Sterlinko Grm Helena, Weber Malte, Elston Rob, McIntosh Pauline, Griffin Heather, Banks Lawrence, Doorbar John
International Centre for Genetic Engineering and Biotechnology, Padriciano 99, I-34012 Trieste, Italy.
J Mol Biol. 2004 Jan 23;335(4):971-85. doi: 10.1016/j.jmb.2003.10.079.
The E6 oncoprotein derived from the tumour-associated human papillomavirus (HPV) types induces the ubiquitin-mediated degradation of several cellular proteins by conjugating them with the cellular ubiquitin ligase E6-AP. This is a HECT domain-containing ligase that was originally identified through its involvement in the E6-mediated degradation of the cellular tumour suppressor protein p53. Here we have investigated, in more detail, the nature of the E6/E6-AP interaction using binding peptides isolated from an E6-specific library. The selected peptides were either predicted or shown to have an alpha-helical core resembling the E6-binding motif on E6-AP, as well as amino acid alterations that increased their affinity for E6. These peptides were potent inhibitors of the E6/E6-AP interaction. Further analysis of the effects of these peptides on the ability of E6 to direct the proteolytic degradation of its various substrates, including p53, Dlg and the MAGI family of proteins, as well as using E6-AP immunodepletion, revealed striking differences in the mechanism by which E6 targets its cellular substrates for degradation. These results suggest that the site on E6 bound by E6-AP is also most likely occupied by other, as yet unidentified, ubiquitin ligases.
源自肿瘤相关人乳头瘤病毒（HPV）类型的E6癌蛋白通过将几种细胞蛋白与细胞泛素连接酶E6-AP缀合，诱导泛素介导的这些细胞蛋白降解。这是一种含HECT结构域的连接酶，最初是通过其参与E6介导的细胞肿瘤抑制蛋白p53的降解而被鉴定出来的。在这里，我们使用从E6特异性文库中分离的结合肽，更详细地研究了E6/E6-AP相互作用的性质。所选肽段要么被预测，要么被证明具有类似于E6-AP上E6结合基序的α-螺旋核心，以及增加其对E6亲和力的氨基酸改变。这些肽是E6/E6-AP相互作用的有效抑制剂。进一步分析这些肽对E6指导其各种底物（包括p53、Dlg和MAGI蛋白家族）进行蛋白水解降解能力的影响，以及使用E6-AP免疫耗竭，揭示了E6将其细胞底物靶向降解的机制存在显著差异。这些结果表明，E6-AP结合的E6位点也很可能被其他尚未鉴定的泛素连接酶占据。
