Carlson Bradley A, Moustafa Mohamed E, Sengupta Aniruddha, Schweizer Ulrich, Shrimali Rajeev, Rao Mahadev, Zhong Nianxin, Wang Shulin, Feigenbaum Lionel, Lee Byeong Jae, Gladyshev Vadim N, Hatfield Dolph L
Molecular Biology of Selenium Section, Laboratory of Cancer Prevention, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
J Biol Chem. 2007 Nov 9;282(45):32591-602. doi: 10.1074/jbc.M707036200. Epub 2007 Sep 11.
Novel mouse models were developed in which the hepatic selenoprotein population was targeted for removal by disrupting the selenocysteine (Sec) tRNA([Ser]Sec) gene (trsp), and selenoprotein expression was then restored by introducing wild type or mutant trsp transgenes. The selenoprotein population was partially replaced in liver with mutant transgenes encoding mutations at either position 34 (34T-->A) or 37 (37A-->G) in tRNA([Ser]Sec). The A34 transgene product lacked the highly modified 5-methoxycarbonylmethyl-2'-O-methyluridine, and its mutant base A was converted to I34. The G37 transgene product lacked the highly modified N(6)-isopentenyladenosine. Both mutant tRNAs lacked the 2'-methylribose at position 34 (Um34), and both supported expression of housekeeping selenoproteins (e.g. thioredoxin reductase 1) in liver but not stress-related proteins (e.g. glutathione peroxidase 1). Thus, Um34 is responsible for synthesis of a select group of selenoproteins rather than the entire selenoprotein population. The ICA anticodon in the A34 mutant tRNA decoded Cys codons, UGU and UGC, as well as the Sec codon, UGA. However, metabolic labeling of A34 transgenic mice with (75)Se revealed that selenoproteins incorporated the label from the A34 mutant tRNA, whereas other proteins did not. These results suggest that the A34 mutant tRNA did not randomly insert Sec in place of Cys, but specifically targeted selected selenoproteins. High copy numbers of A34 transgene, but not G37 transgene, were not tolerated in the absence of wild type trsp, further suggesting insertion of Sec in place of Cys in selenoproteins.
构建了新型小鼠模型，通过破坏硒代半胱氨酸（Sec）tRNA[Ser]Sec基因（trsp）来靶向去除肝脏中的硒蛋白群体，然后通过导入野生型或突变型trsp转基因来恢复硒蛋白表达。在肝脏中，硒蛋白群体被部分替换为在tRNA[Ser]Sec的第34位（34T→A）或第37位（37A→G）编码突变的突变转基因。A34转基因产物缺乏高度修饰的5-甲氧基羰基甲基-2'-O-甲基尿苷，其突变碱基A被转换为I34。G37转基因产物缺乏高度修饰的N(6)-异戊烯基腺苷。两种突变tRNA在第34位（Um34）均缺乏2'-甲基核糖，并且两者都支持肝脏中管家硒蛋白（如硫氧还蛋白还原酶1）的表达，但不支持应激相关蛋白（如谷胱甘肽过氧化物酶1）的表达。因此，Um34负责一组特定硒蛋白的合成，而非整个硒蛋白群体的合成。A34突变tRNA中的ICA反密码子将半胱氨酸密码子UGU和UGC以及Sec密码子UGA解码。然而，用（75）Se对A34转基因小鼠进行代谢标记显示，硒蛋白从A34突变tRNA中掺入标记，而其他蛋白则不然。这些结果表明，A34突变tRNA并非随机地将Sec插入半胱氨酸位置，而是特异性地靶向选定的硒蛋白。在没有野生型trsp的情况下，A34转基因的高拷贝数不被耐受，而G37转基因则不然，这进一步表明在硒蛋白中Sec取代了半胱氨酸。
