利用功能化基因组学去理解GPI锚定蛋白的生物合成

Glycosylphosphatidylinositol (GPI) anchoring of proteins is a conserved post-translational modification in eukaryotes. In mammalian cells, 100 or more proteins are modified by GPI. It is synthesized and transferred to proteins in the endoplasmic reticulum (ER). Phosphatidylinositol is sequentially modified by sugars, a fatty acid and phospho-ethanolamines to generate the complete GPI precursor that is transferred to proteins. GPI-anchored proteins are then transported from the ER to the plasma membrane through the Golgi apparatus. During their transport, the structure of the GPI-anchor is remodeled, which regulates the trafficking and localization of GPI-anchored proteins. To date, more than 25 genes have been shown to be involved in this biosynthetic pathway. Uncovering the biogenesis of GPI is critical to understand the physiological and pathological roles of GPI-anchoring.. In this research proposal, we try to reveal factors required for biosynthesis and transport of glycolipid-modified proteins, named GPI-anchored proteins using a comprehensive forward genetic screening. By combining novel screening methods using “haploid mammalian cells” and “gene-trapping method”, mutant cells defective in biosynthesis and transport of GPI-anchored proteins are enriched. The mutagenized sites from mutant cell populations are then comprehensively identified by “next generation sequencers”. The novel genes listed up from the screening are further confirmed by biochemical and cell biological analysis. Using a series of genome-wide screening, the biological and medical significances of GPI-anchoring of proteins will be clarified.

蛋白质糖基磷脂酰肌醇（GPI）修饰是在真核细胞中高度保守的翻译后修饰过程。哺乳动物细胞中有超过100种的蛋白质都被GPI修饰。GPI在内质网中合成并转移到蛋白质上。磷脂酰肌醇逐步被糖、脂肪酸和磷酸乙醇胺修饰后生成完整的GPI前体并转移到蛋白质上，然后经过高尔基体，从内质网转移到细胞膜上。在转运过程中，GPI进行了重构，从而来调节GPI锚定蛋白的运输和定位。目前有超过25种参与该过程的基因得到了鉴定。揭示GPI锚定蛋白的生物合成过程具有重要的生理医学意义。在本项目中，我们拟利用一种新的基因筛选方法，探寻与GPI锚定蛋白生物合成和转运相关的因素。拟结合 “单倍体动物细胞”和“基因捕获技术”，筛选并富集GPI锚定蛋白合成和转运过程发生突变的细胞。通过“新一代细胞测序技术”对突变细胞进行基因组测序，并对寻找到的基因做进一步分析。运用一系列全基因组技术，揭示对蛋白质的GPI锚定所具有的生理医学意义。

糖基磷脂酰肌醇（GPI）锚修饰是一种保守于真核生物的蛋白质翻译后修饰。哺乳动物中超过150种蛋白被GPI修饰。GPI在内质网中合成并转移到蛋白质上。内质网中合成好的GPI锚定蛋白经由高尔基体转运到细胞膜表明，在这个过程中GPI的结构会发生重构。在这个项目中，我们试图鉴定出参与GPI锚定蛋白合成和转运的蛋白因子。为此，我们建立了一个基于单倍体哺乳动物细胞，基因诱捕和下一代基因测序技术的综合筛选系统。利用该系统我们筛选到了23个参与GPI合成的基因(PLoS ONE (2015))，证明该系统的高效性和可靠性。之后，利用该系统又进行了GPI锚定蛋白转运和质量控制调控因子的筛选，发现高尔基体相关逆转运蛋白（GARP）复合物介导GPI锚定蛋白的高尔基体逆转运(Mol. Biol. Cell (2015))。除此之外还发现蛋白质的N连接糖基化修饰调节GPI的结构重组(J. Cell Biol. (2017))。这些结果再次表明该系统具有很高的实用性。利用该系统我们可以研究其它生化过程。在该项目中，我们鉴定了很多参与GPI锚定蛋白合成和转运的蛋白因子，之后会进一步研究这些因子在GPI合成过程中的具体功能和生物学机制。这个项目中建立的系统和研究方法不仅可以应用到GPI合成的研究中，还可以用来研究其他细胞代谢过程，这都将会加速对细胞生物学机制的研究。