Kiran Shashi, Wilson Briana, Saha Shekhar, Graff Julia Ann, Dutta Anindya
Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, VA 22908, USA.
Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad 500046, India.
Cancers (Basel). 2021 Dec 22;14(1):30. doi: 10.3390/cancers14010030.
E6 from high-risk strains of HPV is well known to transform cells by deregulating p53. We reported that in HPV transformed cell-lines E6 from high-risk HPV can recruit the USP46 deubiquitinase to substrates such as Cdt2 and stabilize the latter, and that USP46 is important for growth of HPV induced tumors in xenografts. Here we show that in cervical cancer biopsies the stabilization of Cdt2 in the HPV-induced cancers leads to the decrease of a CRL4-Cdt2 substrate, the histone H4K20 mono-methyltransferase Set8, and decrease in H4K20me1 or H4K20me3 that can be detected by immunohistochemistry. In HPV-transformed cancer cell lines , knockdown of E6 decreases Cdt2 and increases Set8. Co-knockdown of Set8 shows that some of the gene expression changes produced by E6 knockdown is due to the increase of Set8. EGFR and EGFR regulated genes were identified in this set of genes. Turning to the mechanism by which E6 stabilizes Cdt2, we find that a purified E6:USP46 complex has significantly more de-ubiquitinase activity in vitro than USP46 alone, demonstrating that E6 can directly interact with USP46 in the absence of other proteins and that it can substitute for the known activators of USP46, UAF1 and WDR20. Deletion mapping of Cdt2 shows that there are three discrete, but redundant, parts of the substrate that are essential for stabilization by E6: USP46. The helix-loop-helix region or the WD40 repeat driven beta-propeller structure of Cdt2 are dispensable for the stabilization implying that interaction with DDB1 (and the rest of the CRL4 complex) or with the substrate of the CRL4-Cdt2 E3 ligase is not necessary for E6:USP46 to interact with and stabilize Cdt2. The identification of 50 amino acid stretches in the 731 amino acid Cdt2 protein as being important for the stabilization by E6 underlines the specificity of the process. In summary, E6 activates the deubiquitinase activity of USP46, stabilizes Cdt2 utilizing multiple sites on Cdt2, and leads to degradation of Set8 and changes in gene-expression in HPV-transformed cells.
众所周知，高危型人乳头瘤病毒（HPV）的E6蛋白通过使p53失调控来转化细胞。我们曾报道，在HPV转化的细胞系中，高危型HPV的E6蛋白可将泛素特异性蛋白酶46（USP46）招募至Cdt2等底物，并使后者稳定，且USP46对异种移植中HPV诱导肿瘤的生长很重要。在此我们表明，在宫颈癌活检中，HPV诱导癌症里Cdt2的稳定导致CRL4 - Cdt2底物——组蛋白H4K20单甲基转移酶Set8减少，且免疫组化可检测到H4K20me1或H4K20me3减少。在HPV转化的癌细胞系中，敲低E6会使Cdt2减少而Set8增加。同时敲低Set8表明，E6敲低产生的一些基因表达变化是由于Set8增加所致。在这组基因中鉴定出了表皮生长因子受体（EGFR）及EGFR调控的基因。关于E6使Cdt2稳定的机制，我们发现，纯化的E6:USP46复合物在体外具有比单独的USP46显著更多的去泛素酶活性，这表明E6在无其他蛋白时可直接与USP46相互作用，且它能替代USP46已知的激活剂UAF1和WDR20。对Cdt2的缺失图谱分析表明，底物上有三个离散但冗余的部分对于E6:USP46介导的稳定是必需的。Cdt2的螺旋-环-螺旋区域或WD40重复驱动的β-螺旋桨结构对于稳定并非必需，这意味着与损伤特异性DNA结合蛋白1（DDB1，及CRL4复合物的其余部分）或与CRL4 - Cdt2 E3连接酶的底物相互作用对于E6:USP46与Cdt2相互作用并使其稳定并非必要。在731个氨基酸的Cdt2蛋白中鉴定出50个氨基酸片段对E6介导的稳定很重要，这突出了该过程的特异性。总之，E6激活USP46的去泛素酶活性，利用Cdt2上的多个位点使Cdt2稳定，并导致Set8降解及HPV转化细胞中的基因表达变化。
