Functions of Mammalian Thioredoxin Reductases

描述（由申请人提供）：硫氧还蛋白系统是一种主要的细胞氧化还原系统，存在于所有先前表征的生物体中。硫氧还蛋白还原酶控制硫氧还蛋白的氧化还原状态，从而调节许多细胞过程。硫氧还蛋白还原酶也与其他细胞蛋白和化合物的氧化还原控制有关。哺乳动物硫氧还蛋白还原酶的功能和催化活性依赖于硒半胱氨酸残基，而硒半胱氨酸残基的生物合成又受膳食硒含量的影响。哺乳动物含有三种硒蛋白硫氧还蛋白还原酶同工酶，TR1、TR3和TGR，由于选择性剪接，它们以多种形式出现。本研究的目的是研究哺乳动物硫氧还蛋白还原酶的功能特征，重点研究同工酶及其替代形式在细胞氧化还原调节过程中的具体作用。将使用生物化学、细胞生物学和小鼠模型方法的结合来解决以下具体问题（具体目标）：(i) TGR在男性生殖中的具体作用是什么？我们将测试一个假设，即这种酶能使蛋白质中的二硫键异构化。此外，将开发一个敲除小鼠模型来直接测试这种蛋白质对精子成熟的需求，（ii）硫氧还蛋白还原酶的特定异构体的作用是什么？我们将描述线粒体和细胞质形式TR3的作用，并确定所有三种硫氧还蛋白还原酶的目标。我们还将验证一个假设，即TGR从非规范起始密码子产生线粒体形式。（iii）硒优先供应硫氧还蛋白还原酶的决定因素是什么？我们将检查硫氧还蛋白还原酶基因中负责这种作用的元素。

DESCRIPTION (provided by applicant): The thioredoxin system is a major cellular redox system that is present in all previously characterized organisms. Thioredoxin reductases control the redox state of thioredoxins, which in turn regulate numerous cellular processes. Thioredoxin reductases have also been implicated in the redox control of other cellular proteins and compounds. The function and catalytic activity of mammalian thioredoxin reductases is dependent on a selenocysteine residue, whose biosynthesis in turn is affected by the availability of dietary selenium. Mammals contain three selenoprotein thioredoxin reductase isozymes, TR1, TR3 and TGR, which occur in multiple forms due to alternative splicing. The purpose of the proposed study is to functionally characterize mammalian thioredoxin reductases, with emphasis on the specific roles of isozymes and their alternative forms in redox regulation of cellular processes. A combination of biochemical, cell biology and mouse model approaches will be used to address the following specific questions (specific aims): (i) what is the specific role of TGR in male reproduction? We will test a hypothesis that this enzyme isomerizes disulfide bonds in proteins. In addition, a knockout mouse model will be developed to directly test the requirement of this protein for sperm maturation, (ii) what are the roles of specific isoforms of thioredoxin reductases? We will characterize the roles of mitochondrial and cytosolic forms of TR3 and identify targets of all three thioredoxin reductases. We will also test a hypothesis that TGR generates a mitochondrial form from a non-canonical initiation codon. (iii) What are the determinants for preferential supply of thioredoxin reductases with selenium? We will examine elements in thioredoxin reductase genes responsible for this effect.