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首页> 外文期刊>Biochemistry >The Human Papillomavirus E7−E2 Interaction Mechanism in Vitro Reveals a Finely Tuned System for Modulating Available E7 and E2 Proteins
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The Human Papillomavirus E7−E2 Interaction Mechanism in Vitro Reveals a Finely Tuned System for Modulating Available E7 and E2 Proteins

机译：人乳头瘤病毒E7-E2相互作用的体外机制揭示了微调系统，用于调节可用的E7和E2蛋白

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Transcription of the human papillomavirus E7 oncoprotein is negatively controlled by the viral E2nprotein, and loss of this repression leads to irreversible transformation and carcinogenesis. Here we show thatninteraction of the HPV16 E7 protein with the DNA binding domain of the E2 protein (E2C) leads to ionicnstrength-dependent hetero-oligomerization even at the lowest concentrations measurable. Titration experi-nments followed by light scattering and native gel electrophoresis show insoluble oligomeric complexes with an2000 nm diameter and intermediate soluble complexes 40 and 115 nm in diameter, respectively, formed innexcess of E2C. A discrete oligomeric soluble complex formed in excess of E7 displays a diameter of 12 nm. ThenN-terminal domain of E7 interacts with E2C with a KD of 0.1 μM, where the stretch of residues 25-40 of E7,nencompassing both a PEST motif and phosphorylation sites, is sufficient for the interaction. Displacement ofnthe soluble E7-E2C complex by an E2 site DNA duplex and site-directed mutagenesis indicate that thenprotein-protein interface involves the DNA binding helix of E2. The formation of complexes of differentnsizes and properties in excess of either of the viral proteins reveals a finely tuned mechanism that couldnregulate the intracellular levels of both proteins as infection and transformation progress. Sequestering E2ninto E7-E2 oligomers provides a possible additional route to uncontrolled E7 expression, in addition andnprior to the disruption of the E2 gene during viral integration into the host genome.
机译：人乳头瘤病毒E7癌蛋白的转录受病毒E2n蛋白的负调控，而这种抑制作用的丧失会导致不可逆转的转化和致癌作用。在这里，我们显示HPV16 E7蛋白与E2蛋白（E2C）的DNA结合结构域的互作会导致离子强度依赖性杂聚，即使在可测量的最低浓度下也是如此。滴定实验，随后进行光散射和天然凝胶电泳，发现不溶于E2C的直径不超过2000 nm的不溶性寡聚复合物和直径分别为40和115 nm的中间可溶性复合物。超过E7形成的离散的低聚可溶性复合物的直径为12 nm。然后，E7的N末端结构域以0.1μM的KD与E2C相互作用，其中E7的25-40位残基延伸（既包括PEST图案又包括磷酸化位点）足以进行相互作用。 E2位点DNA双链体置换可溶性E7-E2C复合物并进行定点诱变表明，蛋白-蛋白界面随后涉及E2的DNA结合螺旋。两种病毒蛋白中不同大小和特性的复合物的形成揭示了一种微调的机制，该机制可以随着感染和转化的进行而调节两种蛋白的细胞内水平。将E2nin隔离为E7-E2寡聚体，除了在病毒整合入宿主基因组期间破坏E2基因之外，还提供了不受控制的E7表达的额外途径。

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