Amino-terminal region of human organic anion transporting polypeptide 1B1 dictates transporter stability and substrate interaction
Amino-terminal region of human organic anion transporting polypeptide 1B1 dictates transporter stability and substrate interaction
复制标题
人有机阴离子转运多肽 1B1 的氨基末端区域决定转运蛋白稳定性和底物相互作用
DOI：
10.1016/j.taap.2019.114642
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发表时间：
2019
影响因子：
3.8
通讯作者：
Hong Mei
中科院分区：
文献类型：
--
作者：
Wang Xuyang;Chen Jie;Xu Shaopeng;Ni Chunxu;Fang Zihui;Hong Mei
有机阴离子转运多肽（OATPs）具有广泛的底物特异性、广泛的组织分布和参与药物相互作用，是药物吸收、分布和排泄的关键因素。OATP 1B 1特异性定位于人肝细胞的基底外侧膜，并在药物从体内清除中发挥关键作用。以往的研究表明，跨膜结构域（TM）是OATP正常功能所必需的。在本研究中，进行定点突变以研究OATP 1B 1的TM 1和氨基末端。TM 1中的两个带正电荷的残基K41和K49以及疏水残基I46被鉴定为对转运蛋白的正常功能是重要的。K41 A和K49 A分别在雌酮-3-硫酸酯（ES）的高亲和力和低亲和力结合位点处表现出改变的Km值;而I46的丙氨酸取代显示出改变的Km和Vmax值，用于ES的两种结合组分。K41的额外替换表明，该位置的正电荷性质对于维持OATP 1B 1蛋白水平和功能非常重要;而位置49处赖氨酸的特定侧基结构对于转运蛋白活性是不可替代的。用亮氨酸保守取代I46也恢复了转运蛋白的功能。此外，对OATP 1B 1氨基末端的研究表明，19 - 27位残基对于蛋白质稳定性和底物相互作用至关重要。因此，氨基末端区域（包括TM 1和OATP 1B 1的氨基末端）对膜蛋白的正常功能很重要。
Organic anion transporting polypeptides (OATPs) are key players of drug absorption, distribution and excretion due to their broad substrate specificity, wide tissue distribution and the involvement in drug-drug interaction. OATP1B1 is specifically localized at the basolateral membrane of human hepatocytes and serves a crucial role in the drug clearance from the body. Previous studies have shown that transmembrane domains (TMs) are essential for proper functions of OATPs. In the present study, site-directed mutagenesis was performed to study the TM1 and amino-terminus of OATP1B1. Two positively charged residues, K41 and K49, as well as a hydrophobic residue I46, in TM1 were identified to be important for the proper function of the transporter. K41A and K49A exhibited altered Km value at the high and low affinity binding sites of estrone-3- sulfate (ES), respectively; while alanine substitution of I46 showed altered Km and Vmax values for both binding components of ES. Additional replacement of K41 revealed that the positively charged property at this position is important for maintaining OATP1B1 protein level and function; while the specific side-group structure of lysine at position 49 is irreplaceable for the transporter activity. Conservative replacement of I46 with leucine also recovered the function of the transporter. In addition, studies of the amino-terminus of OATP1B1 revealed that residues ranging from 19 to 27 are essential for protein stability and substrate interaction. Therefore, the amino-terminal region, which includes TM1 and the amino-terminus of OATP1B1, is important for proper function of the membrane protein.