Ubiquitin-mediated proteasomal degradation of non-synonymous SNP variants of human ABC transporter ABCG2
Ubiquitin-mediated proteasomal degradation of non-synonymous SNP variants of human ABC transporter ABCG2
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DOI：
10.1042/bj20071229
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发表时间：
2008-05-01
影响因子：
4.1
通讯作者：
Ishikawa, Toshihisa
中科院分区：
文献类型：
--
作者：
Nakagawa, Hiroshi;Tamura, Ai;Ishikawa, Toshihisa
ABC（ATP结合盒）转运蛋白ABCG 2（ABC亚家族G，成员2）的遗传多态性与药物反应的个体差异之间存在临床相关性。我们通过使用Flp（翻转酶）重组酶系统在Flp-In-293细胞中表达了总共七个非同义SNP（单核苷酸多态性）变体。其中，发现ABCG 2 F208 S和S441 N变体以显著低的水平表达，而它们的mRNA水平与其他SNP变体和ABCG 2 WT（野生型）的mRNA水平相等。有趣的是，当Flp-In-293细胞用蛋白酶体抑制剂MG 132处理时，ABCG 2 F208 S和S441 N变体的蛋白表达水平增加6至12倍。免疫沉淀和免疫印迹分析表明，ABCG 2 F208 S和S441 N变体蛋白在Flp-In-293细胞中内源性泛素化，并且用MG 132处理显著增强了这些泛素化变体的水平。免疫荧光显微镜显示，MG 132在蛋白水平和细胞内分布方面极大地影响了ABCG 2 F208 S和S441 N变体。免疫印迹分析显示，这些变体是N-糖基化的;然而，与ABCG 2 WT上存在的那些相比，它们的寡糖是不成熟的。ABCG 2 F208 S和S441 N变体蛋白似乎不在高尔基体中加工，但在蛋白酶体中经历泛素介导的蛋白质降解，而ABCG 2 WT被分选到质膜，然后通过溶酶体途径降解。本研究提供了第一个证据表明，某些遗传多态性可以影响ABCG 2的蛋白质稳定性。控制ABCG 2的蛋白酶体降解将为癌症化疗提供一种新的方法，以规避人类癌症的多药耐药性。
Clinical relevance is implicated between the genetic polymorphisms of the ABC (ATP-binding cassette) transporter ABCG2 (ABC subfamily G, member 2) and the individual differences in drug response. We expressed a total of seven non-synonymous SNP (single nucleotide polymorphism) variants in Flp-In-293 cells by using the Flp (flippase) recombinase system. Of these, ABCG2 F208S and S441N variants were found to be expressed at markedly low levels, whereas their mRNA levels were equal to those of the other SNP variants and ABCG2 WT (wild-type). Interestingly, protein expression levels of the ABCG2 F208S and S441N variants increased 6- to 12-fold when Flp-In-293 cells were treated with MG132, a proteasome inhibitor. Immunoprecipitation followed by immunoblot analysis showed that the ABCG2 F208S and S441N variant proteins were endogenously ubiquitinated in Flp-In-293 cells, and treatment with MG132 significantly enhanced the level of these ubiquitinated variants. Immunofluorescence microscopy demonstrated that MG132 greatly affected the ABCG2 F208S and S441N variants in terms of both protein levels and intracellular distribution. Immunoblot analysis revealed that those variants were N-glycosylated; however, their oligosaccharides were immature compared with those present on ABCG2 WT. The ABCG2 F208S and S441N variant proteins do not appear to be processed in the Golgi apparatus, but undergo ubiquitin-mediated protein degradation in proteasomes, whereas ABCG2 WT is sorted to the plasma membrane and then degraded via the lysosomal pathway. The present study provides the first evidence that certain genetic polymorphisms can affect the protein stability of ABCG2. Control of proteasomal degradation of ABCG2 would provide a novel approach in cancer chemotherapy to circumvent multidrug resistance of human cancers.