血后24小时（PBM）急剧上升并达到峰值，这与蜕皮甾类激素的高滴度相关；然而，在观察到AaLAP蛋白及其酶活性的峰值水平之前，存在12小时的延迟。这些观察结果表明，高滴度的20-羟基蜕皮酮（20E）可能会阻碍AaLAP mRNA的翻译。在此，我们使用体外器官培养来研究20E对脂肪体中AaLAP蛋白质合成的影响。在PBM 24小时时解剖并培养6或12小时的脂肪体中，AaLAP蛋白水平的增加受到培养基中10^(-5) M 20E的抑制。在无激素培养基中培养不会影响AaLAP蛋白的积累，其积累水平与完整昆虫相当。此外，10^(-5) M 20E对AaLAP积累的影响是可逆的。这些实验支持了20E在翻译水平上介导溶酶体蛋白酶mRNA抑制，从而调节蚊子脂肪体中卵黄生成和卵黄生成后事件的假说。对AaLAP mRNA的5'和3'端非翻译区（UTR）形成的二级结构分析表明，它们也可能有助于mRNA的稳定性和20E介导的翻译抑制。
