Characterization of BAG-1S/C-raf interaction targeting peptide
Characterization of BAG-1S/C-raf interaction targeting peptide
Dosyalar
Tarih
2022-07-25
Yazarlar
Çebi, Ecenur
Süreli Yayın başlığı
Süreli Yayın ISSN
Cilt Başlığı
Yayınevi
Graduate School
Özet
ERK/MAPK cascade is one of the most important cellular pathways, which regulates many distinct physiological functions such as cell proliferation, migration, differentiation, and apoptosis. Since it has vital roles in many cellular functions, dysregulation of its members usually results in uncontrolled proliferation and development of cancer cells such as breast cancer. Ras and Raf serine/threonine protein kinases are mostly deregulated components of this pathway and mutations in both proteins have been identified approximately one-third of all human cancer types. Although there are several inhibitors against oncoproteins, because of the improving drug resistance, there is a need for the development of new drugs. One of the emerging interest is to target protein-protein interactions (PPIs) related with cancers. Human Raf kinase family has three isoforms: A-Raf, B-Raf, and C-Raf. C-Raf and B-Raf are the important members of ERK pathway. Although B-Raf is dysregulated mostly, C-Raf has ability to suppress apoptosis and hence, both of the Raf kinase is vital for the cancer progression. The Raf kinases are regulated by several phosphorylation events and also, interactions with some proteins like BAG-1. Bcl-2 associated athanogene 1 (BAG-1) which is an anti-apoptotic co-chaperone protein is usually overexpressed in several cancer types making it possible oncoprotein. Moreover, its known that the interaction between BAG-1 and C-Raf promotes the C-Raf activation and stabilization. Therefore, targeting this interaction with small molecules and/or peptides would be effective therapeutics against different cancer types. In our previous studies, the interaction surface between BAG-1S and C-Raf was determined and a peptidomimetic which coded as Pep3 was designed against the BAG-1S from natural sequence of C-Raf. The aim of this study was to characterize the interaction between BAG-1S and Pep3 and also, the interaction between BAG-1S and TPep3 which is cell penetrating form of Pep3. Firstly, His-tagged BAG-1S proteins were produced in mammalian and bacterial cells. Then, BAG-1S proteins were purified by Ni-NTA affinity purification in two steps. After first step, His-tag was cleaved with TEV protease and tagless proteins were eluted as flow-through in second step while the impurities and TEV proteases were remained bound to resin. Then, the purities of both proteins (produced in mammalian and bacterial) were calculated as >80% by SDS-PAGE. Secondary structure analysis of proteins was performed with circular dichroism and both of the proteins has been folded and showed primarily ɑ-helix characteristics. After BAG-1S proteins were characterized. Pep3 and TPep3 characterization were performed by mass spectrometry and circular dichroism analysis. Pep3 and TPep3 has been synthesized by Fmoc-based solid phase peptide synthesis and they were dissolved in 20 mM AMBIC and water respectively since the Pep3 shows hydrophobic characteristics. Molecular weights of peptides were measured as 2.066 kDa for Pep3 and 3.653 kDa for TPep3 which were compatible with the theoretical calculations. Both of the peptides showed β-sheet characteristics approximately 30% combined with random coils. The interaction between BAG-1S and peptides were confirmed by crosslinking reaction with medium length crosslinker DSS (disuccinimidyl suberate). BAG-1S proteins were incubated with peptides separately to form interaction. After they formed interaction, the crosslink reaction was performed with 50-fold molar excess of DSS. The reactions were analyzed by immunoblotting. BAG-1S was shifted 2 kDa when interacts with Pep3 and 4 kDa with TPep3 comparing to the BAG-1S only reaction. These results were confirmed the interaction of BAG-1S with two peptides. The binding kinetics of BAG-1S and Pep3 was measured by Surface Plasmon Resonance (SPR) with multi-cycle kinetics method. BAG-1S protein was captured on Protein G chip with Anti-BAG-1 and different concentrations of Pep3 was injected. Binding kinetics was calculated as 68.56 nM by 1:1 binding model showing that Pep3 binds BAG-1S with high affinity. The effects of TPep3 on MAPK pathway and cell viability of MCF-7 breast cancer cells were analysed. MCF-7 cells were treated with different concentrations (0 uM to 50 uM) of TPep3 and total proteins were analysed with immunoblotting. C-Raf and p-C-RAf (S338) levels were decreased with the increasing concentration of TPep3. As a result of p-C-Raf inactivation, p-MEK levels were also decreased which showed the decrease in ERK pathway. Moreover, B-Raf and p-B-Raf (S446) levels were decreased. So, it can be said that peptide does not only affect the activitation of C-Raf but also B-Raf. To see the effects of peptide on other cell survival pathways, Akt and p-Akt (S473) levels were analysed and it was seen that their levels remained unchanged even with the highest peptide concentration. Lastly, the IC50 value was calculated as approximately 18 uM from the cell viability MTT assay. Together with all the results, TPep3 and Pep3 peptides have potential to be a promising therapeutics for cancer types relying on ERK pathway since the activity of pathway was decreased with the peptide treatment.
Açıklama
Thesis (M.Sc.) -- İstanbul Technical University, Graduate School, 2022
Anahtar kelimeler
peptide mimetics,
peptid mimetikleri,
canser,
kanser