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Abstract：
The human ABCG2 multidrug transporter plays a crucial role in the absorption and excretion of xeno- and endobiotics, contributes to cancer drug resistance and the development of gout. In this work, we have analyzed the effects of selected variants, residing in a structurally unresolved cytoplasmic region (a.a. 354-367) of ABCG2 on the function and trafficking of this protein. A cluster of four lysines (K357-360) and the phosphorylation of a threonine (T362) residue in this region have been previously suggested to significantly affect the cellular fate of ABCG2. Here, we report that the naturally occurring K360del variant in human cells increased ABCG2 plasma membrane expression and accelerated cellular trafficking. The variable alanine replacements of the neighboring lysines had no significant effect on transport function, and the apical localization of ABCG2 in polarized cells has not been altered by any of these mutations. Moreover, in contrast to previous reports, we found that the phosphorylation-incompetent T362A, or the phosphorylation-mimicking T362E variants in this loop had no measurable effects on the function or expression of ABCG2. Molecular dynamics simulations indicated an increased mobility of the mutant variants with no major effects on the core structure of the protein. These results may help to decipher the potential role of this unstructured region within this transporter.
摘要：
人ABCG2多药转运体在外源性和内源性生物的吸收和排泄中起着至关重要的作用，有助于癌症耐药和痛风的发展。在这项工作中,我们分析了选定变体的效果，驻留在ABCG2的结构未解析的细胞质区域（a.a.354-367）上，研究该蛋白质的功能和运输。先前已经提出了该区域中的四个赖氨酸簇(K357-360)和苏氨酸(T362)残基的磷酸化显著影响ABCG2的细胞命运。这里,我们报道，人细胞中天然存在的K360del变体增加了ABCG2质膜表达并加速了细胞运输。相邻赖氨酸的可变丙氨酸置换对转运功能没有显著影响，这些突变均未改变ABCG2在极化细胞中的顶端定位。此外，与以前的报告相比，我们发现磷酸化不合格的T362A，或该环中的磷酸化模拟T362E变体对ABCG2的功能或表达没有可测量的影响。分子动力学模拟表明，突变变体的迁移率增加，对蛋白质的核心结构没有重大影响。这些结果可能有助于破译该非结构化区域在该转运蛋白中的潜在作用。
