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

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.

Here, we tested the hypothesis that the transient and dynamic disulfide formation might prevent over-oxidation and irreversible inactivation of PP-1α. First, we used elaborated mass spectrometry (MS) techniques and present an optimized workflow, the so-called "cross-over-read" method, for the identification of disulfide-linked peptides from MS 2 spectra that can be performed on every high-resolution detection system. With this method, we could identify both intra-and inter-molecular disulfide bridges in superoxide dismutase 1 (SOD1) and pyruvate kinase M2 (PKM2). Moreover, we performed a simple combination of experiments by using recombinant PP-1α (rPP-1α) with and without glutathione S-transferase (GST) activity and analysed the formation of transient disulfides in PP-1α upon oxidative stress. We show that the establishment of disulfides in rPP-1α strongly depends on the fast reaction when GSH is present. Our data suggest that the formation of transient disulfide bridges might be a general mechanism of proteins to escape from over-oxidation.

Results
Impact of oxidative stress on the PP-1 activity and its downstream targets. First, we investigated the effect of increasing concentrations of H 2 O 2 on contraction and survival of neonatal rat cardiomyocytes (NRCMs). Live imaging was performed and a matlab-based script, as described previously 18 , was used to generate a heat map of the mean contraction of the cells (Figs 1A, S1). The contraction-relaxation ratios as a measure of myocyte vitality were calculated (Fig. 1A). As expected, the contraction of NRCMs was normal at a concentration of 0.1 mM H 2 O 2 , similar to control NRCMs without H 2 O 2 treatment. With concentrations higher than 1 mM, the contraction was reduced and at 10 mM cells were dying.
To understand the effect of oxidative stress on the PP-1α activity, we investigated the phosphorylation status of its downstream targets phospholamban (PLB) and cardiac myosin binding protein-C (cMyBPC) in NRCMs. We monitored phosphorylation of inhibitor-1 (I-1) that is responsible for the crosstalk between protein kinase A (PKA) and PP-1α signalling (Fig. 1Bi). We observed a slight increase in PLB phosphorylation at Ser 16 (PLB-pSer 16 ) in NRCMs treated with 100 µM H 2 O 2 for 3-6 min and a remarkable decrease in PLB-pSer 16 after 10 min. In contrast, changes in cMyBPC phosphorylation at Ser 282 (cMyBPC-pSer 282 ) showed a bell-shape, peaking after 10 min with 100 µM H 2 O 2 (Fig. 1Bii). These data suggest different kinetics of PKA stimulation and PP-1 inhibition under H 2 O 2 treatment, leading to the site-specific, temporal dynamics of phosphorylation of different substrates, as demonstrated previously 19 . By fixing the incubation time to 3 min and altering the concentrations of H 2 O 2 , we observed a bell-shaped response for I-1 phosphorylation at Thr 35 (I-1-pThr 35 ) with a peak at 100 μM, whereas PLB-pSer 16 and cMyBPC-pSer 282 were not changed much with 100 µM H 2 O 2 . This bell-shaped phosphorylation response of PKA substrates to the increased H 2 O 2 concentrations was already described previously 5 . However, PLB-pSer 16 and cMyBPC-pSer 282 were increased at 10000 μM H 2 O 2 (Fig. 1Biii), suggesting that PKA was still active while PP-1α was inhibited.
Next, we measured phosphatase activity in NRCMs and found the net decrease in phosphatase activity about 25% when we treated NRCMs with 100 µM H 2 O 2 for 3 min (Fig. 1Ci). By using rPP-1α, we established a reproducible in-vitro phosphatase activity assay and observed a maximum effect of H 2 O 2 treatment (200 µM) after 10 min (Fig. 1Cii). By applying increasing concentrations of H 2 O 2 , we showed a reduction of the rPP-1α activity by 50% at 500 µM H 2 O 2 for 10 min (Fig. 1Ciii). Interestingly, the inhibition of rPP-1α activity by H 2 O 2 (200 µM for 15 min) was partially rescued with the mild reducing agent Tris(2-carboxyethyl)phosphine (TCEP) at 100 mM for 5 min (Fig. 1Civ), suggesting that the inactivation of PP-1α by oxidation is reversible.
In-gel dimerization detection of PP-1α. To decipher the mechanism responsible for the reversible inactivation of PP-1α, we investigated whether oxidative stress might induce PP-1α dimerization through surface cysteine modification in NRCMs. We first established non-reducing conditions suitable for the in-gel detection of protein dimerization by analysing PKA and PP-2Ac upon oxidative stress (Fig. S2A). We found an increase in dimerization of PKA at higher concentrations of diamide and H 2 O 2 with consistently reduced monomer formation, but no dimerization of PP-2Ac. In contrast, we observed dimerization of PP-1α in NRCMs without oxidative stress and after treatment with diamide ( Fig. 2A). Quantitative analysis showed that PP-1α monomer expression was already decreased at 1 µM diamide with further reduction up to 10 mM, whereas the dimer expression increased up to 100 µM diamide. At 1 mM diamide, both monomer and dimer expression of PP-1α were reduced (Fig. S2B). At the highest concentration of 10 mM diamide, we observed strong reduction of both PP-1α monomer and dimer due to severe cell damage. Therefore, the dimer/monomer ratio showed a bell-shaped curve peaking at 100 µM diamide (Fig. S2B). These data indicate that the dimerization of PP-1α might not be the primary cause for the reversible inactivation of PP-1α under oxidative stress.

Establishment of new search strategy for detection of disulfide formation in PP-1α.
Based on our in-gel data, we tested whether the reversible Cys-modifications in PP-1α occur under oxidative stress. As illustrated in Fig. 2B, the first step in cysteine oxidation is the formation of sulfenic acid and then different Cys-pools formed with increased concentration of H 2 O 2 : (i) reversible intra-and inter-molecular disulfides, (ii) reversible glutathionylation and (iii) irreversible sulfonic acid. PP-1α owns 13 Cys-residues and can be differently influenced by the solvent. By using the modified search strategy for chemically crosslinked peptides as previously published 20 , we could not get high-quality spectra.
For the identification of disulfide-linked peptides, we established a new database search strategy, which is based on sequence tag search in the PEAKS software. In brief, we applied the concept of searching against linearized databases containing all permutated combinations of Cys-peptides. The databases were either created manually or in an automated way for more complex datasets easily resulting in more than 100,000 combinations. More specificity was obtained by organizing every di-peptide as a separate protein entry and searching without enzyme specificity (Fig. 2C). We introduced a modification for the hidden C-terminus of peptide1 (+18 a.m.u.) to identify the spectra that matched the two database entries peptide1-peptide2 and peptide2-peptide1. The MS/ MS ion series of one peptide stopped at the disulfide link and continued by the ion series of the other peptide in the higher molecular weight region of the spectrum. We hence termed the generation of composite spectrum "cross-over-read" (Fig. 2C).
By applying the new strategy, we searched for free Cys and disulfides in GST-tagged rPP-1α at increasing concentrations of H 2 O 2 (0, 100, 500 µM) in the absence of Mn 2+ . We observed disulfides between Cys 39 and Cys 127 at all concentrations and one homo-di-peptide of Cys 127 at 500 µM (Fig. 2D, Fig. S2C,D). Cys 39 and Cys 127 were not found as free cysteines at all concentrations (Fig. S2D). However, a direct link between Cys 39 and Cys 127 within one monomer of PP-1α is unlikely due to their molecular distance about 15.72 Å (Fig. S3). Because both of them are surface-oriented to the same side, the interaction of two monomers with each other being turned by 90° is possible (Fig. 2E). These data are consistent with the in-gel detection of PP-1α dimerization even without oxidative stress. It is known that the major substrate binding sites of PP-1α, for instance, for spinophilin 21 , include interactions of Cys 140 , Cys 127 , Cys 273 and eventually Cys 202 towards an acidic patch (Fig. S4). Our results suggest that the formation of inter-molecular disulfide bridges between Cys 39 and Cys 127 and between two Cys 127 might protect the active surface of PP-1α when the substrate is absent.

SOD1 as additional model for the detection of disulfide bridges.
To validate if the "cross-over-read" method is suitable for detection of disulfides for other proteins, we analysed bovine SOD1 with only 3 Cys-residues Cys 7 , Cys 56 and Cys 145 (Fig. S5). As expected, we detected the intra-molecular disulfide bridge between Cys 56 and Cys 145 (Fig. S5A,C), which is essentially required for correct folding and metal ion binding 22 . Importantly, we observed disulfide formation between two copies of N-terminally acetylated Cys 7 peptides and between two copies of Cys 145 (Fig. S5A,C). The homo-dimer spectra could be unequivocally identified by high molecular weight fragments (Fig. S5C). We furthermore identified one mixed disulfide with a lower spectrum quality between Cys 7 and Cys 56 (Fig. S5A,C). In contrast, we could not identify any homo-di-peptides of Cys 56 (Fig. S5B), which would have a similar probability due to the molecular distance (15.90 Å). These data indicate that the "cross-over-read" search strategy is suitable to detect both intra-and inter-molecular disulfide bridges.
Transient disulfide formation in PP-1α is enhanced by S-glutathionylation. We next investigated whether the formation of intra-molecular transient disulfides under oxidative stress could cause for the reversible inactivation of PP-1α. The molecular distances of all Cys-residues in non-oxidized PP-1α are summarized in the cross-reactivity scheme (Fig. S3).  Fig. S6A,B).
In contrast to GST-tagged rPP-1α, the only significant disulfide formation in His-tagged rPP-1α was identified between Cys 39 and Cys 155 Cys 158 in the presence of H 2 O 2 but absence of Mn 2+ (Fig. 3E). Apparently, the molecular distance between Cys 39 and Cys 155 with 4.54 Å, the shortest distance among all cysteines (Fig. S3), enables this fast reaction upon H 2 O 2 treatment. Altogether, these data suggest that additional S-glutathionylation might induce the formation of transient disulfide bridges in PP-1α.
Quantitative approach to understand the influence of the H 2 O 2 treatment. To quantify the effect of oxidative stress on cysteine modifications in GST-tagged and His-tagged rPP-1α, we performed spectral count of free, disulfide-linked, sulfonated and glutathionylated cysteines (Fig. 4A, Table S1) and also applied LFQ quantification for the most abundant peptides (Table S2). A much-simplified quantitative response was observed for the His-tagged rPP-1α (Table S2B) compared to GST-tagged rPP-1α (Table S2A). Since Cys 273 and Cys 291 lie on a huge trypsin peptide, they might escape from MS detection, which can explain why we could not detect Cys 273 and Cys 291 in both GST-tagged and His-tagged rPP-1α. Mn 2+ ions have a general protective effect against oxidative stress for both GST-tagged and His-tagged rPP-1α.
As mentioned above, we observed disulfide-linked Cys 155 , Cys 158 , Cys 171 and Cys 172 in both GST-and His-tagged rPP-1α independent of the presence of H 2 O 2 and Mn 2+ . However, the amount of disulfide-linked Cys 155 , Cys 158 , Cys 171 and Cys 172 was much higher in both GST-and His-tagged rPP-1α treated with H 2 O 2 than in their corresponding non-treated rPP-1α. Furthermore, significant amount of disulfide-linked Cys 39 , Cys 62 , Cys 105 , Cys 127 , Cys 140 , Cys 202 and Cys 245 was induced in GST-tagged rPP-1α upon oxidative stress in the absence of Mn 2+ whereas only disulfide-linked Cys 39 was induced in His-tagged rPP-1α (Fig. 4A, Table S1). These data indicate that the intra-molecular disulfide formations in GST-tagged rPP-1α upon oxidative stress depend on the presence of GSH. In GST-tagged rPP-1α, Cys 39 and Cys 127 were completely protected independent of H 2 O 2 and Mn 2+ . In His-tagged rPP-1α, free Cys 39 was observed when Mn 2+ was included to the buffer, which was never detected in the absence of Mn 2+ (Fig. 4A, Table S1). Cys 127 , however, was never observed as a free cysteine in both GST-and His-tagged rPP-1α, showing that Cys 39 and Cys 127 behave completely different in the network.
Interestingly, three cysteines Cys 202 , Cys 245 and Cys 140 in GST-tagged rPP-1α had an outstanding response to the H 2 O 2 treatment: S-glutathionylation (Fig. 4B, Table S1 and S2A). A web-based glutathionylation database proposed Cys 140 , Cys 158 and Cys 245 in PP-1α (PDB 3N5U) as S-glutathionylation sites 23 . Cys 202 is a hitherto unexpected site for GSH modification. Consensus motifs for GSH sites showed acidic Asp/Glu residues in close proximity to the reactive cysteine (Fig. 4B). We also observed sulfone formation for these three cysteines in GST-tagged rPP-1α upon oxidative stress (Table S2A). Their dual response with GSH and sulfone probably reflects the solvent accessibility of Cys 140 , Cys 202 and Cys 245 . In addition, there were more free cysteines for these three cysteines in GST-tagged than in His-tagged rPP-1α upon oxidative stress. In contrast, compared to GST-tagged rPP-1α, His-tagged rPP-1α upon oxidative stress revealed higher amount of sulfone formation of all cysteines except Cys 39 , Cys 155 , Cys 158 , Cys 171 and Cys 172 , which fits quite well with overall less internal disulfide formation in His-tagged rPP-1α (Fig. 4A). These data indicate that significant amount of cysteines in GST-tagged rPP-1α are involved in the reversible disulfide formation and S-glutathionylation upon oxidative stress whereas more irreversible sulfonated cysteines are formed in His-tagged rPP-1α. Oxidations of His and Tyr in the catalytic core of rPP-1α. To further decipher the effect of oxidative stress on the activity and reaction of PP-1α, we performed additional search for the modification of other amino acids. Interestingly, out of 13 Tyr-residues, only Tyr 306 at C-terminus of PP-1α was prone to oxidation. In His-tagged rPP-1α, we observed mono-oxidation (+15.99 Da) of Tyr 306 without H 2 O 2 and Mn 2+ treatment (Fig. S7A), and di-oxidation (+31.99 Da) upon H 2 O 2 treatment (Fig. S7B) (Fig. S7C,D). Although Tyr 306 is the sole tyrosine not covered in the PDB structure (Fig. 5A), we can deduce that it must have a particularly exposed position. An entirely different effect was observed for two out of the four His-residues, His 66 and His 248 , in GST-tagged rPP-1α, which got mono-oxidized (+15.99 Da) under H 2 O 2 treatment (Fig. 5B,C). They were not affected in His-tagged rPP-1α by H 2 O 2 treatment. The X-ray structure of PP-1α strongly suggests that only His 66 , His 125 and His 173 are caging the two Mn 2+ ions and that His 248 is oriented away from the Mn 2+ ions (Fig. 5D). Our data indicate that oxidation of His 66 and His 248 is shielding oxidative stress from the two Mn 2+ ions, or, in return, transferring protons in a controlled manner, thereby controlling the oxidation state of Mn 2+ or Mn 3+ . However, we only observed this effect in the presence of GST-activity and this opens the question if the GST-activity has an influence on the activity of PP-1α catalytic core. Transient disulfides as a general mechanism of proteins escaping from denaturing. To further confirm our hypothesis that formation of transient disulfide bridges might be a general mechanism of proteins to prevent from over-oxidation and irreversible inactivation, we performed the search for other redox-regulated proteins, for example, PKM2 (Fig. S8A). The 3D structure does not show any structurally relevant disulfides although there are cysteines with distances around 10 Å (Fig. S8B). We used PKM2 purified from rabbit muscle and applied H 2 O 2 at increasing concentrations (0, 1, 10, 100, 1000 µM). We detected increasing amounts of potential disulfide precursors in PKM2 (Fig. S8C), represented by the blue dots in the heat-maps (Fig. S8D). These disulfide formations were verified by manual inspection of the spectra (Fig. S8E). Notably, these Cys-combinations are with molecular distances (>20 Å) unlikely for intra-molecular disulfides, indicating that they must be formed between two copies of a protein. However, we could not detect the dimerization in non-denaturing gels. These data suggest that non-specific dimerization of PKM2 upon oxidative stress involving Cys 49 , Cys 317 , Cys 326 and Cys 358 and the other cysteines at the outer domains (Fig. S8A) might protect PKM2 from denaturing.

Discussion
In this study, we first set up the robust search strategy "cross-over-read" for MS/MS-spectra using permutated databases from non-reduced proteomics samples. Applying this method, we show that PP-1α was sensitive to H 2 O 2 treatment and unexpected transient disulfides were formed. We demonstrate that the GST-activity was essentially required for a fast transient disulfide formation in PP-1α. The dimerization of PP-1α involving Cys 39 and Cys 127 is presumably important for the protection of PP-1α active surface in the absence of a substrate. Moreover, we give insight into the potential electron transport around the PP-1α catalytic core. Our data indicate that the reversible inactivation of PP-1α upon oxidative stress is, at least partially, due to transient disulfide formation as an escape mechanism against irreversible Cys-oxidation. Although the strategy using permutated databases was used in the context of sumoylated 24 and chemically crosslinked peptides 25 , we established here for the first time a simple approach that allowed the search of single protein entries for every disulfide-linked peptide without any enzymatic cleavage specificity. We can identify the large-molecular weight fragments, which are disconnected from the low-molecular weight fragments by a gap and normally escape from other database search algorithms, and detect both intra-and inter-molecular disulfide bridges. The PEAKS-based approach requires only MS 2 data generated on Orbitrap XL TM or QExactive TM . The limitation of our approach is that PEAKS identifies the spectra based on either the b-type or the y-type ions and cannot overlap both series into one spectrum. A future task would be to simplify the search workflow by overlap of the two spectrum assignments. By applying the "cross-over-read" workflow, we identified intra-and inter-molecular transient disulfide formations in SOD1 and PKM2. The intra-molecular Cys 56 Cys 145 in bovine SOD1 is essentially required for protein stability, correct folding and metal ion binding 22 . Two homo-di-peptides Cys 7 Cys 7 and Cys 145 Cys 145 identified in bovine SOD1 might be formed upon partial de-folding, which is the first step in complete denaturing and plaque formation of SOD1. In human SOD1, disulfide rearrangement was initiated with the attack of Cys 146 rather than Cys 57 during the intermediate state of unfolding 26,27 . PKM2 is known to exist in the catalytically distinct tetrameric and dimeric states. The dimerization of PKM2 involved transient disulfide formations upon high oxidative stress, which might be presumably important for the inhibition of PKM2 activity and to resist oxidative stress 28 . Increases in cellular ROS resulted in the inhibition of PKM2 activity through oxidation of Cys 358 , allowing the cells to withstand ROS 29 .
It is generally accepted that H 2 O 2 serves as a signalling molecule and that its bioavailability changes modulate physiological signalling by altering the oxidation states of selected target proteins, including the kinases such as PKA, PKG and CaMKII (for details, please see the review 30 ). However, little is known on the oxidative state of PP-1α 11,12 . Oxidative stress is routinely studied by exposing model systems to exogenous H 2 O 2 with 10-1000 μM being directly relevant to biology and required to induce measureable protein oxidation or functional effects reliably 30,31 . Although peroxynitrite and hypochlorite, being at least 100-to 10000-fold more reactive towards thiols than H 2 O 2 32 , could oxidize PP-1α at much lower concentrations in the micromolar (maybe even nanomolar) range, in our study, we did not use them due to additional effects of the reagents as nitration by peroxynitrite-derived radicals or chlorination of molecules. We show that the PP-1α activity is reduced upon H 2 O 2 treatment. In NRCMs, H 2 O 2 treatment resulted in phosphorylation changes of cardiac proteins such as PLB, RyR2, and cMyBPC, which are targets of PKA and PP-1α 3,33 . We also show an effect of H 2 O 2 on I-1 phosphorylation, which is a PP-1 inhibitor and is activated by PKA 34 . H 2 O 2 should theoretically induce phosphorylation of both PLB and cMyBPC due to the activation of PKA and inhibition of PP-1α as summarized in Fig. 1Bi. The discrepancy between PLB and cMyBPC phosphorylation in NRCMs treated with H 2 O 2 might be speculated/ explained by differences of temporal dynamics and localizations of PP-1α and PKA under oxidative stress. Since there is not much known how PP-1α and PKA activities are temporally changed at different cellular compartments under oxidative stress, we might only speculate about the time courses and localizations of PKA stimulation and PP-1α inhibition under H 2 O 2 treatment would be different. We might speculate that PKA activity is decreased after 10 min under 100 μM H 2 O 2 and phosphorylation of both PLB and cMyBPC is reduced. In addition, the PLB-dephosphorylation by PP-1α might be dominant after 10 min although the PP-1α activity is decreased under 100 μM H 2 O 2 . Furthermore, we detected the dimerization of PP-1α in a non-denaturing gel even without oxidative stress. Previous studies showed that the binding surface of PP-1α towards several substrates (spinophilin 21 , MYPT1 35 , NIPP1, PNUTS, RepoMAN 36 and taperin 37 ) involved Cys 202 , Cys 127 and Cys 273 . It is very likely that dimerization between Cys 39 and Cys 127 directly protect the active surface of PP-1α when no substrate is around.
Most importantly, we observed the direct correlation of disulfide formations in PP-1α with increasing concentrations of H 2 O 2 , indicating that disulfides were not formed by chance or disulfide scrambling. Formation of internal transient disulfides in PP-1α under oxidative stress involved mainly either Cys 39 , Cys 127 or one of the three glutathionylated Cys-residues Cys 140 , Cys 202 and Cys 245 . Cys 39 , being located at the surface of the PP-1α protein and together with Cys 155 and Cys 158 being defined as the catalytic centre of PP-1α, might be both the activity regulator and the backdoor cysteine (Fig. 6A). Under harsh oxidative conditions, Cys 39 is not available anymore because it serves as a backdoor cysteine to protect Cys 155 and Cys 158 , therefore, we identified only the homo-di-peptide Cys 127 Cys 127 . Moreover, many mixed disulfides involving Cys 39 and Cys 127 are believed to freeze conformational deformation of PP-1α. In general, the advantages of this structural modification would be that misfolding states are required before the correct folding is established 38 , proteins obtain more thermostability 39 and over-oxidation of catalytically active cysteines is prevented 40 . An oxidoreductase active site, which is highly conserved within the PP-1 subfamily, but not in the PP-2A or -2B subfamilies, was identified in close proximity to the phosphatase active site, suggesting a regulatory control mechanism 41 . Since the active site of PP-1α (Cys 155 , Cys 158 , Pro 192 ) is protected by Cy 39 , we might speculate that the dimerization of PP-1α via Cys 39 is actively controlled and might explain the detection of PP-1α dimerization under all conditions tested.
Notably, we detected S-glutathionylation of Cys 140 , Cys 202 and Cys 245 in GST-tagged rPP-1α upon high oxidative stress. This is in line with previous studies showing that S-glutathionylation occurs through the reversible addition of glutathione to thiolate anions of cysteines in the presence of GST. This modification serves both to protect and to modify structure/function 42 . Previous study showed that both PP-1 and PP-2A activity was inhibited by different oxidizing agents including oxidized glutathione (GSSG) though the kinetics of inactivation of two enzymes were different 43 . Interestingly, the addition of GSH could reactivate both enzymes, which might involve Cys 140 of PP-1 43 . Cys 140 is found as an essential glutathionylation site in our study. It was also reported that PP-2A activity was inhibited in Caco-2 cells treated with 20 µM H 2 O 2 or GSSG and the inhibition was restored by GSH 44 . Therefore, we present here the Cys 140 , Cys 202 and Cys 245 -centered disulfide networks (Fig. 6A) and propose that S-glutathionylation is required as a fast response to oxidative stress by forming mixed disulfide bridges, explaining how PP-1α functions as a redox sensor (Fig. 6B). Cysteines with distances even >20 Å (Fig. 6C) might be bridged upon conformational changes when the additional fast reaction with GSH activates the cysteines for disulfide formation. Moreover, other PTMs might be also involved in the redox regulation of enzyme activities, for example, reversible S-nitrosation of thiolate-containing enzymes involved in providing reducing equivalents is considered a redox-regulated mechanism to keep mitochondria in a protected oxidized state under conditions when an excess of nitric oxide was generated, ultimately leading to the formation of the nitrosating species N 2 O 3 45 . By this way, components of the respiratory chain are reversibly maintained in an oxidized state to transiently protect these complexes from an irreversible modification. We may speculate that both mechanisms Recently the formation of transient disulfides within KIM-like tyrosine phosphatases is also linked with the reversible inactivation of the phosphatase activity 46 . Compared to KIM-like tyrosine phosphatase, PP-1α is different in many aspects, for instance, the dimerization is not oxidative stress dependent, but would rather control the surface availability for binding of substrate proteins. Our data demonstrate that PP-1α is more robust to withstand higher levels of redox stress − 50% inactivation is observed at 500 µM H 2 O 2 for PP-1α, but already at 150 µM H 2 O 2 for the KIM-like tyrosine phosphatases. However, it is largely unknown how oxidative stress-induced disulfide formations in PP-1α could influence its interaction with other proteins and the substrate binding. Previous study showed that oxidative stress led to formation of stable complexes containing PP-1α, GADD34, elF2a and TDP-43 and enhancing the substrate binding 47 .
Furthermore, we propose a mechanism how the electron current is established in/out the catalytic centre of PP-1α. Solely, Cys 273 is only 4.4 Å away from Mn 2+ (Fig. 6A), 3.68 Å from His 66 and 8.55 Å from His 248 and all these residues are located at the entry to the cavity (Fig. 5D). It is likely that GST-mediated reduction of Cys 273 can organize an electron flow in/out of the cavity. Moreover, we found several oxidation states of His 248 and His 66 and oxidation of Tyr 306 . It is known that tyrosines and histidines are prone to oxidation and might also co-act as an electron acceptor/transfer pair 48 . Histidines alone can play a role in the oxidation of metal ions 49 . It was shown that PP-1 activity mainly relied on the di-nuclear metal centre, rather than Cys-redox modifications for catalysis 50,51 . Recently, elevated NOX2 activity in the mouse heart resulted in PP-1α inactivation that involves metal centre oxidation rather than the thiol oxidation 11,12 . Altering the redox state of Mn 2+ -Mn 2+ to Mn 3+ -Mn 2+ or Mn 3+ -Mn 3+ can shorten the bond lengths between the metal ions and the ligands and increase the energy barrier of the related reactions 52 .
One limitation of our study is that we investigated the effect of H 2 O 2 on Cys-modifications of PP-1α using recombinant proteins. Cys-modifications of PP-1α might be different in the heart under oxidative stress. Future study should be conducted in cells or tissues. Another limitation is that we studied only some oxidative modifications of protein thiols. Besides intra-and inter-protein disulfides, S-glutathionylation and S-sulfonation we have studied, protein thiols can form other oxidative modifications, including reversible (S-sulfenation, S-nitrosation, S-sulfhydration, S-sulfenamidation) and irreversible (S-sulfination) redox states. To decipher the mechanism how redox stress regulates PP-1α activity, further studies have to be performed to figure out under which conditions irreversible modifications S-sulfination or S-sulfonation occur, which Cys-residues are involved and how intermediate states such as S-sulfenation, S-nitrosation or S-sulfhydration transition to disulfides 53 .
In conclusion, our study demonstrates that the formation of transient disulfides in PP-1α is involved in sheltering against oxidative stress. For a fast response, GST-activity is required. Thus, the GSH-mediated formation of transient disulfides might help proteins to escape from irreversible cysteine oxidation and to prevent their complete inactivation (Fig. 6B). These data suggest that protein S-glutathionylation might act as a valuable biomarker for oxidative stress, with potential for translation into novel therapeutic strategies 54 .

Material and Methods
Animals. All animal experiments were performed in accordance with the guidelines from Directive 2010/63/ EU of the European Parliament on the protection of animals used for scientific purposes. All procedures involving animals were approved by the Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit (Germany).
Live-cell imaging of NRCMs. NRCMs were isolated at postnatal day 1-3 and cultured in a 6-well plate for 3-4 days. 90 min before live-cell imaging, NRCMs were incubated with 0.1, 1 or 10 mM H 2 O 2 in the climate chamber of the Olympus fluorescence microscope (37 °C, 5% CO 2 ). The video frames were recorded with 57.37% lamp intensity every 20 min with an exposure time of 20 ms for 24 h. A matlab based script was used to generate heat maps of the mean contraction for each given H 2 O 2 concentrations 18 . A resolution of 0.6442 μm/pixel was used and dead floating cells were filtered. The changes in the morphology of NRCMs were analysed with Image J.
Phosphatase activity assay. Total phosphatase activity was measured using the EnzChek Kit (Molecular Probes) as previously described 55 . NRCMs were harvested in a passive lysis buffer (20 mM Tris-HCl pH 7.5, 1 mM Na 2 EDTA, 150 mM NaCl, 1 mM EGTA, 1% Triton and complete protease inhibitor (Roche)). The protein content was measured using Pierce BCA Protein Assay Kit. To analyze phosphatase activity, 6,8-difluoro-4-m ethylumbelliferyl phosphate (DiFMUP) was used as a substrate to mimic phosphorylated proteins, which does not fluoresce. Upon dephosphorylation by phosphatase, DiFMUP changes to DiFMU and becomes highly fluorescent. 100 µM DiFMUP as substrate was pre-mixed with 100 mM sodium acetate (reaction buffer, pH 5.5), and then incubated with 20 µg total protein at RT for 15 min. Fluorescence values of converted DiFMU were read on a Flexstation 3 (Molecular Devices). Linearity of the assay as a function of PPase activity was tested using standards of commercially available recombinant PP-1 (Sigma Aldrich, SRP5338).
In-vitro phosphatase activity assay (Promega) for rPP-1α was performed in 96-well format according to the manufacturer's introduction: (i) reaction of rPP-1α in the buffer containing MgCl 2 and MnCl 2 using non-fluorescent phosphorylated bisamide rhodamine 110 (R110) and 7-amino-4-methylcoumarin (AMC) as substrates; (ii) addition of H 2 O 2 at different concentrations; (iii) stopping reaction after 10 min by adding protease; (iv) digestion of R110 and AMC to generate highly fluorescent R110 and AMC; and (v) calculation of the R110/AMC ratio as a measurement of the PP-1α activity.
Oxidative stress experiments on rPP-1α and PKM2. 100 ng GST-tagged rPP-1α (Sigma-Aldrich PyMOL analysis. PyMOL (Version 2.0.6 Schrödinger, LLC) was used to visualize protein structure and to measure molecular distances of all Cys-residues and Mn 2+ . A PyMOL script file was prepared for selection of several residues and their labelling either as coloured sticks or balls.