Identification of the interaction partners of anti-apoptotic BAG-1M isoform in breast cancer and breast epithelial cells

BAG-1 (Bcl-2 associated athanogene-1) is an anti-apoptotic protein which is a member of BAG family that has been evolutionary conserved in many organisms. BAG-1 is involved in important mechanisms within the cell, as a multifunctional and an adapter protein. BAG-1 functions on the regulation of transcription, hormone activity, cell proliferation, cell motility, tumorigenesis, apoptosis and various cellular signaling pathways via its interactions with heat shock proteins, E3 ligases, nuclear hormone receptors, Raf-1 serine/threonine kinase family, and Bcl-2. When the functions in the cell are taken into account, BAG-1 allows the cell to survive and is overexpressed in many types of cancer. It is thought that BAG-1 has an important role in the development of breast cancer in particular. There are three major isoforms of BAG-1 originated from alternative translation initiation sites on the same mRNA. Both of the three isoforms contain the BAG domain and ULD (ubiquitin like domain) at the C-terminus regions, the difference between the isoforms arise from the N-terminus sites. BAG-1L is the largest isoform, has the nuclear localization signal at the N-terminus and is found in the nucleus, the smallest isoform is the BAG-1S which is known as the most abundant isoform in cell and localized in the cytoplasm. BAG-1M can be found in both cytoplasm and nucleus with a variety of mediator proteins. It is known that BAG-1 isoforms can be specialized to the tissues and cells they are present in, but there is no detailed information in the literature on isoform-specific studies that lighten the proteins that each isoform interacts with and the mechanisms in which it is involved. In this study, it was aimed to detect the interaction partners of BAG-1M isoform in breast cancer and breast epithelial cells. In order to detect the interaction partners of BAG-1M, the vector plasmid that contains the N-terminus TAP-Tagged sequence of BAG-1M was used transfect cells and then BAG-1M was purified from the total protein isolated from the cells transfected. Proteins interacting with BAG-1M were first identified via LC-MS/MS (liquid chromatography-mass spectrometry) analyses for peptide mapping of the digested purified proteins. For verification of identified proteins, 2-DE performed on purified BAG-1M samples and spots of 2-dimensional gel were analyed on MALDI-TOF. For further confirmation, immunoblotting assays were applied on purified samples to check the existance of identified proteins. Pathway analysis and protein-protein interaction networks of the detected proteins were analyzed using KEGG and STRING web servers. For the classification of the identified proteins, GO Enrichment assays were used. In order to illuminate the cellular mechanisms of the complexes formed by BAG-1M and its interacting proteins, native forms of proteins were purified and complexes were xxii separated according to their size by blue native polyacrylamide gel electrophoresis (BN-PAGE). Each complex was cut from the gel, digested to peptides and analyzed on LC-MS/MS for peptide mapping to detect containing proteins. The results obtained in all experimental studies showed that the interacting proteins of BAG-1M as heat shock protein 90 (HSP-90) , heat shock protein family member BiP (HSPA5), protein disulfide isomerase family member A3 (PDIA3), transitional endoplasmic reticulum ATPase (VCP) and UV excision repair protein RAD23 homolog B (RAD23B) which function in folding/refoldig of proteins and the ubiquitin-related proteasomal degradation. Considering of the mechanisms that BAG-1 is involved, known partners and newly identified interacting proteins suggested that interacting with the proteins localized in endoplasmic reticulum, proteins functions in ubiquitination mechanism, proteasome subunits, and chaperons may be related to role of BAG-1M in endoplasmic reticulum associated degradation (ERAD). Prediction of interaction surfaces of the identified proteins with the BAG domain and ubiquitin-like domain of BAG-1 were performed using the PRISM web server and evaluated according to the energy level between two proteins. Structural analyses showed that RAD23B, VCP and PDIA3 are capable to interact with BAG-1, while HSP-90 and HSPA5 interactions were not found as have possible physical interactions. This study identified proteins that interacting with BAG-1M isoform in direct or indirect manner. More detailed studies that will be performed in the light of the experimental findings and structural analyses can contribute to the elucidation of isoform-specific functions of BAG-1, which is seen as a biomarker in breast cancer, and to the development of new therapeutic agents.