To study protein networks containing the translation elongation factor eEF1B gamma (eEF1Bγ) in lung carcinoma cells. Methods. The protein partners of eEF1Bγ in the cytoplasmic fraction of human lung adenocarcinoma A549 cells were identified by co-immunoprecipitation (co-IP) followed by subsequent liquid chromatography-tandem mass spectrometry (LC-MS/MS). The protein interaction network for eEF1Bγ was determined by a Cytoscape 3.2.0 program using a MCODE plugin. Results. 222 high-scored proteins interacting with eEF1Bγ in the cytoplasm of A549 cells have been identified. Possible functional networks involving these protein-protein interactions were predicted using bioinformatic approaches. Conclusions. Five protein networks were identified as possible targets of eEF1Bγ in lung cancer cells. Apart from translation, eEF1Bγ was shown to be potentially involved in cell cycle regulation, nucleosome remodeling, transcription, mRNA splicing and processing, and oxidative stress response.

Translation elongation factor 1Bβ (eEF1Bβ) is a metazoan-specific protein catalyzing the guanine nucleotide exchange on translation elongation factor 1A (eEF1A). eEF1Bβ was reported to form oligomers. Aim. To define the structural region of human eEF1Bβ that mediates its self-association. In addition, the various truncated forms of this protein were tested in the guanine nucleotide exchange assay with two isoforms of mammalian eEF1A. Methods. The truncated forms of eEF1Bβ were generated by PCR, cloned, expressed in Escherichia coli and purified to homogeneity. Their apparent molecular masses were determined by analytical gel filtration and their guanine nucleotide exchange activities were assessed by filter binding assay. Results. Complete deletion of the N-terminal domain of eEF1Bβ does not affect its oligomerization propensity while deletion of the leucine-zipper motif drastically decreases the apparent molecular mass of the truncated form compared to the full-length protein. Also, the leucine-zipper motif of eEF1Bβ fused to glutathione S-transferase causes oligomerization of the chimeric protein. It was demonstrated that all N-terminally truncated forms of eEF1Bβ displayed similar catalytic activity to that of the full-length protein. Weak inhibitory effect on the catalytic activity was observed only for the truncated form with partially deleted central acidic region. Conclusions. The leucine-zipper motif facilitates oligomerization of recombinant eEF1Bβ. Stepwise deletion of the eEF1Bβ N-terminal domain does not significantly affect the guanine nucleotide exchange activity of the truncated proteins. K e y w o r d s: translation elongation factor 1, guanine nucleotide exchange, protein structural domains.

Protein synthesis in eukaryotic cell is spatially and structurally compartmentalized that ensures high efficiency of this process. One of the distinctive features of higher eukaryotes is the existence of stable multi-protein complexes of aminoacyl-tRNA synthetases and translation elongation factors. Here, we report a quaternary organization of the human guanine-nucleotide exchange factor (GEF) complex, eEF1B, comprising α, β and γ subunits that specifically associate into a heterotrimeric form eEF1B(αβγ)3. As both the eEF1Bα and eEF1Bβ proteins have structurally conserved GEF domains, their total number within the complex is equal to six. Such, so far, unique structural assembly of the guanine-nucleotide exchange factors within a stable complex may be considered as a ‘GEF hub’ that ensures efficient maintenance of the translationally active GTP-bound conformation of eEF1A in higher eukaryotes.

Aim.To study the translation elongation factor eEF1B gamma (eEF1Bγ) in the nucleus of lung carcinoma cells. Methods. The protein partners of eEF1Bγin the nuclear fraction of A549 cells were identified by co-immunoprecipitation (co-IP) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The protein interaction network for nuclear eEF1Bγ was determined by Cytoscape 3.2.0 program using the MCODE plugin. Additional analysis of the eEF1Bγ partners was conducted by Map of the cell database. Results. 234 proteins interacting with eEF1Bγ in the nuclear fraction of A549 cells were identified. Possible functional networks involving these contacts were analyzed by two bioinformatic approaches. Conclusions. Splicing of pre-mRNA and regulation of mRNA stability are assumed to be the main processes in which nuclear eEF1Bγ can be involved. We hypothesize that a portion of eEF1Bγ leaves the cytoplasmlocalizede EF1B complex during carcinogenesis and enters the nucleus to regulate certain mRNAs by affecting the splicing of their pre-mRNA and/or stability of mRNA. K e y w o r d s: eEF1Bγ, protein-protein interactions, nucleus, A549 cell Structure and Function of Biopolymers

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