Protein partners of the eEF1Bβ subunit of the translation elongation complex eEF1B in the nuclear fraction of human lung carcinoma cells

Abstract

To identify novel protein partners of translation factor eEF1Bβ in nucleus of human lung carcinoma cells. Methods. Protein partners of eEF1Bβ in the nuclear fraction of A549 cells were identified by co-immunoprecipitation (co-IP) combined with liquid chromatographytandem mass spectrometry (LC-MS/MS). Specific protein partners of eEF1Bβ were further selected by using the results of previously published global, quantitative and dynamic mapping of protein subcellular localization with help of "Mapofthecell" program. Results. 104 highscored proteins interacting with eEF1Bβ in the nuclear fraction of A549 cells have been identified by mass-spectrometry. Among these proteins, 9 partners of eEF1Bβ were confirmed by the co-fractionation approach. Functional analysis of the partners has divided them on the pro-oncogenic (lung-cancer related) and neutral/anti-oncogenic moieties. These two groups are estimated to be spatially separated in human cancer cells. Conclusions. The position of eEF1Bβ as a link between the oncogenic and neutral/tumor-suppressor moieties of its protein partners in nucleus of lung cancer cells is suggested. Deciphering of a possible role of the eEF1Bβ distribution between the pro-cancer or anti-cancer communities of its protein partners can be a subject of further research.

Non-translational Connections of eEF1B in the Cytoplasm and Nucleus of Cancer Cells

“…However, signs of the nuclear localization of eEF1B subunits were displayed in the normal (Drosophila embryos, human cardioesophageal junction, human lung) and cancer (cardioesophageal carcinoma, lung cancer, oral squamous cell carcinoma) tissues as well as in lung adenocarcinoma cells A549 (Fan et al, 2010;Veremieva et al, 2011;Veremieva et al, 2014;Flores et al, 2016). In an attempt to attribute possible noncanonical eEF1B functions to the specific cellular compartments the partners of eEF1Bβ and eEF1Bγ in the cytoplasm and nucleus of human lung carcinoma cells were identified experimentally and analyzed by several bioinformatics approaches (Kapustian et al, 2016(Kapustian et al, , 2017(Kapustian et al, , 2018(Kapustian et al, , 2019. Cytoplasmic eEF1Bβ was predicted to be involved into four protein networks representing cell cycle regulation, DNA replication and repair, chromatin remodeling and chaperoning machinery (Kapustian et al, 2016).…”

Analysis of eEF1Bγ interactome in the nuclear fraction of A549 human lung adenocarcinoma cells

“…Having established a procedure for the cytonucleo fractionation of human lung carcinoma A549 cells [10] we isolated the nuclear fraction and conducted co-immunoprecipitation of the eEF1Bγ protein partners using anti-eEF1Bγ antibodies. Subsequent LC-MS/MS identification of precipitated proteins has delivered 234 interacting partners of eEF1Bγ.…”

“…Precise co-fractionation is considered an alternative way to estimate a possibility of protein-protein interaction in cell [17]. Recently we used this approach to identify the proteins co-fractionated with the eEF1Bβ subunit of eEF1B in human cancer cells [10]. For this aim the Mapofthecell database of the subcellular localization of the proteins in cancer human cells was employed [17].…”

“…Human lung cancer cells A549 were grown up to 7.5×10 6 cells/ml and harvested with Trypsin-EDTA. The nuclear fraction was isolated as in [10]. Briefly, cells were lysed in 1.5 volume of the buffer containing 10 mM HEPES pH7,9; 1,5 mM MgCl 2 ; 10 mM KCl; 0,5 % NP-40; 0,2 mM PMSF; 0,5 mM DTT for 20 min on ice.…”

“…Analysis of the nuclear protein partners which are co-fractionated with eEF1Bγ was done using the Mapofthecell program (http:// www.mapofthecell.org). All protein partners of the nucleus-localized eEF1Bγ identified by co-precipitation studies were tested for a possibility of their co-fractionation with eEF1Bγ as described in [10] for eEF1Bβ.…”