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半胱氨酸，即遗传密码中的“第21”氨基酸。硒代半胱氨酸（Sec）插入蛋白质比其他20种氨基酸要复杂得多，因为硒代半胱氨酸终止密码子必须被重新编码为Sec的意义密码子，而这一过程需要复杂的细胞机制。任何解释在酶中使用Sec的解释都必须解释为什么需要它相对于使用更常用的半胱氨酸（Cys）残基，以证明维持能量昂贵的Sec插入机制是合理的。使用硒的最常见的原因是，它是一种“超级cys”残留物，由于硒相对于硫具有优越的化学反应活性，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。我们正在寻求一个新的假说来解释维持Sec的UGA编码装置的生物压力。这种生物压力是基于硒的优越化学特性（相对于硫），赋予其抗氧化性，因此我们将其命名为Sec存在于酶中的“化学-生物学”原理。Sec能以两种方式抗氧化，而Cys不能。首先，当Sec被氧化为硒酸（Sec- seo2 -）时，它可以相对容易地转化回母体形式（Sec- seh），而Cys的氧化形式（Cys- so2 -）则很难转化为母体形式（Cys- sh）。其次，Sec-SeO2-被进一步氧化为Sec-SeO3-的难度要大得多，而Cys-SO2-则相对容易被氧化为Cys-SO3-。我们相信这两个事实在生化文献中都没有被认识到，我们的实验将解决一个假设，即Sec只发生在酶需要非常抵抗氧化失活的酶中。换句话说，当该酶由于活性位点的Cys残基氧化为亚硫酸（Cys- so2 -）而失去活性时，Sec将替代酶中的Cys。这一主要假设将在本研究中通过展示硒酶硫氧还蛋白还原酶和蛋氨酸亚砜还原酶如何通过体外和体内实验抵抗氧化来解决。在硫氧还蛋白还原酶的案例中，我们将展示这种酶的模块化设计是如何使它自己的内部修复系统将Sec- SeO2-残基还原为Sec- seh。硫氧还蛋白还原酶具有促进细胞增殖和调节细胞凋亡通路的作用，是抗癌药物的主要治疗靶点。蛋氨酸氧化为蛋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，了解蛋氨酸亚砜还原酶如何因氧化而失活对于了解如何最好地使用抗氧化疗法来防止酶失活至关重要。本提案目标的成功完成将为人类和其他生物对硒的营养需求提供一个普遍的化学基础。

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.