The Biological and Chemical Function of Selenium in Enzymes

描述（由申请人提供）：硒酶使用稀有氨基酸硒代半胱氨酸，即遗传密码中所谓的"第21"氨基酸。将硒代半胱氨酸（Sec）插入蛋白质中比其他20种氨基酸复杂得多，因为UGA终止密码子必须被重新编码为Sec的有义密码子，并且该过程需要复杂的细胞机制。任何解释，占在酶中使用的Sec必须解释为什么它是需要相对于使用更常用的半胱氨酸（Cys）残基，以证明维持精力昂贵的Sec插入机器。使用Sec的最常见的原因是它是一种"超级-Cys"残基，由于硒相对于硫的上级化学反应性，它可以"加速反应"。如果这是真的，那么我们可能会发现Sec的使用在自然界中广泛传播，而不是观察到的罕见。我们正在追求一个新的假设，解释生物压力，以维持UGA重新编码装置为SEC。这种生物压力是基于硒（相对于硫）赋予抗氧化性的上级化学性质，因此我们将其命名为酶中存在Sec的"化学-生物学"原理。SEC可以通过两种方式抗氧化，而Cys不能。首先，当Sec被氧化成亚硒酸（Sec-SeO2-）时，它可以相对容易地转化回母体形式（Sec-SeH），相比之下，Cys的氧化形式（Cys-SO2-）可以转化成其母体形式（Cys-SH）非常困难。其次，Sec-SeO2-进一步氧化为Sec-SeO3-要困难得多，而Cys-SO2-相对容易氧化为Cys-SO3-。我们相信这两个事实是未被认可的生物化学文献和我们的实验将解决的假设，即SEC只发生在酶时，酶需要非常耐氧化失活。换句话说，当酶中的Cys酶由于其活性位点Cys残基氧化成亚磺酸（Cys-SO2-）而失活时，Sec将取代该酶中的Cys。在这项研究中，这一主要假设将通过显示硒酶硫氧还蛋白还原酶和甲硫氨酸亚砜还原酶如何使用体外和体内实验抗氧化来解决。在硫氧还蛋白还原酶的情况下，我们将展示酶的模块化设计是如何使得它在自身内携带用于将Sec-SeO 2-残基还原回Sec-SeH的内部救援系统。硫氧还蛋白还原酶由于其在促进细胞增殖和调节细胞凋亡途径中的作用而成为抗癌药物的主要治疗靶点。甲硫氨酸氧化为甲硫氨酸亚砜被怀疑在神经退行性疾病如帕金森病和阿尔茨海默病中起主要作用，并且了解甲硫氨酸亚砜还原酶如何由于氧化而失活对于了解如何最好地使用抗氧化剂疗法来防止酶失活至关重要。该提案目标的成功完成将为人类和其他生物体对硒的营养需求提供普遍的化学基础。 公共卫生相关性：硒是一种必需的微量元素，因为它是需要纳入一套专门的酶-硒酶，使用罕见的氨基酸硒代半胱氨酸。本研究中的主要硒酶硫氧还蛋白还原酶是主要的癌症靶点，因为它在防止细胞凋亡（癌细胞必须避免）和促进细胞增殖中起作用。癌细胞必须快速分裂以引起发病机制，并且需要硫氧还蛋白还原酶的表达增加才能存活。

DESCRIPTION (provided by applicant): Selenoenzymes use the rare amino acid selenocysteine, the so-called "21st" amino acid in the genetic code. Insertion of selenocysteine (Sec) into a protein is much more complicated than the other 20 amino acids because a UGA stop codon must be recoded as a sense codon for Sec and this process requires complex cellular machinery. Any explanation that accounts for the use of Sec in an enzyme must explain why it is needed relative to the use of the more commonly used cysteine (Cys) residue in order to justify maintaining the energetically costly Sec-insertion machinery. The most frequently given reasons for the use of Sec is that it is a type of "super-Cys" residue that can "speed reactions" due to selenium's superior chemical reactivity relative to sulfur. If this were true, then we might expect to find the use of Sec widely spread throughout nature instead of its observed rarity. We are pursuing a new hypothesis that explains the biological pressure to maintain the UGA recoding apparatus for Sec. This biological pressure is based upon the superior chemical property of selenium (relative to sulfur) to confer resistance to oxidation and we thus name it the "chemico-biological" rationale for the presence of Sec in enzymes. Sec can resist oxidation in two ways that Cys cannot. First when Sec is oxidized to seleninic acid (Sec-SeO2-) it can be converted back to the parent form (Sec-SeH) with relative ease compared to the extreme difficulty that the oxidized form of Cys (Cys-SO2-) can be converted to its parent form (Cys-SH). Second, it is much more difficult for Sec-SeO2- to be further oxidized to Sec-SeO3-, while Cys-SO2- can be oxidized to Cys-SO3- relatively easily. We believe both of these facts are unrecognized in the biochemical literature and our experiments will address the hypothesis that Sec only occurs in an enzyme when the enzyme needs to be very resistant to inactivation by oxidation. In other words Sec will substitute for Cys in an enzyme when this Cys-enzyme would otherwise be inactivated due to oxidation of its active-site Cys residue to sulfinic acid (Cys-SO2-). This major hypothesis will be addressed in this study by showing how the selenoenzymes thioredoxin reductase and methionine sulfoxide reductase resist oxidation using both in vitro and in vivo experiments. In the case of thioredoxin reductase we will show how the modular design of the enzyme is such that it carries within itself its own internal rescue system for reducing the Sec- SeO2- residue back to Sec-SeH. Thioredoxin reductase is a major therapeutic target for anti-cancer drugs due to its role in enhancing cell proliferation and regulating cellular apoptotic pathways. Oxidation of methionine to methionine sulfoxide is suspected to play a major role in neurodegenerative disorders such as Parkinson's and Alzheimer's diseases, and understanding how methionine sulfoxide reductase may become inactivated due to oxidation is critical to understanding how antioxidant therapies can best be used to prevent inactivation of the enzyme. The successful completion of the goals of this proposal will provide a universal chemical basis for the nutritional requirement of selenium in humans and other organisms. PUBLIC HEALTH RELEVANCE: Selenium is an essential trace element because it is required for incorporation into a specialized set of enzymes - selenoenzymes that use the rare amino acid selenocysteine. The primary selenoenzyme in this study, thioredoxin reductase is a major cancer target due to its role in preventing apoptosis (which cancer cells must avoid) and promoting cell proliferation. Cancer cells must divide rapidly to cause pathogenesis and require increased expression of thioredoxin reductase to survive.