酵母中新发现的Apd1p铁氧还蛋白及其与Tsa1p抗氧化酶的相互作用的研究

过氧化物氧还蛋白(Prx)是一类对细胞有重要保护功能的抗氧化酶，在抵抗活性氧中间体(ROS)、传递氧化还原信号及维持基因组稳定性方面具有重要作用。小鼠缺失Prx后，基因突变率升高并易患肿瘤。2009年我们发现由一种主要的Prx,TSA1,缺失引起的基因突变率升高的根本原因在于细胞内dNTP过量产生以及DNA损伤关卡的持续激活。随后我们又发现一种新的、可与Tsa1p 结合并具有硫氧还蛋白结构域的铁氧还蛋白Apd1p，其缺失将导致细胞对羟脲敏感。本课题将在此基础上对Apd1p的生物学功能及其与Tsa1p 的相互作用进行深入研究。我们将研究Apd1p的生物化学和生物学特性及其与Tsa1p相互作用的分子基础及生物学意义，并确定Apd1p可否向Tsa1p传递电子。最后，我们将阐明APD1缺失导致基因突变率升高的原因。我们的工作将为Prx、Apd1p及相关氧还调节蛋白结构与功能的研究奠定重要基础。

Reactive oxygen species (ROS) is one of the life-threatening challenges that continuously damage cellular components, thus many antioxidant proteins have been evolved to combat the deleterious effect of ROS. Peroxiredoxins are a family of antioxidant enzymes that are critically involved in cellular defense, redox signaling and maintenance of genome stability. Loss of peroxiredoxin in mice can lead to accumulation of genetic instability and malignant tumors. In 2009, I have uncovered a new role of TSA1, a predominant peroxiredoxin in yeast, in preventing overproduction of dNTPs, which is a root cause of genome instability. Importantly, we have provided the first evidence that the genome instability in tsa1-null cells was tightly associated with constitutive activation of the DNA damage checkpoint as indicated by phosphorylation of Rad9/Rad53p, reduced steady-state amount of Sml1p, and induction of RNR and HUG1 genes. Recently, we have identified 1) Apd1p as a novel binding partner of yeast peroxiredoxin Tsa1p, 2) a synergistic genetic interaction of APD1 and TSA1 in cellular sensitivity to hydroxyurea (HU), a dNTP depleting drug. In this project, we seek to continue our study on the roles and regulation of yeast peroxiredoxins in two areas. First, we will characterize Apd1p and its interaction with Tsa1p. Specifically, we will define the molecular basis of this interaction, characterize the function of Apd1p in antioxidant defense, and investigate whether Apd1p serves as an electron donor for Tsa1p. Second, we will dissect the mechanism of genome instability in apd1? and tsa1?apd1? yeast cells. In particular, we will assess cellular sensitivity to DNA-damaging agents, and determine the competence of DNA checkpoint and RNR machinery. We will also characterize the biochemical properties and biological role of Apd1p. Our studies will derive a better understanding of peroxiredoxins and its redox regulators in oxidative defense and DNA damage signaling, which may have an impact on drug discovery.