Nano-LC based proteomic approach identifies that E6AP interacts with ENO1 and targets it for degradation in breast cancer cells

Abstract

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.