New insights into the functions and regulation of the APC/C ubiquitin ligase

后期促进复合体/环体(APC/C)是一种大分子E3泛素连接酶，通过靶向蛋白质底物的多泛素化和26S蛋白酶体的降解，通过有丝分裂和细胞周期连续的G1期来协调细胞的进程。在E2-泛素结合酶UbcH10和UBE2S的存在下，APC/C E3连接酶的活性可以通过两个相关激活蛋白之一CDC20或CDH1与含有四肽重复序列(TPR)的APC/C亚基(如APC3)的时间协调募集来刺激；CDC20和CDH1也与特定的APC/C亚基结合以结合底物。我们实验室以前的工作已经确定，APC/C-CDc20和APC/C-CDH1的泛素连接酶活性也受到转录共激活子CBP和P300的调节，它们通过腺病毒E1a中保守的相互作用结构域与APC/C亚基APC5和APC7特异性结合。我们还确定了DNA损伤反应和支架蛋白MDC1通过促进CDC20与APC/C的结合来调节有丝分裂中的APC/C-CDc20活性，而转录抑制和肿瘤抑制因子TIF1y也调节有丝分裂中的APC/C-CDc20的活性。目前的项目将使用质谱学来鉴定新的APC/C底物和APC/C调节剂，并确定磷酸化在底物识别和调节功能中的作用。我们还将利用实验室成熟的传统分子和细胞生物学技术，研究新的APC/C底物的细胞周期特定降解以及新型APC/C相互作用蛋白对APC/C功能的调节。

The Anaphase-Promoting Complex/Cyclosome (APC/C) is a macromolecular E3 ubiquitin ligase that, through targeting protein substrates for polyubiquitylation and 26S proteasome-mediated degradation coordinates the progression of cells through mitosis and the successive G1 phase of the cell-cycle. APC/C E3 ligase activity is stimulated, in the presence of the E2-ubiquitin conjugating enzymes UbcH10 and Ube2S, by the temporally coordinated recruitment of one of two related activator proteins, Cdc20 or Cdh1, to tetratricopeptide repeat (TPR)-containing APC/C subunits such as APC3; Cdc20 and Cdh1 also serve in conjunction with particular APC/C subunits to bind substrates. Work from our laboratory has determined previously that the ubiquitin ligase activity of APC/C-Cdc20 and APC/C-Cdh1 is also regulated by the transcriptional co-activators CBP and p300, which bind specifically to APC/C subunits APC5 and APC7, through interaction domains conserved in adenovirus E1A. We have also determined that the DNA damage response, and scaffold protein, MDC1 regulates APC/C-Cdc20 activity during mitosis by promoting Cdc20 association with the APC/C, whilst the transcriptional repressor and tumour suppressor, TIF1y, also regulates APC/C-Cdc20 activity in mitosis. The current project will use mass spectrometry to identify novel APC/C substrates and APC/C regulators, and define the role of phosphorylation in substrate recognition and regulator function. We will also use conventional molecular and cell biology techniques, well established in our laboratory, to study the cell cycle phase-specific degradation of new APC/C substrates and the regulation of APC/C function by novel APC/C-interacting proteins.