Department of Physiology, Membrane Protein Disease Research Group, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.
Department of Oncology, Membrane Protein Disease Research Group, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.
Biochem J. 2018 Oct 31;475(20):3293-3309. doi: 10.1042/BCJ20180543.
Human equilibrative nucleoside transporter 1 (hENT1), the first identified member of the ENT family of integral membrane proteins, is the primary mechanism for cellular uptake of physiologic nucleosides and many antineoplastic and antiviral nucleoside drugs. hENT1, which is potently inhibited by nitrobenzylthioinosine (NBMPR), possesses 11 transmembrane helical domains with an intracellular N-terminus and an extracellular C-terminus. As a protein with 10 endogenous cysteine residues, it is sensitive to inhibition by the membrane permeable sulfhydryl-reactive reagent -ethylmaleimide (NEM) but is unaffected by the membrane impermeable sulfhydryl-reactive reagent -chloromercuriphenyl sulfonate. To identify the residue(s) involved in NEM inhibition, we created a cysteine-less version of hENT1 (hENT1C-), with all 10 endogenous cysteine residues mutated to serine, and showed that it displays wild-type uridine transport and NBMPR-binding characteristics when produced in the oocyte heterologous expression system, indicating that endogenous cysteine residues are not essential for hENT1 function. We then tested NEM sensitivity of recombinant wild-type hENT1, hENT1 mutants C1S to C10S (single cysteine residues replaced by serine), hENT1C- (all cysteine residues replaced by serine), and hENT1C- mutants S1C to S10C (single serine residues converted back to cysteine). Mutants C9S (C416S/hENT1) and S9C (S416C/hENT1C-) were insensitive and sensitive, respectively, to inhibition by NEM, identifying Cys as the endofacial cysteine residue in hENT1 responsible for NEM inhibition. Kinetic experiments suggested that NEM modification of Cys, which is located at the inner extremity of TM10, results in the inhibition of hENT1 uridine transport and NBMPR binding by constraining the protein in its inward-facing conformation.
人嘌呤核苷转运蛋白 1（hENT1）是整联蛋白膜蛋白家族中第一个被鉴定的成员，是细胞摄取生理核苷和许多抗肿瘤和抗病毒核苷药物的主要机制。hENT1 被硝基苄基硫代肌苷（NBMPR）强烈抑制，具有 11 个跨膜螺旋结构域，胞内有 N 端，胞外有 C 端。作为一种含有 10 个内源性半胱氨酸残基的蛋白质，它对膜通透巯基反应性试剂 -乙基马来酰亚胺（NEM）敏感，但不受膜不可透巯基反应性试剂 -氯汞苯磺酸的影响。为了确定涉及 NEM 抑制的残基，我们创建了一个不含半胱氨酸的 hENT1（hENT1C-），其中所有 10 个内源性半胱氨酸残基突变为丝氨酸，并且当在卵母细胞异源表达系统中产生时，它显示出野生型尿苷转运和 NBMPR 结合特性，表明内源性半胱氨酸残基不是 hENT1 功能所必需的。然后，我们测试了重组野生型 hENT1、hENT1 突变体 C1S 至 C10S（单个半胱氨酸残基突变为丝氨酸）、hENT1C-（所有半胱氨酸残基突变为丝氨酸）和 hENT1C-突变体 S1C 至 S10C（单个丝氨酸残基转换回半胱氨酸）对 NEM 的敏感性。突变体 C9S（C416S/hENT1）和 S9C（S416C/hENT1C-）分别对 NEM 的抑制不敏感和敏感，从而确定 Cys 是 hENT1 中负责 NEM 抑制的内表面半胱氨酸残基。动力学实验表明，NEM 修饰位于 TM10 内端的 Cys 会导致 hENT1 尿苷转运和 NBMPR 结合的抑制，从而将蛋白质约束在其内向构象中。
