Department of Pharmaceutical Sciences, University of Colorado Denver, Aurora, Colorado 80045, USA.
J Biol Chem. 2013 Jun 21;288(25):18421-8. doi: 10.1074/jbc.M113.468090. Epub 2013 Apr 29.
The endogenously produced oxidant hypothiocyanous acid (HOSCN) inhibits and kills pathogens but paradoxically is well tolerated by mammalian host tissue. Mammalian high molecular weight thioredoxin reductase (H-TrxR) is evolutionarily divergent from bacterial low molecular weight thioredoxin reductase (L-TrxR). Notably, mammalian H-TrxR contains a selenocysteine (Sec) and has wider substrate reactivity than L-TrxR. Recombinant rat cytosolic H-TrxR1, mouse mitochondrial H-TrxR2, and a purified mixture of both from rat selectively turned over HOSCN (kcat = 357 ± 16 min(-1); Km = 31.9 ± 10.3 μM) but were inactive against the related oxidant hypochlorous acid. Replacing Sec with Cys or deleting the final eight C-terminal peptides decreased affinity and turnover of HOSCN by H-TrxR. Similarly, glutathione reductase (an H-TrxR homologue lacking Sec) was less effective at HOSCN turnover. In contrast to H-TrxR and glutathione reductase, recombinant Escherichia coli L-TrxR was potently inhibited by HOSCN (IC50 = 2.75 μM). Similarly, human bronchial epithelial cell (16HBE) lysates metabolized HOSCN, but E. coli and Pseudomonas aeruginosa lysates had little or no activity. HOSCN selectively produced toxicity in bacteria, whereas hypochlorous acid was nonselectively toxic to both bacteria and 16HBE. Treatment with the H-TrxR inhibitor auranofin inhibited HOSCN metabolism in 16HBE lysates and significantly increased HOSCN-mediated cytotoxicity. These findings demonstrate both the metabolism of HOSCN by mammalian H-TrxR resulting in resistance to HOSCN in mammalian cells and the potent inhibition of bacterial L-TrxR resulting in cytotoxicity in bacteria. These data support a novel selective mechanism of host defense in mammals wherein HOSCN formation simultaneously inhibits pathogens while sparing host tissue.
内源性产生的氧化剂次碘酸（HOSCN）抑制和杀死病原体，但矛盾的是，它被哺乳动物宿主组织很好地耐受。哺乳动物高分子量硫氧还蛋白还原酶（H-TrxR）与细菌低分子量硫氧还蛋白还原酶（L-TrxR）在进化上有很大的不同。值得注意的是，哺乳动物 H-TrxR 含有一个硒代半胱氨酸（Sec），并且比 L-TrxR 具有更广泛的底物反应性。重组大鼠胞质 H-TrxR1、小鼠线粒体 H-TrxR2 以及从大鼠中纯化的两者混合物选择性地使 HOSCN 发生转化（kcat = 357 ± 16 min(-1)；Km = 31.9 ± 10.3 μM），但对相关氧化剂次氯酸没有活性。用半胱氨酸取代 Sec 或删除 H-TrxR 的最后 8 个 C 末端肽降低了 HOSCN 的亲和力和周转率。同样，谷胱甘肽还原酶（一种缺乏 Sec 的 H-TrxR 同系物）在 HOSCN 转化中效率较低。与 H-TrxR 和谷胱甘肽还原酶相反，重组大肠杆菌 L-TrxR 被 HOSCN 强烈抑制（IC50 = 2.75 μM）。同样，人支气管上皮细胞（16HBE）裂解物代谢 HOSCN，但大肠杆菌和铜绿假单胞菌裂解物几乎没有或没有活性。HOSCN 选择性地使细菌产生毒性，而次氯酸对细菌和 16HBE 都具有非选择性毒性。用 H-TrxR 抑制剂 auranoffin 处理可抑制 16HBE 裂解物中 HOSCN 的代谢，并显著增加 HOSCN 介导的细胞毒性。这些发现表明，哺乳动物 H-TrxR 代谢 HOSCN 导致哺乳动物细胞对 HOSCN 的抗性，以及细菌 L-TrxR 的强烈抑制导致细菌的细胞毒性。这些数据支持哺乳动物中宿主防御的一种新的选择性机制，其中 HOSCN 的形成同时抑制病原体，同时保护宿主组织。
