oa Qatar Cultivated Vegetables Enhance Resistance To Protein Modifications Induced By Altered Redox Homeostasis

Oxygen is essential for the survival of all aerobic organisms but it is also, paradoxically, a toxic, mutagenic gas. Molecular oxygen is highly reactive and its partial reduction generates reactive oxygen species (ROS). Under normal physiological conditions, ROS are produced in low amounts as a result of active aerobic metabolism and have an important role as signaling molecules. At higher concentrations, ROS are cytotoxic and damage macromolecules. Proteins are major targets of ROS, resulting in a change in the structure and/or function of the oxidized proteins. Dietary antioxidants have been shown to modulate the extent of protein oxidation in plasma. However, the significance of dietary antioxidants in preventing vital oxidative changes of proteins still remains unknown.Therefore, in the present work we have evaluated the antioxidant significance of cucumber and zucchini extracts in the prevention of oxidative modification of proteins in human keratinocytes. Vegetables were collected from the farm in Qatar and their juice was extracted by a heavy-duty juicer, with a stone mill-like screw, using a low speed of 80 RPMs resulting in minimal heat build up and oxidation of samples. Copper sulphate, a well known inducer of lipid peroxidation, was used to alter redox homeostasis in human keratinocytes. Cells were treated with copper sulphate (LD22.5) and vegetable extracts were used to prevent oxidative damage of proteins. After 24 hours, the ability of extracts to inhibit copper induced cell death was evaluated by MTT assay and the proteins were isolated for redox proteomics analysis. Proteins were then separated by 1-D gel electrophoresis, digested with trypsin and peptides were detected by Orbitrap LC-MS/MS. Afterwards, the MS/MS data were searched using Proteome Discoverer 1.4 against Homo sapiens database incorporating the comprehensive workflow that included several dynamic modifications of peptides, like N-terminal carboxymethyl, C-terminal oxidation and terminal independant oxidation and trioxidation of peptides. The results obtained showed that cucumber and zucchini extracts were able to inhibit copper induced cell death, LD0 and LD12 respectively. Furthermore, the results obtained revealed several peptide modifications that were inhibited by the vegetable extracts and associated with inhibition of copper induced cell death. Finally, future identification and quantification of redox-based changes within the proteome both in redox signaling and under oxidative stress conditions and their inhibition by dietary antioxidants represents new and exciting field that will have a significant impact on a number of pathophysiological conditions and aging