Strategies Aimed at Nox4 Oxidase Inhibition Employing Peptides from Nox4 B-Loop and C-Terminus and p22phox N-Terminus: An Elusive Target
Figure 1
Nox4 B-loop peptides and Nox4 activity. (a) A simplified model of Nox4 oxidase with B- and D-loops located intracellularly. , the only known protein required for Nox4 activity, is also shown. (b) Amino acid sequence of Nox4 B-loop and list of overlapping Nox4 B-loop region synthetic 15-mer and nonamer peptides used to target Nox4 activity. The numbers at the N- and C-terminus of each peptide indicate the location of the corresponding residues in the amino acid sequence of Nox4. (c) H2O2 generated by Nox4 was measured using Amplex Red fluorescence. H2O2 production was initiated by the addition of 36 μM NADPH. The reaction was monitored at 24°C for 40 min. The flavin-containing enzyme inhibitor diphenyleneiodonium (DPI; 50 μM) was used as a positive control. Identification of H2O2 was confirmed by inhibition of fluorescence with catalase (3000 U/mL). For comparison, H2O2 production in nontransfected COS-22 cell lysate is shown. Data represent the mean ± SEM of 3 experiments. indicates significant difference between COS-22 and COS-Nox4 cell lysates. (d) COS-Nox4 cell lysates were preincubated with various concentrations of B90–98 (from 0.1 to 100 μM) for 15 min at 24°C, and H2O2 was measured using Amplex Red fluorescence. Data represent the mean ± SEM of 3 experiments. (e) COS-Nox4 cell lysates were preincubated with the R96E mutant version of B90–98 (PSRRTRELL; 0.3 to 30 μM) for 15 min at 24°C, and H2O2 was measured. Data represent the mean ± SEM of 3 experiments. (f) COS-Nox4 cell lysates were preincubated with peptides from N- and C-terminal ends of the Nox4 B-loop (B77–91, B82–96, and B92–106; 1 to 50 μM) for 15 min at 24°C, and H2O2 was measured. Data represent the means ± SEM of 3 experiments. indicates a significant difference between COS-22 and COS-Nox4 cell lysates. indicates a significant difference between COS-Nox4 + 0 μM peptide and COS-Nox4 + DPI.
