9th Symposium on Structural Proteomics, Göttingen, Almanya, 3 - 06 Kasım 2019, ss.81
BAG-1 is a potent anti-apoptotic molecule and its aberrant expression is prevalent in human malignancies. BAG-1 is expressed as multiple isoforms (-1L, -1M, -1S, -XS), each functioning in an array of distinct key cellular events including proliferation, cell survival, transcription, apoptosis and cell motility. This multi-functionality renders BAG-1 a potential therapeutic target to blockade signal transmission in cancer cells. However, the lack of resolved tertiary structure of BAG-1 isoforms limits the development of molecularlytargeted approaches for therapeutic use. In this study, to define higher-order structure and to understand in more depth the molecular architecture of two major BAG-1 isoforms (BAG-1L, BAG-1S), we utilized hydrogen-deuterium exchange mass spectrometry (HDX-MS) as a structural tool. First, His6TEV-BAG-1S and His6TEV-BAG-1L plasmids were constructed, and MCF-7 cells were transiently transfected with the constructs. Cells were lysed, and BAG-1L and BAG-1S were purified from total cell lysates of transfectants with a tandem NiNTA purification approach through bind/elute and flow-through mode, respectively. The purified BAG-1L and BAG-1S showed an apparent 33-kDa and 50-kDa band in gel electrophoresis, respectively. Sample purity of both proteins was estimated as >90% using ImageJ analysis of SDS-PAGE gel. To monitor the level of deuteration and to analyze conformational variations among human BAG-1L and BAG-1S isoforms, HDX-MS experiments with five time-points that ranged from 12 s to 24 h were carried out. Subsequent HDX-MS experiments revealed the identification of ~150 peptides of BAG-1L and BAG-1S with a >99% sequence coverage. Based on three-dimensional structural model, we found that nuclear localization signal sequence and the more number of copies of acidic-rich repeats located at the N-terminus of BAG-1L resulted in some structural variations at its N-terminus compared to BAG-1S. We report here the first isoform-specific structural variations of BAG-1.