The mechanism of selenium incorporation into selenocysteine in humans.

说明(申请人提供)：硒是唯一的基因编码的饮食微量营养素，对人类健康和生存是必不可少的。硒缺乏和硒蛋白基因突变导致多种病理变化，有强有力的证据表明，硒在预防各种类型的癌症中具有重要作用。当人们考虑到硒只存在于24种人类蛋白质中时，这些效应是显著的。尽管硒作为硒半胱氨酸发挥其生理作用，但只有少数研究旨在解释硒是如何结合到其主要代谢物中并随后进入硒蛋白中的。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，但对于真核生物中的相同过程，一般情况下，特别是在人类中，人们知之甚少。在这里，我们将在人体系统上研究硒结合到硒半胱氨酸中的机制中的重要和尚未探索的步骤。特别是，第一次合成反应和最终合成反应的机理将在结构水平上确定。我们将用生物物理和生物化学方法研究一系列代表硒半胱氨酸形成的不同阶段的二元和三元络合物。硒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都是独立于其tRNA形成的，但硒半胱氨酸是由氨基酸前体(丝氨酸)在一系列反应中合成的，这些反应需要高度特定的酶和硒半胱氨酸tRNA。在第一步反应中，丝氨酸-tRNA合成酶(SerRS)“错误地”将丝氨酸与硒半胱氨酸tRNA配对，而在第二步中，硒半胱氨酸-tRNA激酶(KK)将丝氨酸基磷酸化。在末端反应中，硒半胱氨酸-tRNA合成酶(合酶)在需要硒磷酸的反应中促进磷酸丝氨酸转化为硒半胱氨酸。反过来，硒磷酸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。被FOD摄取或从降解的硒蛋白中提取的硒首先被硒酶硒磷酸盐合成酶2(SPS2)转化为亚硒化物，然后再转化为硒磷酸盐。因此，SerRS的活性可能调节初始反应底物的数量，而合成酶和SPS2可能调节硒插入氨基酸硒半胱氨酸的效率。尽管总体上对硒代谢和硒半胱氨酸的合成具有明显的重要性，但人们对人类SerRs、SPS2和合成酶如何催化各自的反应、如何选择它们的反应底物以及它们的活性是如何调节的知之甚少。在这里，这些机制将在结构层面上确定。这项拟议的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节，并建立新疗法的可能性。

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.