Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Dec 7;8(1):17711.
doi: 10.1038/s41598-018-36267-6.

The reduced activity of PP-1α under redox stress condition is a consequence of GSH-mediated transient disulfide formation

Simranjit Singh  1   2 , Simon Lämmle  1 , Heiko Giese  3 , Susanne Kämmerer  1 , Stefanie Meyer-Roxlau  1 , Ezzaldin Ahmed Alfar  1 , Hassan Dihazi  4 , Kaomei Guan  1 , Ali El-Armouche  5 , Florian Richter  6
Affiliations

The reduced activity of PP-1α under redox stress condition is a consequence of GSH-mediated transient disulfide formation

Simranjit Singh et al. Sci Rep. .

Abstract

Heart failure is the most common cause of morbidity and hospitalization in the western civilization. Protein phosphatases play a key role in the basal cardiac contractility and in the responses to β-adrenergic stimulation with type-1 phosphatase (PP-1) being major contributor. We propose here that formation of transient disulfide bridges in PP-1α might play a leading role in oxidative stress response. First, we established an optimized workflow, the so-called "cross-over-read" search method, for the identification of disulfide-linked species using permutated databases. By applying this method, we demonstrate the formation of unexpected transient disulfides in PP-1α to shelter against over-oxidation. This protection mechanism strongly depends on the fast response in the presence of reduced glutathione. Our work points out that the dimerization of PP-1α involving Cys39 and Cys127 is presumably important for the protection of PP-1α active surface in the absence of a substrate. We finally give insight into the electron transport from the PP-1α catalytic core to the surface. Our data suggest that the formation of transient disulfides might be a general mechanism of proteins to escape from irreversible cysteine oxidation and to prevent their complete inactivation.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Effect of H2O2 treatment on cardiomyocyte survival, the PP-1α activity and phosphorylation of its downstream targets. (A) Pictures and quantification of NRCM mobility under oxidative stress from the time-lapse movie. (B) Quantitative analyses of immunoblots for downstream targets of PP-1α in NRCMs. (i) Mechanism of PKA activation and PP-1α inactivation upon H2O2 treatment. (ii) Time-dependent levels of PLB-pSer16 and cMyBPC-pSer282 after H2O2 treatment (n = 3). (iii) H2O2 concentration-dependent levels of I-1-pThr35, PLB-pSer16 and cMyBPC-pSer282 (n = 3). (C) Total phosphatase activity in NRCMs with and without H2O2 treatment (100 µM, 3 min, n = 5; i), time-dependent rPP-1α activity after H2O2 treatment (200 μM, n = 2-4; ii), H2O2 concentration-dependent rPP-1α activity (10 min, n = 4; iii) and recovery of rPP-1α activity (H2O2 200 μM for 15 min; TCEP 100 mM for 5 min; iv). Data shown in panel Civ represent one experiment out of two independent experiments. Data are presented as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 using one-way ANOVA and Bonferroni’s correction.
Figure 2
Figure 2
Dimerization of PP-1α involves disulfide formations. (A) Shown is the oxidative stress-independent dimerization of PP-1α in NRCMs in non-reducing PAGE. (B) General scheme of the sulfhydryl groups formed upon H2O2 treatment: reversible modifications including sulfenic acid, S-glutathionylation (violet box) and disulfides (green box) as well as irreversible modifications (sulfinic acid and sulfonic acid; red box). (C) A robust search strategy for the identification of disulfide bridges. When searching the spectra in a linear database, disulfide formation would not be detectable. Generation of permutated combination database and the “cross-over-read” mechanism with independent y- and b-type ion series identified from two database entries helps the identification of disulfide-linked MS/MS-spectra. Large molecular-weight fragments are indicated in red. (D) Spectra (left) and ion tables (right) for Cys39Cys127 and Cys127Cys127 disulfides identified at 500 µM H2O2 (y-type: red; b-type: blue). Red lines indicate the end of peptide1. (E) Cysteine interactions indicated with dashed lines between two 90°-turned monomers of PP-1α.
Figure 3
Figure 3
Disulfide formations in rPP-1α under different buffer conditions. (A–D) Cross-reactivity schemes for H2O2-depended formation of mixed (dark green) and one-peptide (light green) disulfides for GST-tagged rPP-1α. Pale green combinations are weak MS/MS spectra. The four buffer conditions with or without Mn2+ (100 µM) and with or without H2O2 (500 µM) were compared. (E) Sole disulfide spectrum (left) and ion table (right) of the Cys39 and Cys155Cys158 peptide identified for His-tagged rPP-1α. Red lines indicate the end of peptide1. Blue circles highlight the low abundant, but still present indicative fragments from the b-type ion series, showing unequivocally that the two peptides are interconnected with each other. The same fragment masses are highlighted in the ion table.
Figure 4
Figure 4
Quantitative analyses of different cysteine pools found in GST- and His-tagged rPP-1α under different conditions. (A) Spectral counts of disulfide-, free, GSH- and sulfone-Cys in GST-tagged and His-tagged r-PP-1α. Black dashed lines show the H2O2 treated samples and the grey solid lines the non-treated samples. (B) Spectra of three S-glutathionylation sites detected and the two consensus motifs they match to. Cys + GSH areas are highlighted in red.
Figure 5
Figure 5
Oxidation of histidine and tyrosine residues under H2O2 treatment in GST-tagged PP-1α. (A) Cartoon view of oxidatively affected His66, His248 and Tyr306 in PP-1α. (B-C) Spectra and ion tables of His66 (B) and His248 (C) as indicated in blue (b-ions) or red (y-ions). (D) Surface view of all His- and Tyr-residues.
Figure 6
Figure 6
The formation of Cys140, Cys202 and Cys245-centered disulfide networks in PP-1α upon oxidative stress is proposed. (A) Cys140/Cys245/Cys202-centered cysteine-networks (up to 14 Å) being S-glutathionylation targets. (B) Connection of S-glutathionylation and formation of transient disulfides in rPP-1α (red arrows). (C) Summary of distances (in Å) of identified disulfides.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Wagner S, Rokita AG, Anderson ME, Maier LS. Redox regulation of sodium and calcium handling. Antioxid Redox Signal. 2013;18:1063–1077. doi: 10.1089/ars.2012.4818. - DOI - PMC - PubMed
    1. Marti-Carvajal AJ, Kwong JS. Pharmacological interventions for treating heart failure in patients with Chagas cardiomyopathy. Cochrane Database Syst Rev. 2016;7:CD009077. doi: 10.1002/14651858.CD009077.pub3. - DOI - PMC - PubMed
    1. Heijman J, Dewenter M, El-Armouche A, Dobrev D. Function and regulation of serine/threonine phosphatases in the healthy and diseased heart. J Mol Cell Cardiol. 2013;64:90–98. doi: 10.1016/j.yjmcc.2013.09.006. - DOI - PubMed
    1. Griendling KK, FitzGerald GA. Oxidative stress and cardiovascular injury: Part I: basic mechanisms and in vivo monitoring of ROS. Circulation. 2003;108:1912–1916. doi: 10.1161/01.CIR.0000093660.86242.BB. - DOI - PubMed
    1. Brennan JP, et al. Oxidant-induced activation of type I protein kinase A is mediated by RI subunit interprotein disulfide bond formation. J Biol Chem. 2006;281:21827–21836. doi: 10.1074/jbc.M603952200. - DOI - PubMed

Publication types