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， 有许多健康的好处已被归因于这种元素，包括预防癌症，心脏病， 疾病和其他心血管和肌肉疾病，抑制病毒表达，延缓 艾滋病在艾滋病毒阳性患者中的发展，减缓衰老过程， 在哺乳动物的发育、雄性生殖和免疫功能中。我们先前提出， 硒的健康益处在很大程度上是由于 硒蛋白作为含硒氨基酸，硒代半胱氨酸（Sec）， 我们知道Sec是如何生物合成的，我们进行了一个项目来阐明这种氨基是如何合成的。 氨基酸，这是遗传密码中的第21个氨基酸，被合成并鉴定， 表征其途径中涉及的每个组分。我们最近建立了 真核生物和古菌中Sec的生物合成途径，现在正在表征 负责其合成的成分。在过去的一年里，我们与 Markus Wahl和Vadim Gladyshev结晶了硒代半胱氨酸（Sec）合酶（SecS）， 是合成Sec on Sec tRNA的酶我们在1.65埃处结晶了SecS 分辨率，有趣的是，两个SecS单体相互作用，在周围产生两个活性位点， 磷酸吡哆醛是活性所需的辅因子。这两个二聚体又进一步 缔合以形成同源四聚体。通过测量SecS对基于机制的 抑制剂，并从结晶研究，我们能够确定一个详细的 SecS的反应机理此外，我们还产生了一种条件性基因敲除小鼠， 靶向去除SECp 43，SECp 43是一种参与Sec tRNA上Um 34甲基化的蛋白质。 这种甲基化酶负责Sec tRNA成熟的最后一步， 在我们的实验室中，已经证明了Sec tRNA的成熟同种型负责 参与胁迫相关现象的硒蛋白亚类的表达 哺乳动物，被称为应激相关硒蛋白。因此，这一条件 敲除小鼠应该阐明Secp 43在甲基化过程中的作用，并帮助我们 进一步阐明我们目前正在表征的Um 34甲基化酶。初始 条件性Secp 43敲除小鼠能够表达Secp 43的第二种同种型， 因此，该蛋白没有被完全消除，并且不能在该小鼠中进行研究。我们有 产生了一种新的条件性敲除Secp 43小鼠，目前正在表征和 阐明Secp 43的靶向去除对Um 34甲基化的作用。而且我们 有证据表明，第二种途径的生物合成的Sec可能存在于哺乳动物。到 为了研究这种可能性，我们产生了Sec合酶条件性敲除小鼠， 也是这只老鼠的特征Sec合酶是负责 在磷酸丝氨酰-tRNA [Sec]上合成Sec以产生硒代半胱氨酸-tRNA [Sec]， 这是哺乳动物细胞中唯一已知的合成Sec的方法。

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), and 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 its pathway. We recently established the biosynthetic pathway of Sec in eukaryotes and archeae and are now characterizing the components responsible for its synthesis. During the past year, we have, in collaboration with Markus Wahl and Vadim Gladyshev, crystallized the selenocysteine (Sec) synthase (SecS) which is the enzyme that synthesizes Sec on Sec tRNA. We crystallized SecS at 1.65 angstroms resolution, and interestingly, two SecS monomers interact to generate two active sites around pyridoxal phosphate which is a required cofactor for activity. The two dimers in turn further associate to form a homotetramer. By measuring the activity of SecS towards mechanism-based inhibitors, and from the crystallization studies, we were able to ascertain a detailed reaction mechanism for SecS. In addition, we generated a conditional knockout mouse that targeted the removal of SECp43, a protein involved in the methylation of Um34 on Sec tRNA. This methylase is responsible for the final step in the maturation of Sec tRNA and the resulting mature isoform of Sec tRNA has been shown in our laboratory to be responsible for the expression of the subclass of selenoproteins involved in stress-related phenomena in mammals which are designated as stress-related selenoproteins. Therefore, this conditional knockout mouse should elucidate the role of Secp43 in the methylation process and help us further elucidate the Um34 methylase that we are currently characterizing. The initial conditional Secp43 knockout mouse was capable of expressing a second isoform of Secp43 and thus this protein was not fully abolished and could not be studied in this mouse. We have generated a new conditional knockout Secp43 mouse and are currently characterizing and elucidating the role of the targeted removal of Secp43 on Um34 methylation. Furthermore, we have evidence that a second pathway for the biosynthesis of Sec may be present in mammals. To investigate this possibility, we generated a Sec synthase conditional knockout mouse and are also currently characterizing this mouse. Sec synthase is the enzyme responsible for the synthesis of Sec on phosphoseryl-tRNA[Sec] to yield selenocysteyl-tRNA[Sec] which is presently the only known means of synthesizing Sec in mammalian cells.