Abstract 9801: Thiol-Disulfide Exchange Reaction Between Thioredoxin-1 (trx1) and Atg7 Promotes Transnitrosylation of Atg7

Abstract

Thioredoxin 1 (Trx1) reduces proteins with disulfide bonds. However, when the oxidoreductase activity of Trx1 is inactivated, Trx1 is S-nitrosylated at Cys73 and acts as a trans-nitrosylase. Glucose deprivation (GD) inactivates the oxidoreductase activity of Trx1 and stimulates autophagy in cardiomyocytes. We investigated how Trx1 mediates S-nitrosylation of Atg7 in response to GD, thereby stimulating autophagy. GD-induced autophagy, evaluated with tandem fluorescent LC3 (mRFP-GFP-LC3), was inhibited in the presence of Trx1C73S, a transnitrosylation defective mutant (Relative GFP/RFP under GD, control 0.75; Trx1C73S 0.95*; p<0.05 vs. control), suggesting that Trx1 mediates GD-induced autophagy via Cys73. Mass spectrometric and biochemical analyses showed that Cys545-548 in Atg7, a thioredoxin motif (CXXC), undergoes thiol-disulfide exchange reactions with Trx1, whereas Atg7 Cys402 is S-nitrosylated by SNO-Trx1. Atg7 C402S impairs Atg7 functions, as evidenced by reduced LC3-II formation with and without bafilomycin (Baf) (relative LC3-II, Atg7: 2.5; Atg7C402S: 1.7*; Atg7 with Baf: 3.1; Atg7C402S with Baf: 1.6*; p<0.05). Thus, S-nitrosylation at Cys402 promotes Atg7 function, thereby promoting autophagy. On the other hand, Atg7 C545/548S inhibited both binding of Atg7 to Trx1 and Atg7 S-nitrosylation, suggesting that Trx1 S-nitrosylates Atg7 at Cys402 during thiol-disulfide exchange reactions between Atg7 Cys545-548 and Trx1. Knockdown of Trx1 prevented whereas overexpression of Trx1 promoted GD-induced Atg7 oxidation. In vitro redox reactions revealed that Atg7 can reduce Trx1. Thus, Trx1 mediates GD-induced autophagy through the following mechanism: 1) GD induces Trx1 oxidation at Cys32-35; 2) oxidized Trx1 is S-nitrosylated at Cys73; 3) Atg7 is S-nitrosylated at Cys402 during thiol-disulfide exchange reactions between Trx1 Cys32-35 and Atg7 Cys545-548; and 4) S-nitrosylated Atg7 at Cys402 promotes autophagy.