Biosynthesis of Selenocysteine and Its Incorporation into Protein

硒是人类和其他哺乳动物饮食中必需的微量营养素，它对健康有许多好处，包括预防癌症、心脏病和其他心血管和肌肉疾病，抑制病毒表达，延缓艾滋病病毒阳性患者的进展，减缓衰老过程，并在哺乳动物发育、雄性繁殖和免疫功能中发挥作用。我们之前提出，硒的健康益处在很大程度上是由于硒蛋白中硒作为含硒氨基酸，硒半胱氨酸（Sec）的存在。由于对Sec是如何生物合成的知之甚少，我们承担了一个项目来阐明这种氨基酸（遗传密码中的第21个氨基酸）是如何合成的，并鉴定和表征该途径中涉及的每个组分。我们在真核生物和古细菌中建立了Sec的生物合成途径（PLoS Biology 5: 96-105, 2007），并继续对Sec合成的组分进行表征。在过去的一年里，我们已经确定了Sec生物合成途径中涉及的酶可以使用硫化物作为底物合成tRNA[Ser]Sec上的Cys。在硒蛋白翻译中，Cys- trna [Ser]Sec可用于解码码字UGA后插入Cys。在哺乳动物细胞硒缺乏的饮食条件下，这种将胱氨酸误合到蛋白质中的途径可能被使用。我们还建立了两个条件敲除小鼠模型，硒磷酸合成酶1 （SPS1）和SECp43敲除小鼠。两种小鼠系都表明，完全敲除相应的基因是胚胎致死的。我们发现SPS1敲除不影响硒蛋白代谢，而SECp43敲除仍在研究中。我们正在对SPS1基因敲除小鼠进行胚胎分析，以阐明SPS1在哺乳动物胚胎发育中的功能。我们还在哺乳动物细胞中发现了一个可能参与tRNA[Ser]Sec修饰的新因子，该修饰可显著增加丝氨酸与tRNA[Ser]Sec的seryl-tRNA合成酶附着。在酵母seryl-tRNA合成酶中没有发现该因子的活性，有趣的是，酵母不编码硒代半胱氨酸的生物合成机制。我们正在纯化该因子，以表征其在修饰tRNA[Ser]Sec中的作用。

Selenium is an essential micronutrient in the diet of humans and other mammals and it has many health benefits have been ascribed to this element including preventing cancer, heart disease and other cardiovascular and muscle disorders, inhibiting viral expression, delaying the progression of AIDS in HIV positive patients, slowing the aging process, and having roles in mammalian development, male reproduction and immune function. We proposed previously that the health benefits of selenium are due in large part to the presence of selenium in selenoproteins as the selenium-containing amino acid, selenocysteine (Sec). Since little was known about how Sec was biosynthesized, we undertook a project to elucidate how this amino acid, which is the 21st amino acid in the genetic code, was synthesized and to identify and characterize each of the components involved in the pathway. We established the biosynthetic pathway of Sec in eukaryotes and archeae (PLoS Biology 5: 96-105, 2007) and are continuing our characterization of the components responsible for Sec synthesis. During the past year, we have identified that the enzymes involved in the biosynthetic pathway of Sec can be used to synthesize Cys on the tRNA[Ser]Sec using sulfide as a substrate. The Cys-tRNA[Ser]Sec can be used to insert Cys upon decoding the codeword, UGA, in selenoprotein translation. This pathway of misincorporating Cys into protein is likely used under dietary conditions of selenium deficiency in mammalian cells. We have also generated two conditional knock-out mouse models, selenophosphate synthetase 1 (SPS1) and SECp43 knockout mice. Both mouse lines have shown that the total knockout of the corresponding gene is embryonic lethal. We have found that the SPS1 liver knockout did not affect selenoprotein metabolism while the SECp43 liver knockout is still being investigated. We are conducting embryo analysis of SPS1 knockout mice to elucidate the function of SPS1 in mammalian embryo development. We also found a new factor in mammalian cells which may be involved in tRNA[Ser]Sec modification, and this modification can dramatically increase seryl-tRNA synthetase attachment of serine to tRNA[Ser]Sec. The activity of this factor was not found to be present in yeast seryl-tRNA synthetase, and interestingly, yeast do not encode the selenocysteine biosynthesis machinery. We are purifying this factor to characterize its role in modifying tRNA[Ser]Sec.