Tryptophanyl-tRNA synthetase mediates high-affinity tryptophan uptake into human cells
Tryptophanyl-tRNA synthetase mediates high-affinity tryptophan uptake into human cells
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DOI：
10.1074/jbc.ra117.001247
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发表时间：
2018-06-01
影响因子：
4.8
通讯作者：
Wakasugi, Keisuke
中科院分区：
文献类型：
--
作者：
Miyanokoshi, Miki;Yokosawa, Takumi;Wakasugi, Keisuke
色氨酸（Trp）转运系统对Trp具有高亲和力和选择性，据报道存在于人和小鼠巨噬细胞中。尽管该系统在干扰素 (IFN-) 处理的细胞和吲哚胺 2,3-双加氧酶 1 (IDO1) 表达的细胞中高度表达，但其身份仍不完全清楚。色氨酰-tRNA 合成酶 (TrpRS) 在 IFN 处理的细胞中也高度表达，并且对 Trp 也具有高亲和力和选择性。在这里，我们研究了人 TrpRS 表达对 IFN 处理的人 THP-1 单核细胞或 HeLa 细胞摄取 Trp 的影响。 TrpRS 特异性 siRNA 对人 TrpRS 表达的抑制减少，而 TrpRS 的过度表达则增加细胞对 Trp 的摄取。值得注意的是，TrpRS介导的摄取系统对Trp的亲和力比已知的System L氨基酸转运蛋白高出一百倍以上，促进低Trp浓度的摄取，并且具有非常高的Trp选择性。此外，定点诱变实验表明，TrpRS 中的 Trp 和 ATP 结合位点（而非 tRNA 结合位点）对于 TrpRS 介导的色氨酸摄取进入人体细胞至关重要。我们进一步证明，在细胞培养基中添加纯化的 TrpRS 可以增加细胞对 Trp 的摄取。总而言之，我们的结果表明 TrpRS 在人类细胞高亲和力 Trp 摄取中发挥着重要作用。
The tryptophan (Trp) transport system has a high affinity and selectivity toward Trp, and has been reported to exist in both human and mouse macrophages. Although this system is highly expressed in interferon- (IFN-)-treated cells and indoleamine 2,3-dioxygenase 1 (IDO1)-expressing cells, its identity remains incompletely understood. Tryptophanyl-tRNA synthetase (TrpRS) is also highly expressed in IFN--treated cells and also has high affinity and selectivity for Trp. Here, we investigated the effects of human TrpRS expression on Trp uptake into IFN--treated human THP-1 monocytes or HeLa cells. Inhibition of human TrpRS expression by TrpRS-specific siRNAs decreased and overexpression of TrpRS increased Trp uptake into the cells. Of note, the TrpRS-mediated uptake system had more than hundred-fold higher affinity for Trp than the known System L amino acid transporter, promoted uptake of low Trp concentrations, and had very high Trp selectivity. Moreover, site-directed mutagenesis experiments indicated that Trp- and ATP-binding sites, but not tRNA-binding sites, in TrpRS are essential for TrpRS-mediated Trp uptake into the human cells. We further demonstrate that the addition of purified TrpRS to cell culture medium increases Trp uptake into cells. Taken together, our results reveal that TrpRS plays an important role in high-affinity Trp uptake into human cells.