Functional Characterization of Human Peptide/Histidine Transporter 1 in Stably Transfected MDCK Cells.
Functional Characterization of Human Peptide/Histidine Transporter 1 in Stably Transfected MDCK Cells.
复制标题
DOI：
10.1021/acs.molpharmaceut.7b00728
复制
发表时间：
2018-02-05
影响因子：
4.9
通讯作者：
Jiang H
中科院分区：
文献类型：
--
作者：
Song F;Hu Y;Wang Y;Smith DE;Jiang H
质子偶联寡肽转运蛋白PHT 1（SLC 15 A4）是一种跨膜转运组氨酸和小分子肽的蛋白质，在细胞内肽稳态和天然免疫应答中起重要作用。然而，它仍然是一个挑战，以阐明功能特性的PHT 1转运蛋白，因为它的亚细胞定位。本研究的目的是诉诸hPHT 1蛋白从亚细胞外细胞膜的MDCK细胞稳定转染人PHT 1突变体，并在这些细胞中的功能活性的特点。使用该模型，通过用d3-l-组氨酸、GlySar和细菌肽聚糖产物MDP和Tri-DAP的细胞摄取研究来评价hPHTl的功能活性。我们发现，两个双亮氨酸基序的破坏是必不可少的hPHT 1转运蛋白优先靶向质膜。hPHT 1对d3-l-组氨酸表现出高亲和力，对GlySar表现出低亲和力，Km值分别为16.3 ± 1.9 µM和1.60 ± 0.30 mM。此外，细菌肽聚糖组分MDP和Tri-DAP最终被证明是hPHT 1底物。hPHT 1对MDP的摄取可被二肽/三肽和肽类药物抑制，但不被甘氨酸和阿昔洛韦抑制。hPHT 1的功能活性也是pH依赖性的，在pH 6.5的缓冲液中具有最佳细胞摄取。综上所述，我们建立了一种新的细胞模型，在体外评估hPHT 1的功能，并证实MDP和Tri-DAP是hPHT 1的底物。我们的研究结果表明，PHT 1可能作为一个潜在的目标，减少免疫反应和药物治疗的炎症性疾病。
The proton-coupled oligopeptide transporter PHT1 (SLC15A4), which facilitates cross-membrane transport of histidine and small peptides from inside the endosomes or lysosomes to cytosol, plays an important role in intracellular peptides homeostasis and innate immune responses. However, it remains a challenge to elucidate functional properties of the PHT1 transporter because of its subcellular localization. The purpose of this study was to resort hPHT1 protein from the subcellular to outer cell membrane of MDCK cells stably transfected with human PHT1 mutants, and to characterize its functional activity in these cells. Using this model, the functional activity of hPHT1 was evaluated by cellular uptake studies with d3-l-histidine, GlySar, and the bacterial peptidoglycan products MDP and Tri-DAP. We found that the disruption of two dileucine motifs was indispensable for hPHT1 transporter being preferentially targeting to plasma membranes. hPHT1 showed high affinity for d3-l-histidine and low affinity for GlySar, with Km values of 16.3 ± 1.9 µM and 1.60 ± 0.30 mM, respectively. Moreover, the bacterial peptidoglycan components MDP and Tri-DAP were shown conclusively to be hPHT1 substrates. The uptake of MDP by hPHT1 was inhibited by di/tripeptides and peptide-like drugs, but not by glycine and acyclovir. The functional activity of hPHT1 was also pH-dependent, with an optimal cellular uptake in buffer pH 6.5. Taken together, we established a novel cell model to evaluate the function of hPHT1 in vitro, and confirmed that MDP and Tri-DAP were substrates of hPHT1. Our findings suggest that PHT1 may serve as a potential target for reducing the immune responses and for drug treatment of inflammatory diseases.
登录
查看更多内容
影响因子：
4.8
作者：
Charriere, Guillaume M.;Ip, W. K. Eddie;Stuart, Lynda M.
通讯作者：
Stuart, Lynda M.
影响因子：
3.9
作者：
Hu, Yongjun;Chen, Xiaomei;Smith, David E.
通讯作者：
Smith, David E.
影响因子：
15.9
作者：
KOPPLE, JD;SWENDSEID, ME
通讯作者：
SWENDSEID, ME
影响因子：
64.8
作者：
Nakamura, Norihiro;Lill, Jennie R.;Mellman, Ira
通讯作者：
Mellman, Ira
影响因子：
4.7
作者：
Hu Y;Xie Y;Keep RF;Smith DE
通讯作者：
Smith DE