Role of Cys-603 in dimer/oligomer formation of the breast cancer resistance protein BCRP/ABCG2
Role of Cys-603 in dimer/oligomer formation of the breast cancer resistance protein BCRP/ABCG2
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DOI：
10.1111/j.1349-7006.2005.00126.x
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发表时间：
2005-12-01
期刊：
影响因子：
5.7
通讯作者：
Sugimoto, Y
中科院分区：
文献类型：
--
作者：
Kage, K;Fujita, T;Sugimoto, Y
乳腺癌耐药蛋白（BCRP/ABCG2）是一种半分子atp结合盒转运蛋白，我们之前提出可能作为二硫键桥接的二聚体起作用。在本研究中，我们进行了半胱氨酸扫描诱变，将Ser替换为Cys，并建立了12个表达BCRP突变体的PA317突变体，其S-S键可能被破坏。来自野生型转染物（PA/WT）的BCRP Western blot分析证实，野生型蛋白在非还原条件下以140 kda的二聚体迁移，而在还原条件下以70 kda的单体迁移。然而，在非还原条件下，BCRP- c603s突变体以70 kda单体和140 kda二聚体的形式迁移，而所有其他突变体BCRP仅以二聚体的形式迁移。转染C603S-BCRP （PA/C603S）的PA317细胞表现出与PA/WT细胞相似或仅略低的SN-38抗性，尽管这些细胞中BCRP二聚体水平降低。此外，在还原条件下，BCRP突变体对SN-38的抗性程度与单体表达水平有关。逆转录-聚合酶链反应分析表明，BCRP mRNA水平在转染物中相似。随后，我们产生了6个BCRP C603X突变体（X = D， H， R， Y， A和W），并进行了western blot分析和药敏试验。结果与PA/C603S细胞的结果相同，但有一些变化再次与单体水平相对应。我们的研究结果表明，Cys-603是BCRP单体之间共价桥的重要残基，但BCRP的功能单元可能不一定需要共价键。
Breast cancer resistance protein (BCRP/ABCG2) is a half-molecule ATP-binding cassette transporter that we have previously suggested might function as a homodimer, bridged by disulfide bonds. In the present study, we carried out cysteine-scanning mutagenesis, substituting Ser for Cys, and established 12 PA317 transfectants expressing BCRP mutants with possible disruptions to their S-S bonds. Western blot analysis of BCRP from the wildtype transfectants (PA/WT) confirmed that the wild-type protein migrates as a 140-kDa dimer under non-reducing conditions, but as a 70-kDa monomer under reducing conditions. However, under non-reducing conditions the BCRP-C603S mutant migrated both as a 70-kDa monomer and a 140-kDa dimer, whereas all other mutant BCRP migrated only as dimers. PA317 cells transfected with C603S-BCRP (PA/C603S) showed either similar or only marginally lower SN-38 resistance than PA/WT cells, despite the reduced levels of BCRP dimer in these cells. Moreover, the degree of SN-38 resistance in the mutant BCRP transfectants was found to be associated with the monomer expression levels under reducing conditions. Reverse transcription-polymerase chain reaction analysis showed that the BCRP mRNA levels were similar in the transfectants. We subsequently generated six C603X mutants of BCRP (X = D, H, R, Y, A and W) and carried out western blot analysis and drug sensitivity assays. The results were equivalent to those from the PA/C603S cells, with some variations that again corresponded to the monomer levels. Our findings suggest that Cys-603 is an important residue in the covalent bridge between BCRP monomers but that a functioning unit of BCRP may not necessarily require covalent linkages.