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Registration Number

0213U006512, 0112U000995 , R & D reports

Title

The A2 isoform of translation elongation factor 1 as a potential oncomarker

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Head

Shalak V.F.,

Registration Date

14-10-2013

Organization

Institute of Molecular Biology and Genetics of NASU

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Translation elongation factor 1A (eEF1A) is a key component of the apparatus of protein synthesis in eukaryotic cells. There are two isoforms of translation elongation factor 1A, eEF1A1 and eEF1A2 in short, which are 92%identical. eEF1A1 is expressed in most tissues and cells, with the exception of skeletal muscle, cardiac muscle and neurons, where eEF1A2 is expressed. In the case of a failure in expression of the second isoform, namely, when in the normal cells the "silent" gene encoding eEF1A2 begins its expression; it may result in malignant transformation of cells. At present, monoclonal antibodies preparations, which recognize both isoforms, are commercially available. Evidently, that the problem of selective determination of eEF1A2 cannot be solved by these antibodies. In the world there is only one laboratory that has own antibody against eEF1A2 and use it for the immunological studies. However, it should be noted that these antibodies are not commercialized and, therefore, are not yet available for use by other researchers. The aim of project was to generate specific antibodies against A2 isoform of translation elongation factor eЕF1, detect this isoform in tumors of different localization and search for signaling pathways by means of which eЕF1А2 may influence the malignant transformation of cell. To attain the aim of the project we choose using of short peptides (about 10-16 aminoacid residues) derived from eEF1А2 sequence as a main component of immunogenic constructions. We compared amino acid sequences of eEF1А1 and eEF1А2 and on the base of three-dimensional models of these factors we selected the peptides which were different and at the same time situated on the protein surface to be accessible to antibodies. Selected peptides of eEF1A2 were conjugated to a carrier protein bovine serum albumin and were used for animal immunization to generate antibodies. These antibodies were purified and their specificity was tested by ELISA, immunoblotting and imunohistochemistry. It was shown that purified antibodies recognized eEF1A2 in the individual state and in the crude cell and tissue extracts and didn't demonstrate any cross-interaction with eEF1A1. High specificity of antibodies against eEF1A2 allowed us to use them for screening of histological samples of tumors of different localization by the method of imunohistochemistry and tumor extracts by immunoblotting. As a result, we found the presence of eEF1A2 protein in 90% of lung cancer samples, approximately 70% of human gastric cancer specimens and in all the samples of human breast cancer. We also used the antibodies against eEF1A2 to identify this protein in the extracts of cells that grow in culture. A clear positive signal on western-blot was obtained in human breast carcinoma cell extracts and mouse fibroblasts. The results indicate that in cancer cell lines, except HeLa cells, eEF1A2 isoform is also present. We failed to detect eEF1A2 in HEK293 cell line extracts, but it should be noted that this cell line is not cancerous but only immortalized. Thus, the results of our study confirm the assumption that eEF1A2 can serve as one of the tumor markers, whose presence in the postoperative samples can confirm the malignant nature of the tumor. To determine the protein-partners of eEF1A2 human breast carcinoma cell line was selected. First, these cells are malignant and, secondly, in the postoperative samples of breast tumors the presence of eEF1A2 was determined by immunohistochemistry. These cells were transfected by created genetic constructs expressing eEF1A1 and eEF1A2 fused to HaloTag. The resulting protein complexes were separated on polyacrylamide gel in denaturing conditions and the protein bands were selected for further identification by mass spectrometry. To study the interaction of both isoforms of translation elongation factor 1A with protein partners in vitro, we chose three proteins involved in cell signaling pathway, in the organization of the cell cytoskeleton and phenomenon of RNA dependent interference. This is, in particular, calmodulin, protein Argonaut, and a new protein Sgt1, whose interaction with translation elongation factor we have recently discovered. We first found that eEF1A2 isoform can form a complex with the protein Argonaut using an antibody specific to eEF1A2. In addition, we determined that eEF1A2 forms a complex with a second protein isoform of Argonaut during mitotic phase of the cell cycle.

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Єльська Г. В.
Вісловух А.А
Власенко Д.О
Негруцький Б.С.
Новосильна О.В
Порубльова Л.В

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2020-04-02