Structure - function analysis of Arabidopsis thaliana CC-type glutaredoxins ROXY1 and ROXY9

电子转移反应在所有有生命的生物体中起着至关重要的作用。例如，呼吸过程中氧气的减少是正在塑造我们星球的复杂生命进化的基础。尽管氧气对有氧代谢是必不可少的，但它也会导致生物分子不必要的氧化。这些都可以通过抗氧化系统的组成部分来减少。谷氧还蛋白(GRX)作为这一保护系统的重要组成部分，催化二硫键的还原。另外或替代地，它们中的一些配位铁-硫(FeS)簇，从而促进FeS簇的合成，转移到靶蛋白，氧化还原和铁的传感。在活性部位含有CP(Y/F)C或CGFS基序变体的GRX在所有类型的生物体中都存在。有趣的是，第三种类型的GRX-以CC(M/L)(C/S)基序为特征-只在陆地植物的基因组序列中发现。与CPYC和CGFS型GRX相比，这些陆地植物特有的CC型GRX的生化和生物物理特征描述得很差。一个标志是它们与TGACG结合蛋白(TGA)转录因子家族成员相互作用和调节的能力。这项提议提出了这样一个问题：CC型谷氧还蛋白是否仍然需要进化中较老的典型GRX的功能来调节TGA活性。我们重点研究了21个拟南芥谷氧还蛋白中的两个(ROXY1和ROXY9)，因为它们很可能通过不同的机制调节其同源TGA。ROXY1负调控TGA因子PAN，以确定花发育过程中正确的花瓣数量。ROXY9可以抑制调节生长和防御反应的冗余因子TGA1和TGA4的活性。关键氨基酸突变的蛋白质将从氧化还原状态、氧化还原酶活性、FeS簇结合能力和植物功能方面进行表征。这些分析将显示，ROXY1和ROXY9的体内活性需要哪些体外性质。

Electron transfer reactions play essential roles in all living organisms. The reduction of oxygen during respiration, for instance, is the basis for the evolution of the complex life that is shaping our planet. Although being essential for aerobic metabolism, oxygen also leads to unwanted oxidation of biomolecules. These can be reduced by components of the anti-oxidative system. Serving as essential components of this protective system, glutaredoxins (GRXs) catalyze the reduction of disulfide bonds. In addition or alternatively, some of them coordinate iron-sulfur (FeS) clusters and thus facilitate FeS cluster synthesis, transfer to target proteins and redox and iron sensing. GRXs containing variants of a CP(Y/F)C or a CGFS motif in the active site occur in all types of organisms. Intriguingly, a third type of GRXs - being characterized by a CC(M/L)(C/S) motif - is only found in the genome sequences of land plants. In contrast to CPYC- and CGFS-type GRXs, the biochemical and biophysical features of these land plant-specific CC-type GRXs are poorly described. A hallmark is their ability to interact with and regulate members of the TGACG-binding protein (TGA) family of transcription factors. This proposal asks the question whether CC-type glutaredoxins still require the functions of the evolutionary older canonical GRXs to regulate TGA activity. We focus on two of the 21 Arabidopsis thaliana glutaredoxins (ROXY1 and ROXY9), since they most likely regulate their cognate TGAs by different mechanisms. ROXY1 negatively regulates TGA factor PAN to determine the correct number of petals during flower development. ROXY9 can repress the activities of the redundant factors TGA1 and TGA4, which regulate growth and defense responses. Proteins with mutations in critical amino acids will be characterized with respect to their redox states, oxidoreductase activities, FeS cluster binding capacities and in planta functions. These analyses will show, which in vitro properties are required for the in vivo activities of ROXY1 and ROXY9.