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Identification and In Silico Analysis of Major Redox Modulated Proteins from Brassica juncea Seedlings Using 2D Redox SDS PAGE (2-Dimensional Diagonal Redox Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis)

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Abstract

The thiol–disulphide exchange regulates the activity of proteins by redox modulation. Many studies to analyze reactive oxygen species (ROS), particularly, hydrogen peroxide (H2O2) induced changes in the gene expression have been reported, but efforts to detect H2O2 modified proteins are comparatively few. Two-dimensional diagonal redox sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) was used to detect polypeptides which undergo thiol–disulphide exchange in Brassica juncea seedlings following H2O2 (10 mM) treatment for 30 min. Eleven redox responsive polypeptides were identified which included cruciferin, NLI [Nuclear LIM (Lin11, Isl-1 & Mec-3 domains)] interacting protein phosphatase, RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) large subunit, and myrosinase. Redox modulation of RuBisCO large subunit was further confirmed by western blotting. However, the small subunit of RuBisCO was not affected by these redox changes. All redox modulated targets except NLI interacting protein (although it contains two cysteines) showed oxidation sensitive cysteines by in silico analysis. Interestingly, interactome of cruciferin and myrosinase indicated that they may have additional function(s) beside their well-known roles in the seedling development and abiotic stress respectively. Cruciferin showed interactions with stress associated proteins like defensing-like protein 192 and 2-cys peroxiredoxin. Similarly, myrosinase showed interactions with nitrilase and cytochrome p450 which are involved in nitrogen metabolism and/or hormone biosynthesis. This simple procedure can be used to detect major stress mediated redox changes in other plants.

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Abbreviations

ROS:

Reactive oxygen species

H2O2 :

Hydrogen peroxide

2D Diagonal Redox SDS PAGE:

2-Dimensional redox sodium dodecyl sulfate polyacrylamide gel electrophoresis

RuBisCO:

Ribulose-1, 5-bisphosphate carboxylase/oxygenase

References

  1. Xie LH, Chen F, Karagueuzian HS, Weiss JN (2009) Oxidative stress-induced after depolarizations and calmodulin kinase II signaling. Circ Res 104:79–86

    Article  CAS  Google Scholar 

  2. Hasanuzzaman M, Nahar K, Alam MM, Roychowdhury R, Fujita M (2013) Physiological, biochemical, and molecular mechanisms of heat stress tolerance in plants. Int J Mol Sci 14(5):9643–9684

    Article  Google Scholar 

  3. Hossain MA, Bhattacharjee S, Armin SM, Qian P, Wang X, Li HY, Burritt DJ, Fujita M, Tran LS (2015) Hydrogen peroxide priming modulates abiotic oxidative stress tolerance: insights from ROS detoxification and scavenging. Front Plant Sci 6:420

    Google Scholar 

  4. Janssen-Heininger YMW, Aesif SW, Velden J, Guala AS, Reiss JN, Roberson EC, Budd RC, Reynaert NL, Anathy V (2010) Regulation of apoptosis through cysteine oxidation: implications for fibrotic lung disease. Ann N Y Acad Sci 1203:23–28

    Article  CAS  Google Scholar 

  5. García-Santamarina S, Boronat S, Hidalgo B (2014) Reversible cysteine oxidation in hydrogen peroxide sensing and signal transduction. BioChemistry 53:2560–2580

    Article  Google Scholar 

  6. Rinalducci S, Murgiano L, Zolla L (2008) Redox proteomics: basic principles and future perspectives for the detection of protein oxidation in plants. J Exp Bot 59:781–801

    Article  Google Scholar 

  7. Nadeau PJ, Charette SJ, Toledano MB, Landry J (2007) Disulfide bond-mediated multimerization of Ask1 and its reduction by thioredoxin-1 regulate H2O2-induced c-Jun NH2-terminal kinase activation and apoptosis. Mol Biol Cell 18:3903–3913

    Article  CAS  Google Scholar 

  8. Reczek CR, Chandel NS (2015) ROS-dependent signal transduction. Cur Opin Cell Biol 33:8–13

    Article  CAS  Google Scholar 

  9. Stroher E, Dietz KJ (2008) The dynamic thiol–disulphide redox proteome of the Arabidopsis thaliana chloroplast as revealed by differential electrophoretic mobility. Physiol Plant 133:566–583

    Article  Google Scholar 

  10. Sethuraman M, Comb Mc, Huang ME, Huang H, Heibeck T, Costello CE, Cohen RA (2004) Isotope-coded affinity tag (ICAT) approach to redox proteomics: identification and quantitation of oxidant-sensitive cysteine thiols in complex protein mixtures. J Proteome Res 6:1228–1233

    Article  Google Scholar 

  11. Liu P, Zhang H, Wang H, Xia Y (2014) Identification of redox-sensitive cysteines in the Arabidopsis proteome using OxiTRAQ, a quantitative redox proteomics method. Proteomics 14:750–762

    Article  Google Scholar 

  12. Wong JH, Balmer Y, Cai N, Tanaka CK, Vensel WH, Hurkman WJ, Buchanan BB (2003) Unraveling thioredoxin-linked metabolic processes of cereal starchy endosperm using proteomics. FEBS Lett 547:151–156

    Article  CAS  Google Scholar 

  13. Wong JH, Cai N, Balmer Y, Tanaka CK, Vensel WH, Hurkman WJ, Buchanan BB (2004) Thioredoxin targets of developing wheat seeds identified by complementary proteomic approaches. Phytochemistry 65:1629–1640

    Article  CAS  Google Scholar 

  14. Alkhalfioui F, Renard M, Vensel WH, Wong J, Tanaka CK, Hurkman WJ, Buchanan BB, Montrichard F (2007) Thioredoxin-linked proteins are reduced during germination of Medicago truncatula seeds. Plant Physiol 144:1559–1579

    Article  CAS  Google Scholar 

  15. Bykova NV, Hoehn B, Rampitsch C, Hu J, Stebbing JA, Knox R (2011) Thiol redox-sensitive seed proteome in dormant and non-dormant hybrid genotypes of wheat. Phytochemistry 10:1162–1172

    Article  Google Scholar 

  16. Alvarez S, Zhu M, Chen S (2009) Proteomics of Arabidopsis redox proteins in response to methyl jasmonate. J Proteomics 73:30–40

    Article  CAS  Google Scholar 

  17. Wang H, Wang S, Lu Y, Alvarez S, Hicks LM, Ge X, Xia Y (2012) Proteomic analysis of early-responsive redox-sensitive proteins in Arabidopsis. J Proteome Res 11:412–424

    Article  CAS  Google Scholar 

  18. Galant A, Koester RP, Ainsworth EA, Hicks LM, Jez JM (2012) From climate change to molecular response: redox proteomics of ozone-induced responses in soybean. New Phytol 1:220–229

    Article  Google Scholar 

  19. Hägglund P, Bunkenborg J, Maeda K, Svensson B (2008) Identification of thioredoxin disulfide targets using a quantitative proteomics approach based on isotope-coded affinity tags. J Proteome Res 12:5270–5276

    Article  Google Scholar 

  20. Sehrawat A, Deswal R (2014) S-nitrosylation analysis in Brassica juncea apoplast highlights the importance of nitric oxide in cold-stress signaling. J Proteome Res 7(12):2599–2619

    Article  Google Scholar 

  21. Muthuramalingam M, Matros A, Scheibe R, Hans-Peter M, Karl-Josef D (2013) The hydrogen peroxide-sensitive proteome of the chloroplast in vitro and in vivo. Front Plant Sci 4:1–14

    Article  Google Scholar 

  22. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–254

    Article  CAS  Google Scholar 

  23. Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76:4350–4354

    Article  CAS  Google Scholar 

  24. EDBCP tool. http://omictools.com. Accessed 11 Aug 2016

  25. DiANNA 1.1 web server. http://clavius.bc.edu. Accessed 11 Aug 2016

  26. CYS_REC program. http://www.softberry.com. Accessed 16 Aug 2016

  27. Trujillo M, Alvarez B, Radi R (2015) One- and two-electron oxidation of thiols: mechanisms, kinetics and biological fates. Free Radic Res 50:150–171

    Article  Google Scholar 

  28. Poole LB (2015) The basics of thiols and cysteines in redox biology and chemistry. Free Radic Biol Med 0:148–157

    Article  CAS  Google Scholar 

  29. Lin HH, Hsu J-C, Hsu Y-N, Pan R-H, Chen Y-F, Tseng L-Y (2013) Disulfide connectivity prediction based on structural information without a prior knowledge of the bonding state of cysteines. Comput Biol Med 43:941–1948

    Article  Google Scholar 

  30. Jones DT (1999) Protein secondary structure prediction based on position-specific scoring matrices. J Mol Biol 292:195–202

    Article  CAS  Google Scholar 

  31. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402

    Article  CAS  Google Scholar 

  32. Ferrè F, Clote P (2005) Disulfide connectivity prediction using secondary structure information and diresidue frequencies. Bioinformatics 21:2336–2346

    Article  Google Scholar 

  33. Principle of DiANNA (2016) Softberry Inc, USA. http://linux1.softberry.com. Accessed 16 Aug 2016

  34. Wittstocka U, Burowb M (2010) Glucosinolate breakdown in Arabidopsis: mechanism, regulation and biological significance. Arabidopsis Book. doi:10.1199/tab.0134

    Google Scholar 

  35. Yano H, Wong JH, Cho MJ, Bob B (2001) Redox changes accompanying the degradation of seed storage proteins in germinating rice. Plant Cell Physiol 42:879–883

    Article  CAS  Google Scholar 

  36. Bassham J, Benson A, Calvin M (1950) The path of carbon in photosynthesis. J Biol Chem 185:781–787

    CAS  Google Scholar 

  37. Berg JM, Tymoczko JL, Stryer L (2002) Biochemistry, 5th edn. W H Freeman, New York

  38. García-Ferris C, Moreno J (1994) Oxidative modification and breakdown of ribulose 1,5-bisphosphate carboxylase/oxygenase induced in Euglena gracilis by nitrogen starvation. Planta 193:208–215

    Article  Google Scholar 

  39. Ferreira RB, Davies DD (1989) Conversion of ribulose-1,5-bisphosphate carboxylase to an acidic and catalytically inactive form by extracts of osmotically stressed Lemna fronds. Planta 179:448–455

    Article  CAS  Google Scholar 

  40. García-Ferris C, Moreno J (1993) Redox regulation of enzymatic activity and proteolytic and susceptibility of ribulose-1,5-bisphosphate carboxylase/oxygenase from Euglena gracilis. Photosynth Res 35:55–66

    Article  Google Scholar 

  41. Moreno J, Penarrubia L, Garcia-Ferris C (1995) The mechanism of redox regulation of ribulose-1,5-bisphosphate carboxylase/oxygenase turnover. A hypothesis. Plant Physiol Biochem 33:121–127

    CAS  Google Scholar 

  42. Schloss JV, Stringer CD, Hartman FC (1978) Identification of essential lysyl and cysteinyl residues in spinach ribulosebisphosphate carboxylase/oxygenase modified by the affinity label N-bromoacetylethanolamine phosphate. J Biol Chem 253:5707–5711

    CAS  Google Scholar 

  43. Julia M-N, Moreno J (2006) Cysteines 449 and 459 modulate the reduction–oxidation conformational changes of ribulose 1·5-bisphosphate carboxylase/oxygenase and the translocation of the enzyme to membranes during stress. Plant Cell Environ 29:898–908

    Article  Google Scholar 

  44. Moreno J, García-Murria MJ, Marín-Navarro J (2008) Redox modulation of Rubisco conformation and activity through its cysteine residues. J Exp Bot 59:1605–1614

    Article  CAS  Google Scholar 

  45. Marcus Y, Altman-Gueta H, Finkler A, Gurevitz M (2003) Dual role of cysteine 172 in redox regulation of ribulose 1,5-bisphosphate carboxylase/oxygenase activity and degradation. J Bacteriol 85:1509–1517

    Article  Google Scholar 

  46. Abat JK, Mattoo AK, Deswal R (2008) S-nitrosylated proteins of a medicinal CAM plant Kalanchoe pinnata—ribulose-1,5-bisphosphate carboxylase/oxygenase activity targeted for inhibition. FEBS J 275:2862–2872

    Article  CAS  Google Scholar 

  47. Abat K, Deswal R (2009) Differential modulation of S-nitrosoproteome of Brassica juncea by low temperature: change in S-nitrosylation of Rubisco is responsible for the inactivation of its carboxylase activity. Proteomics 9:4368–4380

    Article  CAS  Google Scholar 

  48. Job C, Rajjou L, Lovigny Y, Belghazi M, Job D (2005) Patterns of protein oxidation in Arabidopsis seeds and during germination. Plant Physiol 138:790–802

    Article  CAS  Google Scholar 

  49. Thomas N, Dudkina NV, Haase C, Denolf P, Semchonok DA, Boekema EJ, Braun HP, Sunderhaus S (2013) The native structure and composition of the Cruciferin complex in Brassica napus. J Biol Chem 288:2238–2245

    Article  Google Scholar 

  50. Bernardi R, Finiguerra MG, Rossi AA, Palmieri S (2003) Isolation and biochemical characterization of a basic myrosinase from ripe Crambe abyssinica seeds, highly specific for epi-progoitrin. J Agric Food Chem 51:2737–2744

    Article  CAS  Google Scholar 

  51. Burgmeister W, Cottaz S, Driguez H, Iori R, Palmieri S, Henrissat B (1997) The crystal structures of Sinapis alba myrosinase and a covalent glycosyl-enzyme intermediate provide insights into the substrate recognition and active-site machinery of an S-glycosidase. Structure 5:663–675

    Article  Google Scholar 

  52. Jurata LW, Pfaff SL, Gill GN (1998) The nuclear LIM domain interactor NLI mediates homo- and heterodimerization of LIM domain transcription factors. J Biol Chem 273:3152–3157

    Article  CAS  Google Scholar 

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Acknowledgements

Funding for this work was provided by University Grant Commission (UGC), India in the form of Non-Net fellowship and by University of the Delhi, Research & Development Grant.

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  1. Molecular Physiology and Proteomics Laboratory, Department of Botany, University of Delhi, New Delhi, India

    Satya Prakash Chaurasia & Renu Deswal

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  1. Satya Prakash Chaurasia
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Correspondence to Renu Deswal.

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Antibodies were raised using two male rabbits following guidelines from the Canadian Council on Animal Care.

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Chaurasia, S.P., Deswal, R. Identification and In Silico Analysis of Major Redox Modulated Proteins from Brassica juncea Seedlings Using 2D Redox SDS PAGE (2-Dimensional Diagonal Redox Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis). Protein J 36, 64–76 (2017). https://doi.org/10.1007/s10930-017-9698-x

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