Thioredoxin1 (Trx1) is a 12 kD oxidoreductase that reduces proteins with disulfide bonds through thiol-disulfide exchange reactions. Trx1 attenuates mitochondrial dysfunction, pathological hypertrophy, and ischemic injury. Trx1 directly interacts with signaling molecules and transcription factors, and regulates the function of its targets through reduction of disulfide bonds at cysteine residues. We have identified class II HDACs, AMPK, and mTOR as important targets of Trx1 in the heart. Trx1 also modulates protein S-nitrosylation through both transnitrosylation and de-nitrosylation. Covalent modification by nitrosylation reversibly modifies the functions of proteins. Although proteins can be nitrosylated directly by small molecules containing NO, such as S- nitrosoglutathione, they can also receive NO from other proteins through transnitrosylation, which is highly efficient and takes place in a regulated manner. Although Trx1 can act as either a de-nitrosylase or transnitrosylase in non-cardiac cells, the role of endogenous Trx1 in controlling the level of protein S- nitrosylation in the heart remains poorly understood. Furthermore, the molecular mechanism of transnitrosylation is poorly understood. Our goal is to elucidate the role of Trx1 in mediating nitrosylation of downstream targets in the heart. Our preliminary results suggest that Trx1 stimulates autophagy and protects the heart in response to myocardial ischemia by mediating transnitrosylation of cardio-protective proteins, including Atg7, an E1-like enzyme essential for autophagy. Furthermore, our results suggest that Cys73 in Trx1 plays a critical role in the transnitrosylation of Atg7. We hypothesize that Atg7 is S-nitrosylated during myocardial ischemia through Trx1 Cys73-mediated transnitrosylation. S-nitrosylation of Atg7 at Cys294 and Cys402 plays an essential role in mediating autophagy, which in turn protects the heart against myocardial ischemia. We will: 1. Demonstrate that endogenous Trx1 plays an important role in mediating transnitrosylation of cardiac proteins involved in protection against myocardial ischemia. 2. Demonstrate that Trx1 Cys73 plays an important role in mediating S-nitrosylation of Atg7. 3. Determine whether prior intermolecular disulfide bond formation between Trx1 and Atg7 is required for transnitrosylation of Atg7 by Trx1. 4. Demonstrate that Trx1 S-nitrosylates Atg7 at Cys294 and Cys402 and that S-nitrosylation of Atg7 at Cys294 and/or Cys402 plays an important role in mediating autophagy during ischemia in vivo. We have generated cardiac-specific Trx1 knockout mice, Trx1(C73S) knock-in (KI) mice, and Atg7 KI mice in which Cys294 and/or Cys402 is mutated to serine(s). We will use a quantitative proteomic approach in combination with the biotin switch method to identify Trx1 transnitrosylation targets. We will demonstrate a novel role of Trx1 as a transnitrosylase during myocardial ischemia and Atg7 as a novel target of Trx1 whose function is protected by transnitrosylation. Our study will show that it is possible to protect the heart against ischemia by modifying the transnitrosylation activity of Trx1 and its downstream targets, including Atg7.
Lay summary My laboratory has been investigating the molecular mechanisms by which thioredoxin 1 (Trx1), a small anti- oxidant protein, protects the heart against stress. The major mechanism by which Trx1 protects the heart against stress is to alleviate modification of protein functions by oxidative stress, a condition in which highly reactive molecules modified by molecular oxygen are increased. However, we found that Trx1 also modifies the function of proteins in the heart through another mechanism, namely addition of nitric oxide (NO) to cardiac proteins. One such modified protein is Atg7, a key regulator of autophagy, which is an important lysosome- mediated mechanism of protein and organelle degradation. In this study, we will elucidate the role of Trx1 in mediating protection of the heart through delivery of NO. This study may lead to development of a novel intervention to protect the heart against stresses such as myocardial ischemia.
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