Human papillomavirus type 16 E6 induces self-ubiquitination of the E6AP ubiquitin-protein ligase

The E6 protein of the high-risk human papillomaviruses (HPVs) and the cellular ubiquitin-protein ligase E6AP form a complex which causes the ubiquitination and degradation of p53. We show here that HPV16 E6 promotes the ubiquitination and degradation of E6AP itself. The half-life of E6AP is shorter in HPV-positive cervical cancer cells than in HPV-negative cervical cancer cells, and E6AP is stabilized in HPV-positive cancer cells when expression of the viral oncoproteins is repressed. Expression of HPV16 E6 in cells results in a threefold decrease in the half-life of transfected E6AP. E6-mediated degradation of E6AP requires (i) the binding of E6 to E6AP, (ii) the catalytic activity of E6AP, and (iii) activity of the 26S proteasome, suggesting that E6-E6AP interaction results in E6AP self-ubiquitination and degradation. In addition, both in vitro and in vivo experiments indicate that E6AP self-ubiquitination results primarily from an intramolecular transfer of ubiquitin from the active-site cysteine to one or more lysine residues; however, intermolecular transfer can also occur in the context of an E6-mediated E6AP multimer. Finally, we demonstrate that an E6 mutant that is able to immortalize human mammary epithelial cells but is unable to degrade p53 retains its ability to bind and degrade E6AP, raising the possibility that E6-mediated degradation of E6AP contributes to its ability to transform mammalian cells.     

Identification and proteomic analysis of distinct UBE3A/E6AP protein complexes

The E6AP ubiquitin ligase catalyzes the high-risk human papillomaviruses' E6-mediated ubiquitylation of p53, contributing to the neoplastic progression of cells infected by these viruses. Defects in the activity and the dosage of E6AP are linked to Angelman syndrome and to autism spectrum disorders, respectively, highlighting the need for precise control of the enzyme. With the exception of HERC2, which modulates the ubiquitin ligase activity of E6AP, little is known about the regulation or function of E6AP normally. Using a proteomic approach, we have identified and validated several new E6AP-interacting proteins, including HIF1AN, NEURL4, and mitogen-activated protein kinase 6 (MAPK6). E6AP exists as part of several different protein complexes, including the proteasome and an independent high-molecular-weight complex containing HERC2, NEURL4, and MAPK6. In examining the functional consequence of its interaction with the proteasome, we found that UBE3C (another proteasome-associated ubiquitin ligase), but not E6AP, contributes to proteasomal processivity in mammalian cells. We also found that E6 associates with the HERC2-containing high-molecular-weight complex through its binding to E6AP. These proteomic studies reveal a level of complexity for E6AP that has not been previously appreciated and identify a number of new cellular proteins through which E6AP may be regulated or functioning.     

The role of the ubiquitin ligase E6-AP in human papillomavirus E6-mediated degradation of PDZ domain-containing proteins

The E6 oncoprotein of human papillomaviruses associated with cervical cancer targets the tumor suppressor p53 and several other cellular proteins including the human homologs of Dlg and Scribble for degradation via the ubiquitin-proteasome system. Similar to p53 degradation, E6-induced degradation of Scribble is mediated by the ubiquitin ligase E6-AP. In contrast, degradation of Dlg in vitro and within cells has been reported to be independent of E6-AP, suggesting that the E6 oncoprotein has the ability to interact with ubiquitin ligases other than E6-AP. Furthermore, the ability of the E6 oncoprotein to interact with these yet unidentified ubiquitin ligases may be shared by the E6 protein of so-called low risk human papillomaviruses that are not associated with cervical cancer. In this study, we used the RNA interference technology and mouse embryo fibroblasts derived from E6-AP-deficient mice to obtain information about the identity of the ubiquitin ligase(s) involved in E6-mediated degradation of Dlg. We report that, within cells, E6-mediated degradation of Dlg depends on the presence of functional E6-AP and provide evidence that the E6 protein of low risk human papillomaviruses functionally interacts with E6-AP. Based on these data, we propose that, in general, the proteolytic properties of human papillomavirus E6 proteins are mediated by interaction with E6-AP.     

e6-ap与蛋白降解

e6-ap基因是HECTE3蛋白家族成员,根据N端的不同分为3个转录本,编码的3种蛋白均具有降解蛋白的功能。E6-AP的功能复杂,主要包括：①降解抑癌基因如p53、pml、tsc2、pdz家族基因的功能;②诱导htert基因启动子激活、提高端粒酶活性的功能;③与C型肝炎病毒的核心蛋白结合,参与r6基因的降解;④双向调节固醇类激素受体的表达;⑤调节ANNEXINA1蛋白的表达、促进其降解。E6-AP的蛋白表达受泛素及E6的调控,在肿瘤发病机制中起着重要作用。