Mass spectrometric analysis of Xenopus laevis cytoskeletal protein zyxin post-translational modifications

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Abstract

In addition to its involvement in fundamental cellular processes, zyxin, a LIM-domain protein in the cytoskeletal system, is actively studied because it plays an important role in mechanosensory functions, actin polymerization regulation at cell junctions, as well as gene expression regulation. The disruption of zyxin expression and processing has been associated with carcinogenesis and cardiovascular disease. Zyxin plays an important role in the invasion and metastasis of tumors. The post-translational modification of zyxin in mammals regulates its activity and subcellular location. Given that zyxin is an evolutionarily highly conserved protein, we conducted a search for post-translational modifications of the zyxin homolog from Xenopus laevis using chromatographic mass spectrometry. To identify modified peptides, an enrichment method was employed using co-immunoprecipitation of endogenous zyxin from gastrula-stage embryonic cell lysates. As a result, previously unknown modifications of this protein were discovered, specifically N-terminal acetylation at methionine position 1 and phosphorylation at Ser197 and Ser386. To identify zyxin isoforms with different electrophoretic mobilities, separation was performed using polyacrylamide gel electrophoresis. Zyxin was found in bands with electrophoretic mobilities of 70 and 105 kDa. Thus, this study presents entirely new data on the post-translational modifications of zyxin from X. laevis. Since defects in mechanical signal transduction are associated with developmental disorders, oncogenesis, and metastasis, the study of mechanosensitive protein zyxin modifications and processing on the model organism X. laevis opens up opportunities for diagnostic studies at the molecular level, which can be used in the future to determine drugs use prospective in pharmacology.

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About the authors

E. D. Ivanova

Pirogov Russian National Research Medical University

Email: martnat61@gmail.com
Russian Federation, ul. Ostrovitianova 1, Moscow, 117997

R. H. Zyganshin

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences

Email: martnat61@gmail.com
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

E. A. Parshina

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences

Email: martnat61@gmail.com
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

A. G. Zaraisky

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences

Email: martnat61@gmail.com
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

N. Y. Martynova

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences

Author for correspondence.
Email: martnat61@gmail.com
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

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2. Fig. 1. Determination of zyxin isoforms with different electrophoretic mobility. (a) – Peptides that were identified in the bands with electrophoretic mobility of 105 kDa (23 peptides) and 70 kDa (2 peptides). The number of peptides was estimated by the ratio of the area of ​​the chromatographic peak in the mass spectrum (Area) to the sum of the areas of all identified peaks in the sample, in fractions of a percent; (b) – electrophoresis of the embryo lysate in 10% SDS-PAAG with staining of ½ gel with Coomassie and Western blotting with staining with antibodies to zyxin. The gel strips corresponding to the bands stained with antibodies to zyxin, corresponding to the molecular weight of 70 and 105 kDa, were cut out. Additional bands on the Western blotting are the background; (в) – quantitative assessment of zyxin distribution by bands after Western blotting (Image G program). The percentage content of (а) in the peptides identified in the 70 kDa band is an order of magnitude lower than that of similar peptides in the 105 kDa band, which coincides with the Image G program data on the assessment of the intensity of the 70 and 105 kDa bands in Western blotting.

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3. Fig. 2. Scheme of the arrangement of the identified modified residues in the zyxin molecule. The modified amino acid residues Met1, Ser197 and Ser386 are shown in the frame of the scheme. The scheme also contains constant modifications required for the analysis: Cys – carbamidomethylation (C in the scheme), as well as variable modifications: oxidation of Met (O in the scheme).

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