Detection of COP1-interacting proteins using co-immunoprecipitation followed MALDI-TOF mass spectrometry

摘要

In 2004, COP1 was characterized as p53 E3-specific human ubiquitin ligase according to in vitro studies. Later, oncogenic activity of COP1 and correlation of its overexpression with level of p53 protein in clinical tumor samples and cell lines were shown. High expression level of COP1 was described in 25 of 32 cases of breast carcinomas and in 76 out of 171 cases of ovarian carcinomas. High expression level of COP1 was described in hepatocellular carcinoma cell lines (PLC, Hep3B and HepG2, Huh7). In 40 of 55 cases of pancreatic cancer, overexpression of COP1 was also noted. Some types of leukemia, melanoma, breast, lung and prostate cancer contain focal deletions of COP1. COP1 overexpression was also found in non- oncological diseases like Duchenne muscular dystrophy, ischemic cardiomyopathy and juvenile dermatomyositis. Similar to other p53-related E3 -ubiquitin ligases COP1 has different substrates. The selection of target for ubiquitination depends on cell type or differentiation stage. Among the substrates of COP1, besides p53 are: c-Jun, ETV1, ACC, TORC2, FOXO1 and C/EBP@a, FIP200. To identify novel proteins that interact with COP1 we applied proteomics. Proteins co-immunoprecipitated with COP1 were analyzed by MALDI -TOF mass spectrometry. To achieve this, COP1-3xFlag expression vector was generated and transfected into HEK 293T cells by calcium phosphate transfection method. Protein complexes were immunoprecipitated with anti-FLAG beads. Bound proteins were then separated by 1D SDS-PAGE gel electrophoresis and analyzed by means of ESI-LC/MS/MS mass spectrometry. Subsequent bioinformatics analysis of the interacting proteins was employed. About 25% of identified proteins were attributed to the cytoskeleton proteins, almost 20% of proteins had known role in metabolism. According to the literature data, it was not surprising to find several proteins involved in the lipid metabolism. Finally, a significant groups of interactants play role in transcription, cell adhesion, apoptosis, protein modification and transport. It is important to note that several COP1-bound proteins can be strong regulators of cell cycle in G2/M transition. Our data can be used for the subsequent studies of previously unknown roles of COP1 in different diseases. This work was funded by Grants fromRussian Scientific Foundation- Russia (No.114-15-00816) and Grant of Molecular and Cellular Biology of the Presidium of the Russian Academy of Sciences.