Ottaviano Filomena G, Tang Shiow-Shih, Handy Diane E, Loscalzo Joseph
Whitaker Cardiovascular Institute and Evans Department of Medicine, Boston University School of Medicine, Boston, MA, USA.
Mol Cell Biochem. 2009 Jul;327(1-2):111-26. doi: 10.1007/s11010-009-0049-x. Epub 2009 Feb 15.
Plasma glutathione peroxidase (GPx-3) is a selenocysteine-containing extracellular antioxidant protein that catalyzes the reduction of hydrogen peroxide and lipid hydroperoxides. Selenoprotein expression involves the alternate recognition of a UGA codon as a selenocysteine codon and requires signals in the 3'-untranslated region (UTR), including a selenocysteine insertion sequence (SECIS), as well as specific translational cofactors. To ascertain regulatory determinants of GPx-3 expression and function, we generated recombinant GPx-3 (rGPX-3) constructs with various 3'-UTR, as well as a Sec73Cys mutant. In transfected Cos7 cells, the Sec73Cys mutant was expressed at higher levels than the wild type rGPx-3, although the wild type rGPx-3 had higher specific activity, similar to plasma purified GPx-3. A 3'-UTR with only the SECIS was insufficient for wild type rGPx-3 protein expression. Selenocompound supplementation and co-transfection with SECIS binding protein 2 increased wild type rGPx-3 expression. These results demonstrate the importance of translational mechanisms in GPx-3 expression.
血浆谷胱甘肽过氧化物酶（GPx-3）是一种含硒代半胱氨酸的细胞外抗氧化蛋白，可催化过氧化氢和脂质氢过氧化物的还原。硒蛋白的表达涉及将UGA密码子交替识别为硒代半胱氨酸密码子，并且需要3'-非翻译区（UTR）中的信号，包括硒代半胱氨酸插入序列（SECIS）以及特定的翻译辅助因子。为了确定GPx-3表达和功能的调控决定因素，我们构建了具有不同3'-UTR的重组GPx-3（rGPX-3）构建体以及一个Sec73Cys突变体。在转染的Cos7细胞中，Sec73Cys突变体的表达水平高于野生型rGPx-3，尽管野生型rGPx-3具有更高的比活性，类似于血浆纯化的GPx-3。仅具有SECIS的3'-UTR不足以实现野生型rGPx-3蛋白的表达。补充硒化合物并与SECIS结合蛋白2共转染可增加野生型rGPx-3的表达。这些结果证明了翻译机制在GPx-3表达中的重要性。
