Room No. LSS008, Biochemistry Division, CSIR-Central Drug Research Institute, Lucknow, Uttar Pradesh, India.
Academy of Scientific and Innovative Research (AcSIR), CSIR-Central Drug Research Institute, Lucknow, Uttar Pradesh, India.
IUBMB Life. 2019 Dec;71(12):1896-1905. doi: 10.1002/iub.2132. Epub 2019 Jul 22.
E6AP (E6 associated protein) is a HECT domain containing protein having dual E3 ligase and ERα coactivation activity in breast cancer cells. Although E6AP is known to possess antitumorigenic activity, the underlying molecular mechanism is poorly understood. In the present study, we applied nano-LC based proteomics approach to identify E6AP-interacting proteins where we performed GST-pull down using GST-E6AP from whole cell extracts of MCF7 cells, resolved the differentially interacting proteins on 1D-SDS-PAGE, excised the gel bands that were trypsin digested followed by fractionation and spotting on MALDI-TOF/TOF plate through Nano-LC MALDI spotter. Subsequently, fractionated and spotted peptides were identified using MALDI-TOF/TOF. We identified several E6AP interacting proteins including previously reported such as HSP70 and new ones such as Enolase-1. We further confirmed that E6AP and Enolase1 interacted and colocalized more in the cytoplasmic periphery in breast cancer cells and further demonstrated that E6AP also targeted ENO1 for ubiquitin-mediated degradation in these cells.
E6AP（E6 相关蛋白）是一种含有 HECT 结构域的蛋白，在乳腺癌细胞中具有双重 E3 连接酶和 ERα 共激活活性。尽管已知 E6AP 具有抗肿瘤活性，但其潜在的分子机制尚不清楚。在本研究中，我们应用基于纳升液相色谱的蛋白质组学方法来鉴定 E6AP 相互作用蛋白，我们使用 GST-E6AP 从 MCF7 细胞的全细胞提取物中进行 GST 下拉，在 1D-SDS-PAGE 上分离差异相互作用的蛋白，然后将凝胶条切下进行胰酶消化，接着通过纳升液相色谱 -MALDI 斑点仪进行分级和斑点。随后，使用 MALDI-TOF/TOF 鉴定分级和斑点的肽。我们鉴定了几种 E6AP 相互作用蛋白，包括先前报道的 HSP70 和新的 Enolase-1 等。我们进一步证实 E6AP 和 Enolase1 在乳腺癌细胞的细胞质周围相互作用和共定位更多，并进一步表明 E6AP 还靶向 ENO1 进行这些细胞中的泛素介导降解。
