Functional Studies of Cytosolic Antioxidant Proteins

描述（由申请人提供）：我们寻求了解真核细胞线粒体中抗氧化保护的分子机制。特别是，我们计划探索线粒体抗氧化酶、氧化应激和金属代谢之间的关系，并开发新的工具来可视化体内细胞状态。我们的研究将无机化学、光谱学和其他生物学方法与分子生物学、酵母遗传学和其他生物学方法相结合，详细阐明活细胞中氧化还原平衡、氧化应激和金属离子代谢之间的化学关系。我们将继续以出芽酵母酿酒酵母（Saccharomyces cerevisiae）为模型系统进行体内研究，同时利用分离的抗氧化蛋白开展相关的生化和生物物理研究。在这项提议中，我们遵循几个主要的研究路线，重点了解居住在线粒体膜间空间的蛋白质的详细机制和生物学作用-铜锌超氧化物歧化酶（Sod1p）， CCS （Sod1p的铜伴侣）和细胞色素c过氧化物酶。首先，我们提出了一系列的生物学和遗传学实验，旨在探索这些蛋白质在保护线粒体基质（以及膜间空间）免受氧化损伤中的作用。包括代谢改变的研究，这些研究对缺乏Sod1p的突变体有益，涉及第四种IMS蛋白乳酸：细胞色素c氧化还原酶。此外，我们解决了为什么基质酶锰超氧化物歧化酶（MnSOD）在高超氧化物通量条件下不能完全保护线粒体基质的问题。其次，我们研究了允许离子锰在功能上替代酵母中缺乏这种蛋白质的Sod1p的化学作用，包括细胞色素c过氧化物酶的潜在作用。最后，我们对CCS活化Sod1的机理提出并验证了一个新的假设，即CCS的本质活性是在Sod1p中形成二硫键，而不是通常认为的铜的插入。在后面的这些实验中，我们将详细描述二硫键在Sod1p中的作用。总之，这些研究将揭示所有真核生物用来保护线粒体免受超氧化物介导损伤的小分子和蛋白质的复杂相互作用。

DESCRIPTION (provided by applicant): We seek to understand the molecular mechanisms of antioxidant protection in the mitochondria of eukaryotic cells. In particular, we plan to explore the relationships between the mitochondrial antioxidant enzymes, oxidative stress, and metal metabolism and to develop new tools to visualize cellular status in vivo. Our studies integrate the tools of inorganic chemistry, spectroscopy, and other biological methods with those of molecular biology, yeast genetics, and other biological methodology to elucidate in detail the chemical relationships between redox balance, oxidative stress, and metal ion metabolism in living cells. We will continue to use the budding yeast Saccharomyces cerevisiae as a model system for in vivo studies, and, in parallel, carry out related biochemical and biophysical studies using isolated antioxidant proteins. In this proposal, we follow several major lines of investigation focused on understanding the detailed mechanisms and the biological roles of proteins residing in the mitochondrial intermembrane space--copper- zinc superoxide dismutase (Sod1p), CCS (copper chaperone for Sod1p), and cytochrome c peroxidase. First, we propose a series of biological and genetic experiments designed to explore the roles of these proteins in protecting the mitochondrial matrix (as well as the intermembrane space) from oxidative damage. Included are studies on metabolic alterations that are beneficial for mutants that lack Sod1p involving a fourth IMS protein, lactate:cytochrome c oxidoreductase. In addition, we address the question of why the matrix enzyme manganese superoxide dismutase (MnSOD) does not fully protect the mitochondrial matrix under conditions of high superoxide flux. Second, we study the chemistry that allows ionic manganese functionally to substitute for Sod1p in yeast lacking this protein, including a potential role for cytochrome c peroxidase. Finally, we put forth and test a new hypothesis on the mechanism by which CCS activates Sod1, i.e., that the essential activity of CCS is formation of the disulfide bond in Sod1p, rather than insertion of copper as is commonly thought. In the course of these latter experiments, we will do a detailed characterization of the role of the disulfide bond in Sod1p. Together, these studies will shed light on the complex interactions of small molecules and proteins that all eukaryotes use to protect their mitochondria from superoxide-mediated damage.