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 1

Trx1 Cys73 regulates cell survival during glucose deprivation (GD).

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Trx1 Cys73 regulates cell survival during glucose deprivation (GD). (A) ...
(A) Recombinant Trx1 was S-nitrosylated using GSNO in vitro. The NO group was replaced with biotin-HPDP in biotin switch assays. LC-MS/MS analyses were performed. The MS/MS spectrum of m/z 789.36 resulting from a doubly charged ion (m/z 788.86) with an isotope space (0.5) corresponds to the peptide sequence of 73CMPTFQFFK81 with a biotin-HPDP (+428.19 Da) modification on Cys73 in Trx1. The b- and y-ion series correspond to the fragment ions of peptides from the N- and C-terminus, respectively, which confirms the peptide sequence of Trx1(73–81). The b-ion series (from the N-terminus), but not y-ion series (from the C-terminus), correspond to the fragment ions with a biotin-HPDP, indicating covalent binding of biotin-HPDP to Cys73. (B) Cys73 of Trx1 is S-nitrosylated. Cardiomyocytes were transduced with Ad-LacZ, Ad-Flag-Trx1 WT-HA, or Ad-Flag-Trx1(C73S)-HA for 48 hours. A biotin switch assay was performed, followed by pull-down with streptavidin-agarose to detect S-nitrosylated Trx1. The ratio of S-nitrosylated Trx1/total Trx1 is shown. *P < 0.05 vs. Trx1 WT, n = 3. Error bars represent SEM. (C) Cys73 is conserved among vertebrates. (D) Trx1 is oxidized during GD. Cardiomyocytes were lysed with biotin-labeled iodoacetamide (BIAM) after indicated periods of GD. The BIAM-labeled reduced form of Trx1 was pulled down with streptavidin-agarose. n = 6. (E) Trx1 is S-nitrosylated in response to GD in cardiomyocytes. SNO-Trx1 was detected by biotin switch assay. n = 5. (F) Trx1 is S-nitrosylated in response to GD in cardiac fibroblasts. (G) Cys73 of Trx1 promotes cell survival under GD conditions. Cardiomyocytes were transduced with lacZ, Flag-Trx1 WT-HA, Flag-Trx1(C73S)-HA, and shTrx1 adenoviruses for 48–96 hours, after which they were incubated with normal or glucose-free medium for 24 hours. Cell death was assessed using CellTiter-Blue. n = 12. *P < 0.05 by 2-tailed Student’s t test (B, E, and F) or 1-way ANOVA (D and G)