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

后期促进复合物/环体（APC/C）是一种大分子E3遍在蛋白连接酶，通过靶向蛋白质底物进行聚遍在蛋白化和26 S蛋白酶体介导的降解，协调细胞通过有丝分裂和细胞周期连续G1期的进展。在E2-泛素结合酶UbcH 10和Ube 2S存在的情况下，通过两种相关激活蛋白Cdc 20或Cdh 1中的一种在时间上协调地募集到含有三肽重复序列（TPR）的APC/C亚基（如APC 3）来刺激APC/C E3连接酶活性; Cdc 20和Cdh 1也与特定APC/C亚基结合以结合底物。我们实验室的工作已经确定APC/C-Cdc 20和APC/C-Cdh 1的泛素连接酶活性也受到转录共激活因子CBP和p300的调节，CBP和p300通过腺病毒E1 A中保守的相互作用结构域特异性结合APC/C亚基APC 5和APC 7。我们还确定了DNA损伤反应和支架蛋白MDC 1通过促进APC/C与Cdc 20的结合来调节有丝分裂期间APC/C-Cdc 20的活性，而转录抑制因子和肿瘤抑制因子TIF 1 y也调节有丝分裂中APC/C-Cdc 20的活性。目前的项目将使用质谱法来鉴定新的APC/C底物和APC/C调节剂，并确定磷酸化在底物识别和调节剂功能中的作用。我们还将使用传统的分子和细胞生物学技术，以及建立在我们的实验室，研究新的APC/C底物的细胞周期阶段特异性降解和APC/C功能的调节新的APC/C相互作用蛋白。

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.