Higher-Order Structural Analysis of Pro-survival BAG-1S through HDX-MS


Tatlı Ö. , Türk M., Dinler Doğanay G.

67th ASMS Conference on Mass Spectrometry and Allied Topics, Georgia, Amerika Birleşik Devletleri, 31 Mayıs - 04 Haziran 2019, ss.1

  • Basıldığı Şehir: Georgia
  • Basıldığı Ülke: Amerika Birleşik Devletleri
  • Sayfa Sayısı: ss.1

Özet

Introduction

Acquired drug resistance causes to the failure in cancer treatment and recurrence of disease. Global burden of drug resistance calls for seeking new therapeutic targets against cancer. Anti-apoptotic BAG-1 is a multifunctional protein and frequently overexpressed in breast carcinoma. BAG-1 involves in a wide variety of cellular processes through direct interaction with Hsp70, Raf-1 kinase, the anti-apoptotic Bcl-2, nuclear hormone receptors and ubiquitylation/proteasome machinery components. Hence, BAG-1, with its three functionally distinct and differentially localized isoforms, is a candidate protein to target signaling hubs in cancerous cells. Here, we adopted a hydrogen deuterium exchange mass-spectrometry (HDX-MS)-guided structural study to ultimately understand structural aspects of full-length, human BAG-1S as a potential therapeutic target.

Methods

BAG-1S was first cloned into pcDNA3.1(+) mammalian expression vector, along with an N-terminal hexahistidine tag and a TEV cleavage site. Generated recombinant plasmid was transfected into MCF-7 epithelial breast cancer cell line and total protein was isolated from transfectants. BAG-1S was purified from protein lysate using Ni-NTA affinity purification chromatography through the incorporated hexahistidine tag. The tag was removed subsequently with TEV protease to eliminate any change in protein conformation and loss or alteration of biological activity. A second Ni-NTA purification was conducted with a flow/through mode, to remove His-tagged TEV protease enzyme from the reaction mixture. HDX-MS experiments were carried out and the level of deuteration was monitored to map higher-order structure of anti-apoptotic BAG-1S.

Preliminary data

Three major isoforms of BAG-1, each with a distinct N-termini, include BAG-1S (33 kDa), BAG-1M (46 kDa) and BAG-1L (52 kDa). All BAG-1 isoforms exist with an integrated ubiquitin-like domain (ULD) to contact with proteasome components for degradation of specific target proteins and a COOH-terminal evolutionarily conserved BAG domain to interact and modulate the activity of Hsp70. The lack of resolved 3D structure of full-length BAG-1 limits the development of molecularly-targeted approaches for therapeutic purposes. Here, we aimed to precisely define tertiary protein structure and domain architecture of full-length, human BAG-1S. The study comprises purification of BAG-1S and higher-order structural analysis through HDX-MS. The purified BAG-1S showed an apparent 33-kDa band in gel electrophoresis. Sample purity was estimated as >90% using ImageJ analysis of SDS-PAGE gel. For confirmation, the protein product was analyzed by western blotting. Subsequent HDX-MS experiments revealed the identification of 145 peptides of BAG-1S with a 99.1% of sequence coverage. The level of deuteration was monitored at 0.5, 1, 2, 5, 15 and 30 min. Time-course measurements of H/D exchange of BAG-1S, which provides solvent accessibility information, were evaluated. The HDX-MS data on BAG-1S indicated rapid incorporation of deuterium at the earlier time points in the majority of peptides, and the H/D exchange levels stayed constant throughout the time course of the experiment. Peptide-specific deuterium uptake rates were projected onto the modelled structure of BAG-1S and deuterium incorporation was evaluated on the overall structure. BAG domain exhibited more solvent-protected and stabilized structure compared to UBL domain.

Novel Aspect

We report here the first purification and structural characterization of full-length, native BAG-1S from mammalian cells