Vats Arushi, Braga Luca, Kavcic Nezka, Massimi Paola, Schneider Edoardo, Giacca Mauro, Laimins Laimonis A, Banks Lawrence
Tumour Virology, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy.
Department of Microbiology-Immunology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA.
mBio. 2025 Feb 5;16(2):e0278324. doi: 10.1128/mbio.02783-24. Epub 2024 Dec 17.
Previous studies have shown that E6 interacts with the E6-associated protein (E6AP) ubiquitin-protein ligase and directs its ubiquitylation activity toward several specific cellular proteins, one of the most important of which is p53. Interestingly, E6AP not only aids in the E6-directed degradation of cellular substrates but also stabilizes the E6 protein by protecting it from proteasome-mediated degradation. However, there is no information available about the ubiquitin ligases that regulate the stability and activity of the human papillomavirus (HPV) E6 oncoprotein in the absence of E6AP. Therefore, to identify these novel ubiquitin ligases, we performed high-throughput human siRNA library screen against ubiquitin ligases in clustered regularly interspaced palindromic repeat (CRISPR)-edited E6AP-knockout human embryonic kidney (HEK) 293 cells, stably expressing green fluorescent protein (GFP)-tagged HPV-18E6. We found a number of ubiquitin ligases that increase the expression of GFP-tagged 18E6 upon their knockdown in the absence of E6AP. Upon validation of the interaction of 18E6 with these ubiquitin ligases in cervical cancer-derived cell lines, we found that the knockdown of ubiquitin ligase F-box protein 4 (FBXO4), together with E6AP knockdown, leads to a dramatic increase in the levels of endogenous HPV-18E6 oncoprotein. Furthermore, our data demonstrate that the combined knockdown of FBXO4 and E6AP not only rescues the protein levels of E6 but also induces high levels of cell death in a p53-dependent manner in the HPV-positive cervical cancer cell line, HeLa. These results indicate a close interplay between FBXO4, E6AP, and p53 in the regulation of cell survival in HPV-positive cervical tumor-derived cells.
E6-associated protein (E6AP)-mediated stabilization of human papillomavirus (HPV) E6 plays a crucial role in the development and progression of cervical and other HPV-associated cancers. This study, for the first time, identifies a novel ubiquitin ligase, FBXO4 that targets the degradation of HPV E6 oncoprotein in the absence of E6AP in cervical cancer-derived cell lines. This may have significant implications for our understanding of HPV-associated cancers by providing deeper insights into the intricate interplay between viral proteins and host cellular machinery and the development of targeted therapies.
先前的研究表明，E6与E6相关蛋白（E6AP）泛素蛋白连接酶相互作用，并将其泛素化活性导向几种特定的细胞蛋白，其中最重要的一种是p53。有趣的是，E6AP不仅有助于E6介导的细胞底物降解，还通过保护E6蛋白免受蛋白酶体介导的降解来使其稳定。然而，在没有E6AP的情况下，关于调节人乳头瘤病毒（HPV）E6癌蛋白稳定性和活性的泛素连接酶尚无相关信息。因此，为了鉴定这些新型泛素连接酶，我们在稳定表达绿色荧光蛋白（GFP）标记的HPV - 18E6的成簇规律间隔短回文重复序列（CRISPR）编辑的E6AP基因敲除人胚肾（HEK）293细胞中，针对泛素连接酶进行了高通量人siRNA文库筛选。我们发现了一些泛素连接酶，在没有E6AP的情况下，它们的敲低会增加GFP标记的18E6的表达。在验证18E6与这些泛素连接酶在宫颈癌衍生细胞系中的相互作用后，我们发现泛素连接酶F - 盒蛋白4（FBXO4）的敲低与E6AP的敲低一起，会导致内源性HPV - 18E6癌蛋白水平急剧增加。此外，我们的数据表明，FBXO4和E6AP的联合敲低不仅能挽救E6的蛋白水平，还能在HPV阳性的宫颈癌细胞系HeLa中以p53依赖的方式诱导高水平的细胞死亡。这些结果表明FBXO4、E6AP和p53在HPV阳性宫颈肿瘤衍生细胞的细胞存活调节中存在密切的相互作用。
E6相关蛋白（E6AP）介导的人乳头瘤病毒（HPV）E6稳定在宫颈癌和其他HPV相关癌症的发生和发展中起着关键作用。本研究首次鉴定出一种新型泛素连接酶FBXO4，它在宫颈癌衍生细胞系中，在没有E6AP的情况下靶向HPV E6癌蛋白的降解。这可能通过深入了解病毒蛋白与宿主细胞机制之间的复杂相互作用以及靶向治疗的开发，对我们理解HPV相关癌症具有重要意义。
