CUG Start Codon Generates Thioredoxin/Glutathione Reductase Isoforms in Mouse Testes
CUG Start Codon Generates Thioredoxin/Glutathione Reductase Isoforms in Mouse Testes
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DOI：
10.1074/jbc.m109.070532
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发表时间：
2010-02-12
影响因子：
4.8
通讯作者：
Gladyshev, Vadim N.
中科院分区：
文献类型：
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作者：
Gerashchenko, Maxim V.;Su, Dan;Gladyshev, Vadim N.
哺乳动物细胞质和线粒体硫氧还蛋白还原酶是控制硫氧还蛋白功能的必需的含硒代半胱氨酸的酶。硫氧还蛋白/谷胱甘肽还原酶 (TGR) 是该酶家族的第三个成员。它具有额外的谷氧还蛋白结构域，并在睾丸中表现出最高表达。在此，我们发现人类和其他几种哺乳动物的 TGR 基因缺乏任何可以在翻译起始中发挥作用的 AUG 密码子。尽管小鼠和大鼠 TGR 具有此类密码子，但我们通过免疫印迹分析和直接蛋白质组分析检测了它们上游的蛋白质序列。进一步的基因工程和表达分析表明，位于先前认为启动翻译的序列上游的 CUG 密码子是小鼠 TGR 中的实际起始密码子。该密码子的使用依赖于 Kozak 共有序列和核糖体扫描机制。然而，CUG 作为一个低效的起始密码子，允许下游起始，从而在体内和体外产生两种酶同工型。 CUG 的使用在哺乳动物 TGR 中进化而来，并且在其中一些生物体中，使用 GUG 代替。新发现的较长 TGR 形式显示出培养细胞中的胞质定位，并在小鼠睾丸的精子细胞中表达。这项研究表明，CUG 密码子被用作低效的起始密码子，在小鼠中生成蛋白质亚型。
Mammalian cytosolic and mitochondrial thioredoxin reductases are essential selenocysteine-containing enzymes that control thioredoxin functions. Thioredoxin/glutathione reductase (TGR) is a third member of this enzyme family. It has an additional glutaredoxin domain and shows highest expression in testes. Herein, we found that human and several other mammalian TGR genes lack any AUG codons that could function in translation initiation. Although mouse and rat TGRs have such codons, we detected protein sequences upstream of them by immunoblot assays and direct proteomic analyses. Further gene engineering and expression analyses demonstrated that a CUG codon, located upstream of the sequences previously thought to initiate translation, is the actual start codon in mouse TGR. The use of this codon relies on the Kozak consensus sequence and ribosome-scanning mechanism. However, CUG serves as an inefficient start codon that allows downstream initiation, thus generating two isoforms of the enzyme in vivo and in vitro. The use of CUG evolved in mammalian TGRs, and in some of these organisms, GUG is used instead. The newly discovered longer TGR form shows cytosolic localization in cultured cells and is expressed in spermatids in mouse testes. This study shows that CUG codon is used as an inefficient start codon to generate protein isoforms in mouse.