Abstract
Protein S-glutathionylation is an important reversible post-translational modification implicated in redox signaling. Oxidative modifications to protein thiols can alter the activity of metabolic enzymes, transcription factors, kinases, phosphatases, and the function of contractile proteins. However, the extent to which muscle contraction induces oxidative modifications in redox sensitive thiols is not known. The purpose of this study was to determine the targets of S-glutathionylation redox signaling following fatiguing contractions. Anesthetized adult male CB6F1 (BALB/cBy × C57BL/6) mice were subjected to acute fatiguing contractions for 15 min using in vivo stimulations. The right (stimulated) and left (unstimulated) gastrocnemius muscleswere collected 60 min after the last stimulation and processed for redox proteomics assay of S-glutathionylation. Using selective reduction with a glutaredoxin enzyme cocktail and resin-assisted enrichment technique, we quantified the levels of site-specific protein S-glutathionylation at rest and following fatiguing contractions. Redox proteomics revealed over 2200 sites of S-glutathionylation modifications, of which 1290 were significantly increased after fatiguing contractions. Muscle contraction leads to the greatest increase in S-glutathionylation in the mitochondria (1.03%) and the smallest increase in the nucleus (0.47%). Regulatory cysteines were significantly S-glutathionylated on mitochondrial complex I and II, GAPDH, MDH1, ACO2, and mitochondrial complex V among others. Similarly, S-glutathionylation of RYR1, SERCA1, titin, and troponin I2 are known to regulate muscle contractility and were significantly S-glutathionylated after just 15 min of fatiguing contractions. The largest fold changes (> 1.6) in the S-glutathionylated proteome after fatigue occurred on signaling proteins such as 14-3-3 protein gamma and MAP2K4, as well as proteins like SERCA1, and NDUV2 of mitochondrial complex I, at previously unknown glutathionylation sites. These findings highlight the important role of redox control over muscle physiology, metabolism, and the exercise adaptive response. This study lays the groundwork for future investigation into the altered exercise adaptation associated with chronic conditions, such as sarcopenia.
Highlights
Oxidative modifications are implicated in both the pathological damage of oxidative stress and the physiological and adaptive responses to redox signaling
Glutathione is the most abundant antioxidant molecule within cells, especially the mitochondria [3,4]. It is an essential cofactor of glutathione peroxidase, glutathione S-transferase, and glutaredoxin for the scavenging of hydrogen peroxide and the reversal of oxidative modifications to proteins [3]
Many of the PSSG sites identified in muscle are known to alter actin-myosin bridge formation, calcium homeostasis, and metabolism, and contribute to fatigue [40]
Summary
Oxidative modifications are implicated in both the pathological damage of oxidative stress and the physiological and adaptive responses to redox signaling. The thiol group on the amino acid cysteine is a major target for oxidative modification of proteins [1]. Glutathione is the most abundant antioxidant molecule within cells, especially the mitochondria [3,4]. It is an essential cofactor of glutathione peroxidase, glutathione S-transferase, and glutaredoxin for the scavenging of hydrogen peroxide and the reversal of oxidative modifications to proteins [3]. Reduced or oxidized glutathione can react with oxidized protein thiols or thiolate anions to form protein S-glutathionylation (P-SSG) modifications through several enzymatic and non-enzymatic mechanisms [5]. Glutathione can react with oxidized derivatives of protein cysteines, such as sulfenic acid (-SOH), thiyl radicals (-S.), or S-nitroso
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have