Non-canonical interactions of the β subunit of the translation elongation complex eEF1B and analysis of their possible functional role

Abstract

To predict protein networks which may comprise the β subunit of the translation elongation complex eEF1B in lung carcinoma cell line. Methods. The protein partners of eEF1Bβ from cytoplasmic extract of A549 cells were identified by co-immunoprecipitation (co-IP) combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The molecular interaction network for eEF1Bβ was predicted and visualized by a Cytoscape 3.2.0 program using an MCODE plugin. GO analysis of cellular distribution was performed by a STRAP program. Results. 162 high-scored proteins interacting with eEF1Bβ in the cytoplasm of lung carcinoma cells A549 have been identified by mass-spectrometry. Possible functional networks involving these contacts were predicted bioinformatically. Conclusions. Four protein networks are identified as possible targets of eEF1Bβ in lung cancer cells. These groups are involved in the cell cycle regulation; DNA replication and repair; chromatin remodeling; chaperoning and signal transduction. The data allow to narrow down further search for non-canonical cancer-related function of eEF1Bβ.

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).…”

“…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). Nuclear eEF1Bβ interacted with proteins involved into RNA transcription and splicing, microRNA turnover, degradation of mRNA and proteins, DNA damage response.…”

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“…As these proteins obviously belong to different functional classes we attempted to classify the nuclear molecular networks which could involve eEF1Bβ, by using MCODE plugin in the Cytoscape 3.2.0 program [10]. This approach has been successfully used by us recently for clustering the cytoplasmic partners of eEF1Bβ [12]. Surprisingly, Cytoscape 3.2.0 was not capable to reveal any functional cluster of the 104 protein partners of eEF1Bβ in nucleus.…”

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

“…The nuclear extract incubated with plain G-Sepharose was used as a control of nonspecific binding. The electrophoretic bands that were not present in the control or were much more extensive than in the control were cut and processed for mass spectrometry analysis at the Mass Spectrometry Laboratory of the Institute of Biochemistry and Biophysics (Warsaw, Poland) as described before [12].…”

“…These clusters have been shown to represent protein complexes and/or parts of pathways [14]. For the sake of clarity, the known protein partners of eEF1Bγ: eEF1A1, eEF1A2 and UBC (polyubiquitin-C) were excluded from the database [12]. Also, we limited analysis by the first (direct) partners of the eEF1Bγ partners.…”