Abstract 20500: Trx1 Promotes Trans-Nitrosylation of Cellular Proteins to Stimulate Autophagy and Cell Survival

Abstract

Thioredoxin-1 (Trx1) is cardioprotective during oxidative stress, mainly through its antioxidant activity. Trx1 is also S-nitrosylated and, in turn, trans-nitrosylates other proteins. However, the role of Trx1-dependent S-nitrosylation in cardiomyocytes (CMs) is unknown. Here, we investigated the role of Trx1-mediated protein S-nitrosylation in the regulation of CM survival during stress in vitro. Using biotin-switch assays, we found that wild-type Trx1 (Trx1WT) is S-nitrosylated at baseline, but the extent of S-nitrosylation was attenuated in Trx1C73S, suggesting that Trx1 is S-nitrosylated at Cys73. Trx1WT and Trx1C73S do not differ in their redox activity, as determined by Amplex Red assays. Cellular protein S-nitrosylation levels were increased after 4 hours of glucose deprivation (GD), an energy stress condition (1.64±0.27 fold, p<0.05), as determined by biotin switch assays. Overexpression of Trx1WT increased (3.94-fold), whereas knockdown of Trx1 (0.66±0.01 fold, p<0.01) or overexpression of Trx1C73S (0.77±0.02 fold, p<0.01) decreased, total protein S-nitrosylation in response to GD. These results suggest that Trx1C73 regulates protein S-nitrosylation in CMs during GD. Overexpression of Trx1 increased CM survival after 24 hours of GD (1.42±0.08 fold vs LacZ, p<0.05), as evaluated with propidium iodide assays. Conversely, shTrx1 (2.13±0.05 fold vs control, p<0.01) or Trx1C73S (1.73±0.034 fold vs LacZ, p<0.01) increased cell death during GD. Either knockdown of Trx1 (LC3-II/Tubulin: 0.55 fold vs control) or overexpression of Trx1C73S (vs LacZ: LC3-II/Tubulin, 0.60 fold; autophagosomes, 0.83±0.16-fold, p<0.005; autolysosomes, 0.62±0.13-fold, p<0.005) significantly decreased autophagy during GD. Mechanistically, Trx1 co-immunoprecipitates with Atg7, an E1-like protein which plays a critical role in mediating autophagy. Using mass spectroscopy analyses, we found that SNO-Trx1 can trans-nitrosylate Atg7 in vitro. These results suggest that Trx1 trans-nitrosylates Atg7 during GD. Taken all together, our results indicate that Trx1 promotes trans-nitrosylation of cellular proteins, including Atg7, and autophagy, thereby promoting cell survival during energy stress in CMs.