Analysis of the mechanism of activation and co-translational insertion of an essential trace element, selenium, into polypeptide

硒是一种人体必需的微量元素，以硒代半胱氨酸的形式存在于硒蛋白的特定位置，在生物体内发挥着多种重要作用。本研究对硒代谢相关蛋白的功能进行了研究。1.硒代半胱氨酸裂解酶（Selenocysteine lyase，SCL）催化L-硒代半胱氨酸分解为L-丙氨酸和硒。我们采用酵母双杂交系统，以小鼠SCL为诱饵，寻找与SCL相互作用的蛋白质。我们确定主要尿蛋白（MUP）作为SCL的潜在伙伴。结果表明，SCL抑制了MUP与2-萘酚的相互作用。2.小鼠SCL基因在拟南芥细胞质或叶绿体中表达。3.采用硒定量和定量RT-PCR方法分别测定了硒和硒蛋白mRNA在小鼠脑中的分布; 4.将针对SCL的siRNA转染人HeLa细胞和小鼠A1细胞，以敲低SCL的表达。我们发现针对SCL的siRNA降低了两种细胞中硒蛋白的表达。结果表明，SCL参与硒蛋白的生物合成。5.对大鼠SCL进行了结晶，并解析了其三维结构。结构显示SCL在延伸的叶上具有催化必需的半胱氨酸残基。

Selenium, an essential trace element, is incorporated into a specific position of selenoprotein in the form of selenocysteine residues and plays a various important roles in organisms. In this study, we investigated the function of proteins involved in the metabolism of selenium.1.Selenocysteine lyase (SCL) catalyzes the decomposition of L-selenocysteine into L-alanine and selenium. We employed the yeast two-hybrid system using mouse SCL as a bait to search for a protein(s) that interacts with SCL. We identified major urinary protein (MUP) as a potential partner of SCL. Our analysis revealed that SCL inhibits the interaction between MUP and 2-naphtol in vitro.2.The mouse SCL gene was expressed in the cytosol or chloroplast of Arabidopsis thaliana. The selenium-tolerance of the plant was greatly improved, indicating that SCL is useful for phyroremediation.3.The distribution of selenium and selenoprotein mRNAs in moue brain were determined by quantification of selenium and quantitative RT-PCR, respectively.4.siRNAs targeted to SCL were transfected with human HeLa cells and mouse Al cells to knock-down the expression of SCL. We found that siRNAs against SCL decreases the expression of selenoproteins in both cells. The results suggest that SCL is involved in the biosynthesis of selenoprotein.5.Rat SCL was crystallized and its three-dimensional structure was solved. The structure revealed that SCL has a catalytically essential cysteine residue on a extended lobe.