Biopolym. Cell. 2023; 39(4):277-282.
http://dx.doi.org/10.7124/bc.000AA5
Genomics, Transcriptomics and Proteomics
The use of the in situ proximity ligation assay for validating S6 kinase 1 CoAlation under oxidative stress.
1Bdzhola A. V., 1Filonenko V. V., 1, 2Gout I. T., 1, 2Malanchuk O. M.
  1. Institute of Molecular Biology and Genetics, NAS of Ukraine
    150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03143
  2. University College London
    Gower Street, London WC1E 6BT, UK

Abstract

Aim. To verify CoAlation of ribosomal protein S6 kinase 1 (S6K1) in the HEK293/Pank1β cells exposed to oxidative stress using proximity ligation assay (PLA). Methods. In situ proximity ligation assay. Results. S6 kinase 1 undergoes CoA covalent modification in cellular response to oxidative stress. Conclusions. The previously developed mass spectrometry-based methodology allowed us to identify over 2100 CoAlated proteins in the cells exposed to oxidative or metabolic stress. Many protein kinases were found to be CoAlated, including S6K1. In this study, the proximity ligation assay was used to demonstrate a significant increase of S6K1 CoAlation under oxidative stress compared to untreated cells.
Keywords: CoA, S6 kinase 1, in situ proximity ligation assay, post-translation modification, oxidative stress, signal transduction, confocal microscopy, cell growth, antibodies
Full text: (PDF, in English)

References

[1] Leonardi R, Zhang YM, Rock CO, Jackowski S. Coenzyme A: back in action. Prog Lipid Res. 2005; 44(2-3):125-53.
[2] Malanchuk OM, Panasyuk GG, Serbin NM, Gout IT, Filonenko VV. Generation and characterization of monoclonal antibodies specific to Coenzyme A. Biopolym Cell. 2015; 31(3):187-92.
[3] Malanchuk OM, Bdzhola AV, Tykhonkova IO, Gout IT, Filonenko VV. Monoclonal antibodies to Coenzyme A. Biopolym Cell. 2022; 38(4):215-23.
[4] Tossounian MA, Baczynska M, Dalton W, Newell C, Ma Y, Das S, Semelak JA, Estrin DA, Filonenko V, Trujillo M, Peak-Chew SY, Skehel M, Fraternali F, Orengo C, Gout I. Profiling the Site of Protein CoAlation and Coenzyme A Stabilization Interactions. Antioxidants (Basel). 2022; 11(7):1362.
[5] Tsuchiya Y, Peak-Chew SY, Newell C, Miller-Aidoo S, Mangal S, Zhyvoloup A, Bakovic J, Malanchuk O, Pereira GC, Kotiadis V, Szabadkai G, Duchen MR, Campbell M, Cuenca SR, Vidal-Puig A, James AM, Murphy MP, Filonenko V, Skehel M, Gout I. Protein CoAlation: a redox-regulated protein modification by coenzyme A in mammalian cells. Biochem J. 2017; 474(14):2489-508.
[6] Gout I. Coenzyme A, protein CoAlation and redox regulation in mammalian cells. Biochem Soc Trans. 2018; 46(3):721-8.
[7] Tsuchiya Y, Zhyvoloup A, Baković J, Thomas N, Yu BYK, Das S, Orengo C, Newell C, Ward J, Saladino G, Comitani F, Gervasio FL, Malanchuk OM, Khoruzhenko AI, Filonenko V, Peak-Chew SY, Skehel M, Gout I. Protein CoAlation and antioxidant function of coenzyme A in prokaryotic cells. Biochem J. 2018; 475(11):1909-37.
[8] Tsuchiya Y, Byrne DP, Burgess SG, Bormann J, Baković J, Huang Y, Zhyvoloup A, Yu BYK, Peak-Chew S, Tran T, Bellany F, Tabor AB, Chan AE, Guruprasad L, Garifulin O, Filonenko V, Vonderach M, Ferries S, Eyers CE, Carroll J, Skehel M, Bayliss R, Eyers PA, Gout I. Covalent Aurora A regulation by the metabolic integrator coenzyme A. Redox Biol. 2020; 28:101318.
[9] Magnuson B, Ekim B, Fingar DC. Regulation and function of ribosomal protein S6 kinase (S6K) within mTOR signalling networks. Biochem J. 2012; 441(1):1-21.
[10] Hegazy M, Cohen-Barak E, Koetsier JL, Najor NA, Arvanitis C, Sprecher E, Green KJ, Godsel LM. Proximity Ligation Assay for Detecting Protein-Protein Interactions and Protein Modifications in Cells and Tissues in Situ. Curr Protoc Cell Biol. 2020; 89(1):e115.