Česká společnost pro hmotnostní spektrometrii

(CEEPC/IPM/CMSC 2022 - ThO-02)
Global SEC-PCP-SILAC mapping reveals protein complexes mediating NF-κB activation in breast cancer

Petr Lapčík ^1 *, Greg Stacey ², David Potěšil ³, Leonard Foster ^2,4, Pavel Bouchal ¹

1. Masaryk University, Faculty of Science, Department of Biochemistry, Brno
2. University of British Columbia, Michael Smith Laboratories, Vancouver
3. Masaryk University, Central European Institute of Technology, Brno
4. University of British Columbia, Department of Biochemistry and Molecular Biology, Vancouver

Abstrakt

NF-κB has essential role in immune response and is associated with lymph node metastasis of luminal A breast tumors [1]. Analysis of protein interactome and its changes in response to NF-κB modulation could uncover pro-metastatic mechanisms related to NF-κB. We apply metabolic isotope labeling SILAC, size exclusion chromatography (SEC) and protein correlation profiling (PCP) [2] to construct a network of interactome rearrangement in response to NF-κB modulation in MCF-7 breast cancer cells.
We generated two co-fractionation datasets consisting of 80 fractions from SILAC-labeled and 80 fractions from label-free native MCF-7 lysates with inhibited or native NF-κB activity. LC-MS/MS analysis of SEC fractions using Orbitrap Lumos and Bruker Impact II mass spectrometers quantified 3308 and 5460 protein groups in SILAC and label-free datasets, respectively (FDR = 0.01). Interactome reconstruction using PrinCE [3] detected 7568 interactions among 1520 proteins. Co-elution of subunits of known complexes, such as ribosome, proteasome and MCM, was observed. Modulation of NF-κB was linked to interactome changes of proteins involved in immune response, cell cycle and DNA replication. NF-κB factor RELA interacted with proteins co-eluting with activators of NF-κB and these interactions were modulated by NF-κB inhibition.
Our interaction network represents a complex insight into dynamics of MCF-7 protein interactome associated with NF-κB pathway and could serve as a basis for future studies characterizing NF-κB in breast cancer.

* Korespondující autor: lapcik@mail.muni.cz

Literatura

1. Bouchal P. et al.: Mol Cell Proteomics, 14(7):1814-30. (2015)
2. Kristensen A.R. et al.: Nat Methods, 9(9):907-9. (2012)
3. Stacey R.G. et al.: BMC Bioinformatics, 23;18(1):457 (2017)

Poděkování:

This work was supported by Ministry of Health of the Czech Republic (grant No. NU22-08-00230) and by the project National Institute for Cancer Research (Programme EXCELES, ID Project No. LX22NPO5102) - Funded by the European Union - Next Generation EU.