Thioredoxin 1 promotes autophagy through transnitrosylation of Atg7 during myocardial ischemia
Narayani Nagarajan, … , Hong Li, Junichi Sadoshima
Narayani Nagarajan, … , Hong Li, Junichi Sadoshima
Published December 8, 2022
Citation Information: J Clin Invest. 2023;133(3):e162326. https://doi.org/10.1172/JCI162326.
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Research Article Cardiology Cell biology

Thioredoxin 1 promotes autophagy through transnitrosylation of Atg7 during myocardial ischemia

Abstract

Modification of cysteine residues by oxidative and nitrosative stress affects structure and function of proteins, thereby contributing to the pathogenesis of cardiovascular disease. Although the major function of thioredoxin 1 (Trx1) is to reduce disulfide bonds, it can also act as either a denitrosylase or transnitrosylase in a context-dependent manner. Here we show that Trx1 transnitrosylates Atg7, an E1-like enzyme, thereby stimulating autophagy. During ischemia, Trx1 was oxidized at Cys32-Cys35 of the oxidoreductase catalytic center and S-nitrosylated at Cys73. Unexpectedly, Atg7 Cys545-Cys548 reduced the disulfide bond in Trx1 at Cys32-Cys35 through thiol-disulfide exchange and this then allowed NO to be released from Cys73 in Trx1 and transferred to Atg7 at Cys402. Experiments conducted with Atg7 C402S–knockin mice showed that S-nitrosylation of Atg7 at Cys402 promotes autophagy by stimulating E1-like activity, thereby protecting the heart against ischemia. These results suggest that the thiol-disulfide exchange and the NO transfer are functionally coupled, allowing oxidized Trx1 to mediate a salutary effect during myocardial ischemia through transnitrosylation of Atg7 and stimulation of autophagy.

Authors

Narayani Nagarajan, Shin-ichi Oka, Jihoon Nah, Changgong Wu, Peiyong Zhai, Risa Mukai, Xiaoyong Xu, Sanchita Kashyap, Chun-Yang Huang, Eun-Ah Sung, Wataru Mizushima, Allen Sam Titus, Koichiro Takayama, Youssef Mourad, Jamie Francisco, Tong Liu, Tong Chen, Hong Li, Junichi Sadoshima

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Figure 6

Trx1 transnitrosylates Atg7 at Cys294 and Cys402.

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Trx1 transnitrosylates Atg7 at Cys294 and Cys402. (A) Rat cardiomyocytes...
(A) Rat cardiomyocytes transduced with Ad-Atg7 were cultured in normal or glucose-free medium for 4 hours. A biotin switch assay was performed, followed by MS analysis to determine sites of S-nitrosylation in Atg7. (B) Recombinant Trx1 treated with GSNO was incubated with recombinant human Atg7 protein for 30 minutes. A biotin switch assay followed by MS analysis was performed to determine sites of transnitrosylation in Atg7. A representative MS spectrum is shown. (A and B) The MS/MS spectra of the peptides 283LPEMAFSPDC*PK296 and 394QPLYEFEDC*LGGGKPK409 with a biotin-HPDP (+428.19 Da) modification on C294 and C402 are shown. The MS/MS spectrum of m/z 881.9 corresponds to the peptide sequence of Atg7 (283–296) with a biotin-HPDP. That of Atg7 (394–409) with a biotin-HPDP was not detected due to complete fragmentation of the precursors. The y- and b-ion series confirmed the peptide sequence and the biotin-HPDP modification on the cysteines. The rat Atg7 peptides 283–296 and 394–409 correspond to human Atg7 289–300 and 398–413, respectively. (C) Cardiomyocytes were transduced with Ad-LacZ, Ad-Trx1 WT, or Ad-Trx1(C73S). A biotin switch assay followed by Western blotting was carried out to measure S-nitrosylation of Atg7. Representative immunoblots and quantification analysis of S-NO Atg7/total Atg7 are shown. n = 3. (D) Cardiomyocytes were transduced with Ad-Atg7 WT or Ad-Atg7 CC294/402SS in the presence of Ad-LacZ or Ad-Trx1 WT. A biotin switch assay followed by Western blotting was performed to measure S-nitrosylation of Atg7. Representative immunoblots and quantification analysis of SNO-Atg7/total Atg7 are shown. n = 3. *P < 0.05 by Kruskal-Wallis test (C) or 2-tailed Student’s t test (D).