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

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Resumo

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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Sobre autores

E. Ivanova

Pirogov Russian National Research Medical University

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

R. Zyganshin

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

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

E. Parshina

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

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

A. Zaraisky

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

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

N. Martynova

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

Autor responsável pela correspondência
Email: martnat61@gmail.com
Rússia, ul. Miklukho-Maklaya 16/10, Moscow, 117997

Bibliografia

  1. Beckerle M.C. // Bioessays. 1997. V. 19. V. 949−957. https://doi.org/10.1002/bies.950191104
  2. Hirata H., Tatsumi H., Sokabe M. // Commun. Integr. Biol. 2008. V. 1. P. 192–195. https://doi.org/10.4161/cib.1.2.7001
  3. Hirata H., Tatsumi H., Sokabe M. // J. Cell Sci. 2008. V. 121. P. 2795−2804. https://doi.org/10.1242/jcs.030320
  4. Nix D.A., Beckerle M.C. // J. Cell Biol. 1997. V. 138. P. 1139−1147. https://doi.org/10.1083/jcb.138.5.1139
  5. Moody J.D., Grange J., Ascione M.P., Boothe D., Bushnell E., Hansen M.D. // Biochem. Biophys. Res. Commun. 2009. V. 378. P. 625–628. https://doi.org/10.1016/j.bbrc.2008.11.100
  6. Zhou J., Zeng Y., Cui L., Chen X., Stauffer S., Wang Z., Yu F., Lele S.M., Talmon G.A., Black A.R., Chen Y., Dong J. // Proc. Natl. Acad. Sci. USA. 2018. V. 115. P. E6760−E6769. https://doi.org/10.1073/pnas.1800621115
  7. Zhao Y., Yue S., Zhou X., Guo J., Ma S., Chen Q. // J. Biol. Chem. 2022. V. 298. P. 101776. https://doi.org/10.1016/j.jbc.2022.101776
  8. Siddiqui M.Q., Badmalia M.D., Patel T.R. // Int. J. Mol. Sci. 2021. V. .22. P. 2647. https://doi.org/10.3390/ijms22052647
  9. Nix D.A., Fradelizi J., Bockholt S., Menichi B., Louvard D., Friederich E., Beckerle M.C. // J. Biol. Chem. 2001. V. 276. P. 34759−34767. https://doi.org/10.1074/jbc.M102820200
  10. Uemura A., Nguyen T.N., Steele A.N., Yamada S. // Biophys. J. 2011. V. 101. P. 1069−1075. https://doi.org/10.1016/j.bpj.2011.08.001
  11. Drees B.E., Andrews K.M., Beckerle M.C. // J. Cell Biol. 1999. V. 147. P. 1549−1560. https://doi.org/10.1083/jcb.147.7.1549
  12. Li B., Trueb B. // J. Biol. Chem. 2001. V. 276. P. 33328− 33335. https://doi.org/10.1074/jbc.M100789200
  13. Drees B., Friederich E., Fradelizi J., Louvard D., Beckerle M.C., Golsteyn R.M. // J. Biol. Chem. 2000. V. 275. P. 22503−22511. https://doi.org/10.1074/jbc.M001698200
  14. Golsteyn R.M., Beckerle M.C., Koay T., Friederich E. // J. Cell. Sci. 1997. V. 110. P. 1893−1906. https://doi.org/10.1242/jcs.110.16.1893
  15. Smith M.A., Hoffman L.M., Beckerle M.C. // Cell Biol. 2014. V. 24. P. 575−583. https://doi.org/10.1016/j.tcb.2014.04.009
  16. Martynova N.Y., Parshina E.A., Ermolina L.V., Zaraisky A.G. // Biochem. Biophys. Res. Commun. 2018. V. 504. P. 251–256. https://doi.org/10.1016/j.bbrc.2018.08.164
  17. Martynova N.Y., Ermolina L.V., Ermakova G.V., Eroshkin F.M., Gyoeva F.K., Baturina N.S., Zaraisky A.G. // Dev. Biol. 2013. V. 380. P. 37−48. https://doi.org/10.1016/j.ydbio.2013.05.005
  18. Li N., Goodwin R.L., Potts J.D. // Microsc. Microanal. 2013. V. 19. P. 842−854. https://doi.org/10.1017/S1431927613001633
  19. Hoffman L.M., Nix D.A., Benson B., Boot-Hanford R., Gustafsson E., Jamora C., Menzies A.S., Goh K.L., Jensen C.C., Gertler F.B., Fuchs E., Fässler R., Beckerle M.C. // Mol. Cell Biol. 2003. V. 23. P. 70−79. https://doi.org/10.1128/MCB.23.1.70−79.2003
  20. Rauskolb C., Pan G., Reddy B.V., Oh H., Irvine K.D. // PLoS Biol. 2011. V. 9. P. e1000624. https://doi.org/10.1371/journal.pbio.1000624
  21. Gaspar P., Holder M.V., Aerne B.L., Janody F., Tapon N. // Curr. Biol. 2015. V. 25. P. 679−689. https://doi.org/10.1016/j.cub.2015.01.010
  22. Martynova N.Y., Eroshkin F.M., Ermolina L.V., Ermakova G.V., Korotaeva A.L, Smurova K.M., Gyoeva F.K., Zaraisky A.G. // Dev. Dyn. 2008. V. 237. P. 736−749. https://doi.org/10.1002/dvdy.21471
  23. Martynova N.U., Ermolina L.V., Eroshkin F.M., Zarayskiy A.G. // Bioorg. Khim. 2015. V. 41. P. 744− 748. https://doi.org/10.1134/s1068162015060102
  24. Parshina E.A., Eroshkin F.M., Оrlov E.E., Gyoeva F.K., Shokhina A.G., Staroverov D.B., Belousov V.V., Zhigalova N.A., Prokhortchouk E.B., Zaraisky A.G., Martynova N.Y. // Cell Rep. 2020. V. 33. P. 108396. https://doi.org/10.1016/j.celrep.2020.108396
  25. Ivanova E.D., Parshina E.A., Zaraisky A.G. Martynova N.Y. // Russ. J. Bioorg. Chem. 2024. V. 50. P. 723–732. https://doi.org/10.1134/S1068162024030026
  26. Aebersold R., Mann M. // Nature. 2003. V. 422. P. 198– 207. https://doi.org/10.1038/nature01511
  27. Mann M., Wilm M. // Anal. Chem. 1994. V. 66. P. 4390− 4399. https://doi.org/10.1021/ac00096a002
  28. Eng J.K., Searle B.C., Clauser K.R., Tabb D.L. // Mol. Cell Proteomics. 2011. V. 10. P. R111.009522. https://doi.org/10.1074/mcp.R111.009522
  29. Mann M., Ong S.E., Grønborg M., Steen H., Jensen O.N., Pandey A. // Trends Biotechnol. 2002. V. 20. P. 261−268. https://doi.org/10.1016/s0167−7799(02)01944−3
  30. Groen A., Thomas L., Lilley K., Marondedze C. // Methods Mol. Biol. 2013. V. 1016. P. 121−137. https://doi.org/10.1007/978-1-62703-441-8_9
  31. Maynard J.C., Chalkley R.J. // Mol. Cell Proteomics. 2021. V. 20. P. 100031. https://doi.org/10.1074/mcp.R120.002206
  32. Shevchenko A., Tomas H., Havlis J., Olsen J.V., Mann M. // Nat. Protoc. 2006. V. 1. P. 2856−2860. https://doi.org/10.1038/nprot.2006.468
  33. Ma B., Zhang K., Hendrie C., Liang C., Li M., DohertyKirby A., Lajoie G. // Rapid Commun. Mass Spectrom. 2003. V. 17. P. 2337−2342. https://doi.org/10.1002/rcm.1196
  34. Rappsilber J., Mann M., Ishihama Y. // Nat. Protoc. 2007. V. 2. P. 1896−1906. https://doi.org/10.1038/nprot.2007.261
  35. Nguyen K.T., Mun S.H., Lee C.S., Hwang C.S. // Exp. Mol. Med. 2018. V. 50. P. 1−8. https://doi.org/10.1038/s12276-018-0097-y
  36. Arnaudo N., Fernández I.S., McLaughlin S.H., PeakChew S.Y., Rhodes D., Martino F. // Nat. Struct. Mol. Biol. 2013. V. 20. P. 1119−1121. https://doi.org/10.1038/nsmb.2641
  37. Fujita Y., Yamaguchi A., Hata K., Endo M., Yamaguchi N., Yamashita T. // BMC Cell Biol. 2009. V. 27. P. 10− 16. https://doi.org/10.1186/1471-2121-10-6

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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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