Delivery of superoxide dismutase by TAT and abalone peptides for the protection of skin cells against oxidative stress

Abstract

Trichoderma reesei superoxide dismutase (TrSOD) is a well-characterized enzyme being stable between 30 and 90 • C for 1 h with activity at pH between 2.6 and 9.0. This work aimed to clone, express, purify, and evaluate the protective effect antioxidant of this enzyme on skin cells when fused to transactivator of transcription (TAT) protein transduction domain of HIV-1 and abalone (Ab) peptides to allow cell penetration. TrSOD, TAT-TrSOD-Yfp (fused to yellow fluorescent protein), and Ab-TrSOD were expressed in E. coli and purified as soluble proteins. The cytotoxicity of the enzymes, at the concentrations of 1, 3, and 6 μmol/L, was evaluated for a period of 24 and 48 h of incubation, with no cytotoxic effect on 3T3 fibroblasts. The 3T3 cells were exposed to the oxidant agent tert-butyl hydroperoxide and evaluated for reactive oxygen species (ROS) generation, in the presence or not of the recombinant enzymes. TAT-TrSOD-Yfp was able to decrease the generation of ROS by 15% when used in the concentrations of 3 and 6 μmol/L in comparison to the control, but there was no difference in relation to the effect of TrSOD. Ab-TrSOD, when compared to TrSOD, promoted a decrease in the formation of ROS of 19% and 14% at the concentrations of 1 and 6 μmol/L, respectively, indicating that this recombinant form was more effective in reducing oxidative stress compared to SOD without the cell-penetrating peptide (CPP). Together, these results indicate that the fusion of SOD with these CPP increased the antioxidant capacity of fibroblasts, identified by the reduction in the generation of ROS. In addition, such molecules, in the concentrations initially used, were not toxic to the cells, opening perspectives for the development

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“…In vitro toxicity: Vaccine cytotoxicity was evaluated according to [19]. Briefly, 3T3 fibroblasts, CALU-3 lung cells, primary human fibroblasts (HLF), and rat lung cells (pneumocytes) were added to flat-bottom 96-well plates (Corning, Salt Lake City, UT, USA) at a concentration of 8000 cells per well.…”

Tragacanth Gum and Linseed Gum as Adhesives Improved the Survival, Digestive Function, Antioxidant Enzyme Activities, and Immunity in Large Yellow Croaker (Larimichthys crocea) Larvae

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Neuroprotective Peptides and New Strategies for Ischemic Stroke Drug Discoveries

“…CPPs typically contain more than five positively charged amino acids. Most cationic CPPs are derived from natural TAT (YGRKKRRQRRR) and penetratin (RQIKIWFQNRRMKWKK) peptides [53,66].…”