Abstract 16845: Peroxiredoxin 1 Protects DDAH Activity During Oxidative Stress

Abstract

Peroxiredoxins (Prdxs) are a family of small nonseleno peroxidases that catalyze the reduction of H 2 O 2 , organic hydroperoxides and peroxynitrite. Prx 1 is the most abundant and ubiquitously distributed member of mammalian Prx. In macrophages, nitric oxide (NO) increases Prx1 expression, inhibits Prx overoxidation, and increases Sulfiredoxin (SRX) expression (SRX is the major macrophage sulfinyl reductase that regenerates oxidized 2-cys Prxs to the active form). DDAH1 is the major enzyme that degrades the endogenous NOS inhibitor ADMA and thereby plays an important role in maintaining normal endothelial function. Using co-immunoprecipitation and mass spectrometry we found that Prx1 binds to DDAH1. On the basis of this finding, we hypothesized that as a result of degrading ADMA, increasing NOS activity and NO production, DDAH1 causes increased SRX expression and regenerates active Prx; the association of Prx1 and DDAH1 could thus protect DDAH1 from loss of activity caused by oxidative stress. In HUVEC, the NO donor DETA-NO (100μM) increased SRX expression and decreased Prx overoxidation (Prx-SO2). Comparing aortic endothelial cells from DDAH1 global KO (KO cells) and wild type control mice (wt cells), we found higher Prx-SO2 levels in KO cells. Furthermore, treatment with ADMA (100 μM) inhibited SRX expression in KO cells but not in wt cells. These results suggest that DDAH1 increases SRX expression, and facilitates regeneration of the Prx active form. Since DDAH1 activity can be inhibited by oxidative stress, we studied the effect of hydroperoxide on the Prx1 and DDAH1 interaction. When we treated HUVEC overexpressing DDAH1 with tert-Butyl hydroperoxide (t-BH 50 μM, 30 min), DDAH activity decreased 15% ± 2.9% (p<0.05), while the level of Prx1 and DDAH1 interaction increased as shown by DDAH1 immunoprecipitation and Prx1 western blot. The findings suggest that under conditions of oxidative stress, increased Prx1 binding to DDAH1 acts to protect DDAH activity.