Lipid is one of the important components of living organisms. The precise regulation and homeostasis maintenance of lipid metabolism are essential to human health. The ubiquitination pathway regulates lipid metabolism by degrading lipid-related proteins. Ppa encodes an F-box protein, which is a member of the SCF ubiquitination complex. Previous studies reported that Ppa regulated the body segmentation and the correct localization of centromere histones, while its function in lipid metabolism has not been reported. In this study, Drosophila melanogaster was used to explore the function of Ppa in lipid storage. The subcellular localization of PPA was detected by fusion with green fluorescent protein. The deletion mutant of Ppa was constructed via CRISPR/Cas9 technology. The morphological changes of lipid droplets in deletion mutants and Ppa overexpression flies were analyzed by BODIPY 493/503 or Nile red staining. Further, Ppa was overexpressed in the deletion mutant to verify its function. The results showed that PPA-GFP fusion protein were localized in the nuclei of salivary gland and fat body. Compared with the control flies, the lipid droplets in Ppa deletion mutants became smaller, and overexpression of Ppa exhibited larger lipid droplets. Overexpression of Ppa in the deletion mutant could restore the lipid droplets to normal state. In summary, this study demonstrated that Ppa could promote lipid storage in Drosophila.
脂质是构成生物体的重要成分之一,脂质代谢的精确调节和稳态维持对人类健康至关重要。泛素化途径通过降解脂质相关蛋白来调控脂质代谢。Ppa (partner of paired)编码一种F-box蛋白,后者是SCF (Skp1-Cullin1-F-box)泛素化复合体成员之一。已有的研究表明Ppa在调控果蝇体节的正常发育和着丝粒组蛋白的正确定位方面发挥重要的作用,但在脂肪代谢方面的功能研究却未见报道。本研究以黑腹果蝇(Drosophila melanogaster)作为研究对象,探究了Ppa在脂肪储存中的功能。通过与绿色荧光蛋白融合表达,检测PPA的亚细胞定位;应用CRISPR/Cas9技术,构建Ppa的缺失突变体;通过BODIPY 493/503或Nile red脂滴染色,对缺失突变体和超量表达Ppa果蝇的脂滴形态改变进行分析。随后,在缺失突变体中超量表达Ppa以验证其功能。结果表明,PPA-GFP融合蛋白定位于唾液腺和脂肪体的细胞核中。与对照组果蝇相比,Ppa缺失突变体表现为脂滴变小;超量表达Ppa显示出脂滴变大。在突变体中超量表达Ppa能够将脂滴回复至正常状态。综上所述,本研究揭示了Ppa在果蝇中具有促进脂肪储存的功能。.
Keywords: CRISPR/Cas9; Ppa; lipid droplets; lipid storage.