Functional Studies of Cytosolic Antioxidant Proteins

描述(申请人提供)：我们试图了解真核细胞线粒体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的细胞状态。我们的研究结合了无机化学、光谱学和其他生物学方法，以及分子生物学、酵母遗传学和其他生物学方法，详细阐明了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。我们将继续使用发芽酵母作为体内研究的模型系统，并同时使用分离的抗氧化蛋白进行相关的生化和生物物理研究。在这个建议中，我们遵循几条主要的研究路线，重点了解线粒体膜间间隙中蛋白质的详细机制和生物学作用--铜锌超氧化物歧化酶(Sod1p)、铜伴侣(Sod1p)和细胞色素c过氧化物酶。首先，我们提出了一系列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(以及膜间隙)免受氧化损伤中的作用。包括对代谢改变的研究，这些研究对缺乏Sod1p的突变体有利，涉及第四种IMS蛋白，乳酸：细胞色素c氧化还原酶。此外，我们还讨论了为什么在高超氧通量条件下，基质酶锰超氧化物歧化酶(MnSOD)不能完全保护线粒体基质的问题。其次，我们研究了在缺乏这种蛋白的酵母中允许离子锰在功能上取代Sod1p的化学，包括细胞色素C过氧化物酶的潜在作用。最后，我们提出并验证了一个关于CCS激活SOD1机理的新假说，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。在后面的实验过程中，我们将详细描述二硫键在Sod1P中的作用。总之，这些研究将阐明小分子和蛋白质之间的复杂相互作用，所有真核生物都利用这些相互作用来保护它们的线粒体免受超氧化物介导的损害。

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.