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Registration Number

0225U004156, (0120U102238) , R & D reports

Title

Structural and functional studies of translation elongation factors of higher eukaryotes

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Порівняльне дослідження просторової організації ізоформи eEF1A1 із частково невпорядкованою структурою і онкогенної ізоформи eEF1A2 в GDP- і GTP- зв’язувальній формі методом воднево-дейтерієвого обміну із подальшою мас-спектрометрією.

Head

Negrutskyii Borys S., Доктор біологічних наук

Registration Date

20-11-2025

Organization

Institute of Molecular Biology and Genetics of NAS of Ukraine

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The aim of this study is to analyze the spatial organization and function of mammalian translation elongation factor 1 (eEF1A and the multi-protein eEF1B complex). To achieve this, the following tasks are planned. First, to obtain experimental data that will allow us to build atomic models of the spatial organization of the subunits of the eEF1B translation complex and the architecture of the complex as a whole. Second, to conduct experimental studies and to build the model of spatial organization of isoform 1 of elongation factor 1A based on them. Third, to determine the potential functional significance of methylation of lysine residues in the molecule of elongation factor 1A (eEF1A), in particular, to test the hypothesis experimentally that the methylation of some lysine residues in eEF1A may affect the interaction of this protein with partners , such as the subunits of the eEF1B complex, which may reveal the molecular mechanism of the recently discovered effect of eEF1A methylation on oncogenic cell transformation. Fourthly, describe the molecular mechanisms of action of novel eEF1A inhibitors with potential antitumor effect.

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Objects of study – the human translation factor complex eEF1B, the translation elongation factor eEF1A, its isoforms eEF1A1 and eEF1A2, and the elongation factors eEF1Bα, eEF1Bβ, and eEF1Bγ. Objective of the work – to elucidate the spatial organization of translation complexes and their components, and to investigate the potential role of post-translational modifications in the functioning of translation factors. Research methods – hydrogen–deuterium exchange followed by mass spectrometry (HDX-MS), analytical ultracentrifugation, protein mutagenesis, chromatographic purification of individual proteins, analytical gel filtration, bioluminescence resonance energy transfer (BRET) system, Western blotting, co-immunoprecipitation, protein co-precipitation, confocal microscopy, mass spectrometry, polymerase chain reaction (PCR), molecular biology methods for DNA manipulation, kinetic methods, bioinformatic analysis, molecular modeling, and molecular docking. Using HDX-MS analysis, it was shown that the isoform eEF1A1 of the translation elongation factor exhibits a significantly higher rate of hydrogen–deuterium exchange than the eEF1A2 isoform. Specific regions of the protein structures were identified that differ in exchange dynamics in the presence of various guanosine phosphates. Based on the HDX-MS data, a general mechanism of the negative impact of neurodegenerative eEF1A2 mutations was proposed, which involves disruptions in the local compactness of the protein’s spatial organization.

Product Description

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Novosylna Oleksandra V.
Hanna V. Yelska
Porublova Larysa V.
Tetiana V. Bondarchuk
Вячеслав Ф. Шалак
Oleksandr Y. Tsuvariev
Kolodka Nazar Tarasovych
Ganna Lukyanenko

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2025-11-20