GPI锚定蛋白前体C-端附着信号的疏水性决定其内质网相关蛋白质降解途径  

Hydrophobicity of the C-terminal GPI-anchor Attachment Signals Determines ER-associated Degradation Pathway of Precursor Proteins

作　　者：张楠 柳艺石 藤田盛久 高晓冬 ZHANG Nan;LIU Yi-Shi;MORIHISA Fujita;GAO Xiao-Dong(Key Laboratory of Carbohydrate Chemistry and Biotechnology,Ministry of Education,School of Biotechnology,Jiangnan University,Wuxi 214122,Jiangsu,China)

机构地区：[1]江南大学生物工程学院糖化学与生物技术教育部重点实验室,江苏无锡214122

出　　处：《中国生物化学与分子生物学报》2022年第10期1351-1358,共8页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金(No.32071278,31770853,31971216);山东省重大科技创新工程(No.2019JZZY011006)资助。

摘　　要：目前,已知超过150种糖基磷脂酰肌醇锚定蛋白(glycosylphosphatidylinositol anchored protein,GPI-anchored protein)在哺乳动物细胞中表达,并参与免疫识别、细胞通讯与信号转导等多种生理过程。当蛋白质无法被GPI修饰时,前体蛋白质通过内质网相关蛋白质降解(endoplasmic reticulum associated degradation,ERAD)途径降解。然而,GPI锚定蛋白ERAD的降解机制尚不清楚。为了探究GPI锚定蛋白前体的ERAD途径的具体机制,本研究敲除人胚胎肾细胞293细胞株(HEK293)的GPI转酰胺酶复合物亚基PIGS基因,进而敲除E3泛素连接酶HRD1和GP78基因,之后随机在PIGS-KO,PIGS-HRD1-KO和PIGS-GP78-KO过表达16种GPI锚定蛋白质(以亲本PIGS-KO细胞株作为对照组),Western印迹结果证明,GPI锚定蛋白前体在细胞株PIGS-HRD1-KO中的蛋白质积累量(I_(PHK))和PIGS-GP78-KO中的蛋白质积累量(I_(PGK))体现出明显的差异性,例如LYPD2的I_(PHK)是I_(PGK)的28倍,NEGR1的I_(PHK)是I_(PGK)的0.12倍,这说明GPI锚定蛋白前体的降解主要依赖于2种ERAD途径:ERAD-L和ERAD-M。且HRD1与GP78的2种E3泛素连接酶被选择性地用于GPI锚定蛋白前体的降解。朊病毒(prion)和CD59嵌合构建体表明,GPI前体蛋白C-端GPI附着信号决定了降解途径(朊病毒I_(PHK)/I_(PGK)由突变前的0.33改变为突变后的23.42。相反的是,突变前CD59的I_(PHK)/I_(PGK)是11.45,突变后变为2.81)。接着,通过对C-端附着信号疏水性的计算,我们发现,这种选择性差异由前体蛋白质C-端GPI附着信号疏水性的不同造成。本研究初步解释了未被GPI锚修饰的GPI锚定蛋白前体在ERAD中的降解机制。More than 150 glycosylphosphatidylinositol(GPI)-anchored proteins(GPI-APs) are expressed in mammalian cells and involved in various physiological processes such as immune recognition,cell communication and signal transduction.GPI is transferred to proteins in the endoplasmic reticulum(ER).When GPI-anchoring is impaired,precursor proteins are thought to be degraded through ER-associated degradation(ERAD).However,the mechanism of their degradation in ERAD remains unclear.To investigate the impact of ERAD pathways on degradation of GPI precursor proteins,we used series of knockout(KO) human embryonic kidney 293(HEK293) cells defective in PIGS gene,which encodes a GPI transamidase complex subunit,combined with KO in HRD1(PIGS-HRD1-KO) or GP78(PIGS-GP78-KO),which encodes the E3 ubiquitin ligases for the ERAD pathways.We compared the stability of 16 GPI precursor proteins in the ERAD-deficient cells with the parental PIGS-KO cells.Western blotting data showed that the GPI precursor proteins were stabilized in either PIGS-HRD1-KO(I_(PHK)) or PIGS-GP78-KO(I_(PGK)),whereas the behaviors of each GPI precursor protein were different in two KO cells,suggesting that both HRD1-dependent ERAD-L and GP78-depemdent ERAD-M are selectively utilized for GPI precursor degradation depending upon each protein.For example,the ratio of intensity between PIGS-HRD1-KO and PIGS-GP78-KO cells(I_(PHK)/I_(PGK)) of LYPD2 was 28 times,while that of NEGR1 was 0.12.The chimeric constructs between Prion and CD59 indicate that the C-terminal GPI attachment signal of GPI precursor proteins determines the degradation pathways(The I_(PHK)/I_(PGK) of Prion protein was changed from 0.33 to 23.42 by replacing its GPI attachment signal with CD59’s one.On contrary,the I_(PHK)/I_(PGK) of CD59 was changed from 11.45 to 2.81) by replacing the GPI attachment signal with Prion’s one.Furthermore,hydrophobicity of the C-terminal GPI attachment signalon precursor proteins was correlated to the selection of the ERAD pathways.Our results explain the mechanism of

关 键 词：糖基磷脂酰肌醇锚定蛋白 内质网相关蛋白质降解 蛋白质疏水性 

分 类 号：Q291[生物学—细胞生物学]