A single cysteine post-translational oxidation suffices to compromise globular proteins kinetic stability and promote amyloid formation

Reactive oxygen species (ROS) contribute to the etiology of multiple muscle-related diseases. There is emerging evidence that cellular stress can lead to destabilization of sarcomeres, the contractile unit of muscle. However, it is incompletely understood how cellular stress induces structural destabilization of sarcomeres. Here we report that glutathionylation of SMYD2 contributes to a loss of myofibril integrity and degradation of sarcomeric proteins mediated by MMP-2 and calpain 1. We used a clickable glutathione approach in a cardiomyocyte cell line and found selective glutathionylation of SMYD2 at Cys13. Biochemical analysis demonstrated that SMYD2 upon oxidation or glutathionylation at Cys13 loses its interaction with Hsp90 and N2A, a domain of titin. Upon dissociation from SMYD2, N2A or titin is degraded by activated MMP-2, suggesting a protective role of SMYD2 in sarcomere stability. Taken together, our results support that SMYD2 glutathionylation is a novel molecular mechanism by which ROS contribute to sarcomere destabilization.

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“…15a ). 41 , 42 In vitro binding assays showed that similar levels of SMYD2 WT and C13D were bound to Hsp90 and N2A (Supplementary Fig. 15c–d ).…”

Section: Resultsmentioning

confidence: 94%

SMYD2 glutathionylation contributes to degradation of sarcomeric proteins

et al. 2018

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“…The benzothiazole dye ThT, which displays enhanced fluorescence when it binds to beta sheet-rich structures in amyloids, is widely used as a probe for protein unfolding and aggregation [ [46] , [47] , [48] ]. Native AN showed a high fluorescence emission intensity at 495 nm consistent with ThT binding, and the presence of hydrophobic patches and a beta-sheet structure on the protein surface ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Peroxynitrous acid (ONOOH) modifies the structure of anastellin and influences its capacity to polymerize fibronectin

Becares

Thulstrup

et al. 2020

Redox Biology

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Anastellin (AN), a fragment of the first type III module in fibronectin (FN), initiates formation of superfibronectin, a polymer which resembles the native cell-derived fibrillar FN found in the extracellular matrix of many tissues, but which displays remarkably different functional properties. Here we demonstrate that exposure of AN to the biologically-important inflammatory oxidant, peroxynitrous acid (ONOOH), either as a bolus or formed at low levels in a time-dependent manner from SIN-1, impairs the capability of AN to polymerize FN. In contrast, exposure of FN to ONOOH does not seem to affect superfibronectin formation to the same extent. This oxidant-induced loss-of-function in AN occurs in a dose-dependent manner, and correlates with structural perturbations, loss of the amino acid tyrosine and tryptophan, and dose-dependent formation of modified amino acid side-chains (3-nitrotyrosine, di-tyrosine and 6-nitrotryptophan). Reagent ONOOH also induces formation of oligomeric species which decrease in the presence of bicarbonate, whereas SIN-1 mainly generates dimers. Modifications were detected at sub-stoichiometric (0.1-fold), or greater, molar excesses of oxidant compared to AN. These species have been localized to specific sites by peptide mass mapping. With high levels of oxidant (>100 times molar excess), ONOOH also induces unfolding of the beta-sheet structure of AN, thermal destabilization, and formation of high molecular mass aggregates. These results have important implications for the understanding of FN fibrillogenesis in vivo , and indicates that AN is highly sensitive to pathophysiological levels of oxidants such as ONOOH.

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“…Recently, the decrease of GSH level has been shown to induce cognitive decline and neuronal death during aging and neurodegenerative diseases [53,54]. Interestingly, P2X7R is a therapeutic target in the treatment of epilepsy [12][13][14]55].…”

Section: Discussionmentioning

confidence: 99%

Deletion of P2X7 Receptor Decreases Basal Glutathione Level by Changing Glutamate-Glutamine Cycle and Neutral Amino Acid Transporters

Park

Kim

2020

Cells

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Glutathione (GSH) is an endogenous tripeptide antioxidant that consists of glutamate-cysteine-glycine. GSH content is limited by the availability of glutamate and cysteine. Furthermore, glutamine is involved in the regulation of GSH synthesis via the glutamate–glutamine cycle. P2X7 receptor (P2X7R) is one of the cation-permeable ATP ligand-gated ion channels, which is involved in neuronal excitability, neuroinflammation and astroglial functions. In addition, P2X7R activation decreases glutamate uptake and glutamine synthase (GS) expression/activity. In the present study, we found that P2X7R deletion decreased the basal GSH level without altering GSH synthetic enzyme expressions in the mouse hippocampus. P2X7R deletion also increased expressions of GS and ASCT2 (a glutamine:cysteine exchanger), but diminished the efficacy of N-acetylcysteine (NAC, a GSH precursor) in the GSH level. SIN-1 (500 μM, a generator nitric oxide, superoxide and peroxynitrite), which facilitates the cystine–cysteine shuttle mediated by xCT (a glutamate/cystein:cystine/NAC antiporter), did not affect basal GSH concentration in WT and P2X7R knockout (KO) mice. However, SIN-1 effectively reduced the efficacy of NAC in GSH synthesis in WT mice, but not in P2X7R KO mice. Therefore, our findings indicate that P2X7R may be involved in the maintenance of basal GSH levels by regulating the glutamate–glutamine cycle and neutral amino acid transports under physiological conditions, which may be the defense mechanism against oxidative stress during P2X7R activation.

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A single cysteine post-translational oxidation suffices to compromise globular proteins kinetic stability and promote amyloid formation

Cited by 27 publications

References 47 publications

SMYD2 glutathionylation contributes to degradation of sarcomeric proteins

SMYD2 glutathionylation contributes to degradation of sarcomeric proteins

Peroxynitrous acid (ONOOH) modifies the structure of anastellin and influences its capacity to polymerize fibronectin

Deletion of P2X7 Receptor Decreases Basal Glutathione Level by Changing Glutamate-Glutamine Cycle and Neutral Amino Acid Transporters

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