Furukawa Tomoka, Wakabayashi Kanako, Tamura Ai, Nakagawa Hiroshi, Morishima Yoshihiro, Osawa Yoichi, Ishikawa Toshihisa
Department of Biomolecular Engineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259-B-60 Nagatsuta, Midori-ku, Yokohama, 226-8501, Japan.
Pharm Res. 2009 Feb;26(2):469-79. doi: 10.1007/s11095-008-9752-7. Epub 2008 Oct 29.
Single nucleotide polymorphisms (SNPs) of the ATP-binding cassette (ABC) transporter ABCG2 gene have been suggested to be a significant factor in patients' responses to medication and/or the risk of diseases. We aimed to evaluate the impact of the major non-synonymous SNP Q141K on lysosomal and proteasomal degradations.
ABCG2 WT and the Q141K variant were expressed in Flp-In-293 cells by using the Flp recombinase system. Their expression levels and cellular localization was measured by immunoblotting and immunofluorescence microscopy, respectively.
The protein level of the Q141K variant expressed in Flp-In-293 cells was about half that of ABCG2 WT, while their mRNA levels were equal. The protein expression level of the Q141K variant increased about two-fold when Flp-In-293 cells were treated with MG132. In contrast, the protein level of ABCG2 WT was little affected by the same treatment. After treatment with bafilomycin A1, the protein levels of ABCG2 WT and Q141K increased 5- and 2-fold in Flp-In-293 cells, respectively.
The results strongly suggest that the major non-synonymous SNP Q141K affects the stability of the ABCG2 protein in the endoplasmic reticulum and enhances its susceptibility to ubiquitin-mediated proteasomal degradation.
三磷酸腺苷结合盒（ABC）转运蛋白ABCG2基因的单核苷酸多态性（SNP）被认为是影响患者药物反应和/或疾病风险的重要因素。我们旨在评估主要非同义SNP Q141K对溶酶体和蛋白酶体降解的影响。
利用Flp重组酶系统在Flp-In-293细胞中表达ABCG2野生型（WT）和Q141K变体。分别通过免疫印迹和免疫荧光显微镜检测它们的表达水平和细胞定位。
在Flp-In-293细胞中表达的Q141K变体的蛋白质水平约为ABCG2 WT的一半，而它们的mRNA水平相等。用MG132处理Flp-In-293细胞后，Q141K变体的蛋白质表达水平增加了约两倍。相比之下，相同处理对ABCG2 WT的蛋白质水平影响很小。用巴弗洛霉素A1处理后，Flp-In-293细胞中ABCG2 WT和Q141K的蛋白质水平分别增加了5倍和2倍。
结果强烈表明，主要非同义SNP Q141K影响ABCG2蛋白在内质网中的稳定性，并增强其对泛素介导的蛋白酶体降解的敏感性。
