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首页> 外文期刊>Biochemistry >Human Concentrative Nucleoside Transporter 3 (hCNT3, SLC28A3) Forms a Cyclic Homotrimer
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Human Concentrative Nucleoside Transporter 3 (hCNT3, SLC28A3) Forms a Cyclic Homotrimer

机译：人浓缩核苷转运蛋白3（HCNT3，SLC28A3）形成环状的同性计

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摘要

Many anticancer and antiviral drugs are purine or pyrimidine analogues, which use membrane transporters to cross cellular membranes. Concentrative nucleoside transporters (CNTs) mediate the salvage of nucleosides and the transport of therapeutic nucleoside analogues across plasma membranes by coupling the transport of ligands to the sodium gradient. Of the three members of the human CNT family, CNT3 has the broadest selectivity and the widest expression profile. However, the molecular mechanisms of the transporter, including how it interacts with and translocates structurally diverse nucleosides and nucleoside analogues, are unclear. Recently, the crystal structure of vcCNT showed that the prokaryotic homologue of CNT3 forms a homotrimer. In this study, we successfully expressed and purified the wild type human homologue, hCNT3, demonstrating the homotrimer by size exclusion profiles and glutaraldehyde cross-linking. Further, by creating a series of cysteine mutants at highly conserved positions guided by comparative structure models, we cross-linked hCNT3 protomers in a cell-based assay, thus showing the existence of hCNT3 homotrimers in human cells. The presence and absence of cross-links at specific locations along TM9 informs us of important structural differences between vcCNT and hCNT3. Comparative modeling of the trimerization domain and sequence coevolution analysis both indicate that oligomerization is critical to the stability and function of hCNT3. In particular, trimerization appears to shorten the translocation path for nucleosides across the plasma membrane and may allow modulation of the transport function via allostery.
机译：许多抗癌和抗病毒药物是嘌呤或嘧啶类似物，其使用膜转运蛋白来交叉细胞膜。浓缩核苷转运蛋白（CNT）通过将配体的传输与钠梯度偶联来介导核苷的拯救和在血浆膜上运输治疗核苷类似物。在人类CNT家族的三个成员中，CNT3具有最广泛的选择性和最宽的表达谱。然而，传送器的分子机制，包括如何与其与结构不同的核苷和核苷类似物相互作用，尚不清楚。最近，vccnt的晶体结构表明CNT3的原核同源物形成同种素。在这项研究中，我们成功地表达和纯化了野生型人类同源物HCNT3，通过尺寸排除型和戊二醛交联来证明同性计。此外，通过在比较结构模型引导的高度保守位置产生一系列半胱氨酸突变体，我们在基于细胞的测定中交联HCNT3重组，从而显示存在于人细胞中HCNT3均方格的存在。沿TM9的特定位置的存在和不存在交叉链路通知我们VCCNT和HCNT3之间的重要结构差异。三聚化结构域和序列共区分分析的对比建模表明，寡聚化对HCNT3的稳定性和功能至关重要。特别地，三聚化似乎缩短了血浆膜上的核苷的易位路径，并且可以允许通过构象调节运输功能。

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  • 来源
    《Biochemistry》 |2017年第27期|共9页
  • 作者

    Stecula Adrian; Schlessinger Avner; Giacomini Kathleen M.; Sali Andrej;

  • 作者单位

    Univ Calif San Francisco Dept Bioengn &

    Therapeut Sci San Francisco CA 94158 USA;

    Icahn Sch Med Mt Sinai Dept Pharmacol Sci New York NY 10029 USA;

    Univ Calif San Francisco Dept Bioengn &

    Therapeut Sci San Francisco CA 94158 USA;

    Univ Calif San Francisco Dept Bioengn &

    Therapeut Sci San Francisco CA 94158 USA;

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  • 正文语种 eng
  • 中图分类 生物化学;
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