The mechanism of selenium incorporation into selenocysteine in humans.

描述（由申请人提供）：硒是唯一一种基因编码的膳食微量营养素，对人类健康和生存至关重要。硒缺乏和硒蛋白基因突变导致多种病理，有强有力的证据表明硒在预防各种类型的癌症方面很重要。考虑到硒只存在于二十多种人体蛋白质中，这些效果就显得非常显著了。尽管硒发挥其作为硒半胱氨酸的生理作用，但只有少数研究旨在解释硒如何被纳入其主要代谢物并随后进入硒蛋白。此外，虽然原核生物模型系统中的生化研究已经很好地描述了这一过程中的事件顺序，但对真核生物，特别是人类的相同过程知之甚少。在这里，重要的和尚未探索的步骤硒纳入硒半胱氨酸的机制将在人体系统研究。特别是，第一和末端合成反应的机理将在结构水平上确定。一系列代表硒代半胱氨酸形成不同阶段的二元和三元配合物将通过生物物理和生化方法进行研究。硒代半胱氨酸在氨基酸中是独一无二的，不仅因为它含有一种必需的微量营养素，还因为它是在其tRNA上形成的。换句话说，虽然所有其他氨基酸的形成都独立于它们的tRNA，但硒代半胱氨酸是由氨基酸前体（丝氨酸）通过一系列反应合成的，这些反应需要高度特异性的酶和硒代半胱氨酸tRNA。在第一个反应中，丝氨酸-tRNA合成酶（SerRS）错误地将丝氨酸与硒代半胱氨酸tRNA配对，而在第二步中，硒代半胱氨酸-tRNA激酶（激酶）使丝氨酸磷酸化。在末端反应中，硒代半胱氨酸- trna合成酶（合酶）在需要硒代磷酸盐的反应中促进磷酸丝氨酸转化为硒代半胱氨酸。而磷酸硒则是人体内主要的硒供体，连接着硒半胱氨酸的合成和降解途径。从食物中摄取的硒或从降解的硒蛋白中提取的硒首先通过硒酶硒磷酸合成酶2 （SPS2）转化为硒化物，然后转化为硒磷酸。因此，SerRS活性可能调节初始反应底物的数量，而合成酶和SPS2可能调节硒插入硒半胱氨酸的效率。尽管对硒代谢，特别是硒代半胱氨酸合成具有明显的重要性，但对于人类SerRS、SPS2和合成酶如何催化各自的反应，它们如何选择反应底物以及它们的活性如何被调节，我们知之甚少。在这里，这些机制将在结构层面确定。该研究将为未来全细胞模型系统的研究奠定基础，在全细胞模型系统中，将研究临床相关硒蛋白合成的调控，并建立新疗法的潜力。

DESCRIPTION (provided by applicant): Selenium, the only genetically encoded dietary micronutrient, is essential for human health and survival. Selenium deficiency and mutations in selenoprotein genes lead to numerous pathologies and there is strong evidence that selenium is important in preventing various types of cancer. These effects are remarkable when one considers that selenium is found in only two dozens of human proteins. Although selenium exerts its physiological role as selenocysteine, only a handful of studies have been aimed at explaining how selenium is incorporated into its major metabolite and subsequently into selenoproteins. Also, while the sequence of events during this process has been well described by biochemical studies in prokaryotic model systems, very little is known about the same process in eukaryotes, in general, and in humans, in particular. Here, important and yet unexplored steps in the mechanism of selenium incorporation into selenocysteine will be studied on the human system. In particular, the mechanisms of the first and terminal synthetic reactions will be determined at the structural level. A series of binary and ternary complexes that represent distinct stages in selenocysteine formation will be studied by biophysical and biochemical methods. Selenocysteine is unique amongst amino acids not only because it contains an essential micronutrient, but also because it is formed on its tRNA. In other words, while all other amino acids are formed independent of their tRNAs, selenocysteine is synthesized from an amino-acid precursor (serine) in a series of reactions that require highly specific enzymes and selenocysteine tRNA. In the first reaction, seryl-tRNA synthetase (SerRS) 'erroneously' pairs serine with selenocysteine tRNA, whereas in the second step, selenocysteine-tRNA kinase (kinase) phosphorylates the seryl group. In the terminal reaction, selenocysteine-tRNA synthase (synthase) promotes the conversion of phosphoserine into selenocysteine in a reaction that requires selenophosphate. Selenophosphate, in turn, is the main selenium donor in humans that links the synthetic and degradation pathways of selenocysteine. Selenium that is either ingested with fod or extracted from degraded selenoproteins is converted first into selenide and then into selenophosphate by a selenoenzyme selenophosphate synthase 2 (SPS2). Thus, SerRS activity may regulate the amount of the initial reaction substrate, whereas both synthase and SPS2 may regulate how efficiently selenium is inserted into the amino acid selenocysteine. Despite the obvious importance for selenium metabolism, in general, and selenocysteine synthesis, in particular, very little is known about how human SerRS, SPS2 and synthase catalyze respective reactions, how they select their reaction substrates and how their activities are regulated. Here, these mechanisms wil be determined at the structural level. The proposed study wil serve as a foundation for future studies in whole cell model systems in which the regulation of the synthesis of the clinically relevant selenoproteins will be studied and the potential for novel therapies established.