title: 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).
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 H2O2modified 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.
authors.affiliation: Molecular Physiology and Proteomics Laboratory, Department of Botany, University of Delhi, New Delhi, India.
authors.forename: Satya Prakash
authors.initials: SP
authors.lastname: Chaurasia
authors.affiliation: Molecular Physiology and Proteomics Laboratory, Department of Botany, University of Delhi, New Delhi, India. rdeswal@botany.du.ac.in.
authors.forename: Renu
authors.initials: R
authors.lastname: Deswal
mesh_terms: D015180:Electrophoresis, Gel, Two-Dimensional; D009149:Mustard Plant / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry / Q000378:metabolism*; D036226:Seedlings / Q000737:chemistry / Q000378:metabolism*
chemical_list: D012273:Ribulose-Bisphosphate Carboxylase
keywords: Brassica juncea; Cruciferin; Hydrogen peroxide; Myrosinase; Sodium dodecyl sulfate polyacrylamide gel electrophoresis; Thiol–disulfide exchange