Functional Characterization of Arabidopsis thaliana DCX E3 ligase activities

Cullins是真核生物E3连接酶的中心支架亚基，在泛素蛋白酶体途径中促进靶蛋白的泛素化。在初步工作中，我们已经证明了潜在的拟南芥DCX E3连接酶的复杂组装，该连接酶由CULLIN 4 （AtCUL4）、dna损伤结合1a （AtDDB1a）蛋白和RING-finger蛋白AtRBX1组成。此外，我们还可以证明AtDDB1a与潜在底物蛋白AtDDB2,1的相互作用。在动物和人类中，dcx复合物和DDB2在发育和紫外线依赖性DNA修复中起着关键作用。尽管对这些蛋白的功能和生物学作用知之甚少，但对AtCUL4突变体植物的初步分析表明，cullin对整个植物的发育非常重要。本项目的目标是解释DCX-E3连接酶在拟南芥中的功能，这需要完成五个关键的工作要点：1)建立AtCUL4、AtDDB1a和AtDDB2的相互作用伙伴蛋白，1,2)详细分析dcx亚基和选定底物的表达，3)研究亚细胞蛋白定位及其动力学，4)证明E3泛素连接酶活性和触发这些E3连接酶活性的调控机制，5)表征单和多突变表型，重点研究发育和应激反应的变化。

Cullins are central scaffolding subunits in eukaryotic E3 ligases that facilitate ubiquitination of target proteins within the ubiquitin proteasome pathway. In preliminary works we have demonstrated complex assembly of a potential Arabidopsis thaliana DCX E3 ligase that consists of the CULLIN 4 (AtCUL4), the DNA-Damaged Binding 1a (AtDDB1a) protein and the RING-finger protein AtRBX1. In addition we could also demonstrate interaction of AtDDB1a with AtDDB2,1, a potential substrate protein. In animals and human, the DCX-complex and DDB2 play critical roles in development and UV-dependent DNA repair. Although very little is known about the function and biological roles of these proteins preliminary analysis in plants of AtCUL4 mutants show that the cullin is highly important for whole plant development. The goal of this project is to explain DCX-E3 ligase function in Arabidopsis, and this shall be achieved by accomplishing five crucial work points: 1) Establishing interacting partner proteins of AtCUL4, AtDDB1a, and AtDDB2,1, 2) Detailed expression analysis of DCX-subunits and selected substrates, 3) Studying sub cellular protein localization and their dynamics, 4) Demonstrating E3 ubiquitin ligase activity and regulatory mechanisms triggering these E3 ligase activities, and 5) Characterization of single and multiple mutant phenotypes with a focus on development and changed stress response.