Proximity-based Protein Thiol Oxidation by H2O2-scavenging Peroxidases
2009; Elsevier BV; Volume: 284; Issue: 46 Linguagem: Inglês
10.1074/jbc.m109.059246
ISSN1083-351X
AutoresMarcus Gutscher, Mirko C. Sobotta, Guido H. Wabnitz, Seda Ballikaya, Andreas J. Meyer, Yvonne Samstag, Tobias P. Dick,
Tópico(s)Genomics, phytochemicals, and oxidative stress
ResumoH2O2 acts as a signaling molecule by oxidizing critical thiol groups on redox-regulated target proteins. To explain the efficiency and selectivity of H2O2-based signaling, it has been proposed that oxidation of target proteins may be facilitated by H2O2-scavenging peroxidases. Recently, a peroxidase-based protein oxidation relay has been identified in yeast, namely the oxidation of the transcription factor Yap1 by the peroxidase Orp1. It has remained unclear whether the protein oxidase function of Orp1 is a singular adaptation or whether it may represent a more general principle. Here we show that Orp1 is in fact not restricted to oxidizing Yap1 but can also form a highly efficient redox relay with the oxidant target protein roGFP (redox-sensitive green fluorescent protein) in mammalian cells. Orp1 mediates near quantitative oxidation of roGFP2 by H2O2, and the Orp1-roGFP2 redox relay effectively converts physiological H2O2 signals into measurable fluorescent signals in living cells. Furthermore, the oxidant relay phenomenon is not restricted to Orp1 as the mammalian peroxidase Gpx4 also mediates oxidation of proximal roGFP2 in living cells. Together, these findings support the concept that certain peroxidases harbor an intrinsic and powerful capacity to act as H2O2-dependent protein thiol oxidases when they are recruited into proximity of oxidizable target proteins. H2O2 acts as a signaling molecule by oxidizing critical thiol groups on redox-regulated target proteins. To explain the efficiency and selectivity of H2O2-based signaling, it has been proposed that oxidation of target proteins may be facilitated by H2O2-scavenging peroxidases. Recently, a peroxidase-based protein oxidation relay has been identified in yeast, namely the oxidation of the transcription factor Yap1 by the peroxidase Orp1. It has remained unclear whether the protein oxidase function of Orp1 is a singular adaptation or whether it may represent a more general principle. Here we show that Orp1 is in fact not restricted to oxidizing Yap1 but can also form a highly efficient redox relay with the oxidant target protein roGFP (redox-sensitive green fluorescent protein) in mammalian cells. Orp1 mediates near quantitative oxidation of roGFP2 by H2O2, and the Orp1-roGFP2 redox relay effectively converts physiological H2O2 signals into measurable fluorescent signals in living cells. Furthermore, the oxidant relay phenomenon is not restricted to Orp1 as the mammalian peroxidase Gpx4 also mediates oxidation of proximal roGFP2 in living cells. Together, these findings support the concept that certain peroxidases harbor an intrinsic and powerful capacity to act as H2O2-dependent protein thiol oxidases when they are recruited into proximity of oxidizable target proteins. IntroductionBy now, it is well accepted that hydrogen peroxide (H2O2) acts as a signaling molecule. In a variety of physiological situations, it is generated in a controlled manner and leads to the selective posttranslational modification of cysteine residues on target proteins (1.D'Autréaux B. Toledano M.B. Nat. Rev. Mol. Cell Biol. 2007; 8: 813-824Crossref PubMed Scopus (2399) Google Scholar). Reversible thiol oxidation, in particular disulfide bond formation, changes the functional properties of affected proteins. One prominent example is the transient inactivation of protein tyrosine phosphatases in receptor tyrosine kinase signaling (2.Tonks N.K. Nat. Rev. Mol. Cell Biol. 2006; 7: 833-846Crossref PubMed Scopus (1231) Google Scholar).It is less clear how H2O2 actually oxidizes its target proteins. It is frequently assumed that a low pKa is sufficient to make protein thiols directly reactive toward H2O2. However, it has been pointed out that a low pKa by itself will not lead to a high reaction rate as the rate-limiting step is still impeded by a high activation barrier (3.Winterbourn C.C. Hampton M.B. Free Radic. Biol. Med. 2008; 45: 549-561Crossref PubMed Scopus (929) Google Scholar). To overcome kinetic inhibition, thiol-based peroxidases are equipped with highly evolved catalytic mechanisms (triads or tetrads (4.Tosatto S.C. Bosello V. Fogolari F. Mauri P. Roveri A. Toppo S. Flohé L. Ursini F. Maiorino M. Antioxid. Redox Signal. 2008; 10: 1515-1526Crossref PubMed Scopus (130) Google Scholar)) to bring about the reaction between the peroxidatic thiol and H2O2, thus achieving high rate constants (e.g. 2 × 107 m−1s−1 for Prx2 2The abbreviations and trivial names used are: PrxperoxiredoxinTrxthioredoxinTrxSthioredoxin systemroGFPredox-sensitive green fluorescent proteinGpxglutathione peroxidaseCys-SOHcysteine sulfenic acidH2DCFDA2′,7′-dichlorodihydrofluorescein diacetateDCFdichlorofluoresceinDTTdithiothreitolPBSphosphate-buffered salinedimedone5,5-dimethyl-1,3-cyclohexadionewtwild type. (5.Peskin A.V. Low F.M. Paton L.N. Maghzal G.J. Hampton M.B. Winterbourn C.C. J. Biol. Chem. 2007; 282: 11885-11892Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar)). Specialized catalytic mechanisms for “self-oxidation” are unlikely to exist in redox-regulated proteins, thus explaining their slow reaction with H2O2 when probed in vitro (e.g. 20 m−1s−1 for PTP1B) (3.Winterbourn C.C. Hampton M.B. Free Radic. Biol. Med. 2008; 45: 549-561Crossref PubMed Scopus (929) Google Scholar). It has been argued recently that under the intracellular conditions of kinetic competition, given the high reactivity and abundance of peroxiredoxins, oxidant-sensitive redox-regulated proteins like PTP1B are not likely to be oxidized directly by H2O2 (6.Winterbourn C.C. Nat. Chem. Biol. 2008; 4: 278-286Crossref PubMed Scopus (1687) Google Scholar). A plausible alternative to direct oxidation is a mechanism whereby a peroxidase acts as a primary oxidant acceptor and then passes on the oxidation to a target protein. Oxidative redox relays may also explain the observed selectivity of H2O2 in redox signaling. In support of this idea, a few specific observations of peroxidase-based redox relays have been made in recent years.The best known example is the Orp1-Yap1 redox relay in yeast (7.Toledano M.B. Delaunay A. Monceau L. Tacnet F. Trends Biochem. Sci. 2004; 29: 351-357Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Orp1 (also known as Gpx3) is a seleno-independent member of the glutathione peroxidase (GPx) family. Orp1 is thioredoxin (Trx)-dependent and therefore functionally classified as a peroxiredoxin (8.Maiorino M. Ursini F. Bosello V. Toppo S. Tosatto S.C. Mauri P. Becker K. Roveri A. Bulato C. Benazzi L. De Palma A. Flohé L. J. Mol. Biol. 2007; 365: 1033-1046Crossref PubMed Scopus (98) Google Scholar). Upon encountering H2O2, Orp1 forms a sulfenic acid (Cys-SOH) at its peroxidatic cysteine (Cys36). In the conventional catalytic cycle, Cys36-SOH rapidly condenses with the resolving cysteine of Orp1 (Cys82) to generate an intramolecular disulfide bond that is subject to reduction by Trx. In its redox relay mode, however, Orp1 mediates oxidation of the transcription factor Yap1 rather than oxidation of Trx. In a first step, Orp1 forms an intermolecular mixed disulfide bond with Yap1. It is thought that Cys36-SOH, instead of condensing with Cys82, directly reacts with the target thiol of Yap1 (Cys598). In a second step, the Orp1-Yap1 intermolecular disulfide is attacked by Cys303 of Yap1 and thus exchanged into a Yap1 intramolecular disulfide bond. The disulfide form of Yap1 accumulates in the nucleus and initiates a transcriptional response.The case of Orp1-Yap1 clearly demonstrates the possibility of peroxidase-mediated protein oxidation. However, the Orp1 redox relay may be a unique adaptation, restricted to a specific pairing of proteins in the context of the yeast cell. Therefore, in this study, we asked whether Orp1, as well as other GPx-type peroxidases, may harbor a general intrinsic ability to promote the oxidation of other proteins.Overall, two mechanisms of peroxidase-mediated protein oxidation are conceivable. Firstly, as suggested for Yap1, a target protein thiol may react directly with Cys-SOH (or Sec-SeOH in selenocysteine-based peroxidases), creating an intermolecular disulfide, which is then rearranged into a target intramolecular disulfide by virtue of a second target thiol. Secondly, following the conversion of Cys-SOH into a disulfide bond (either within or between peroxidase subunits or between peroxidase and glutathione), the target protein may be oxidized by a conventional thiol-disulfide exchange reaction. Either scenario implies that proximity and spatial orientation are important. Only if peroxidase and target protein are close to each other can the target protein thiol be expected to intercept either the Cys-SOH or the disulfide state of the peroxidase cycle, both of which are highly transient. Inside the cell, proximity and alignment may be established in a specific manner by adaptor or scaffolding proteins. In fact, the redox relay between Orp1 and Yap1 is known to depend on an additional protein, Ybp1 (9.Veal E.A. Ross S.J. Malakasi P. Peacock E. Morgan B.A. J. Biol. Chem. 2003; 278: 30896-30904Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar), which appears to act as a scaffold.We first addressed the question whether Orp1 is generally able to promote oxidation of thiol proteins when they come into close proximity. As a model target protein, we used redox-sensitive green fluorescent protein (roGFP), which is equipped with an oxidizable dithiol pair on its surface and allows fluorometric real-time measurements of its redox state (10.Dooley C.T. Dore T.M. Hanson G.T. Jackson W.C. Remington S.J. Tsien R.Y. J. Biol. Chem. 2004; 279: 22284-22293Abstract Full Text Full Text PDF PubMed Scopus (597) Google Scholar, 11.Gutscher M. Pauleau A.L. Marty L. Brach T. Wabnitz G.H. Samstag Y. Meyer A.J. Dick T.P. Nat. Methods. 2008; 5: 553-559Crossref PubMed Scopus (601) Google Scholar). We demonstrate that Orp1 does indeed promote the oxidation of roGFP2 in a proximity-dependent manner, both in vitro and inside living cells. In vitro, close proximity of the two proteins in a fusion protein leads to near quantitative conversion of H2O2 molecules into roGFP disulfide bridges. Inside living cells, expression of a roGFP2-Orp1 fusion protein enables specific and sensitive real-time measurements of intracellular H2O2 under physiologically relevant conditions, thus demonstrating the extraordinary efficiency of proximity-based peroxidase redox relays. The mechanism of the Orp1-roGFP2 redox relay in living cells was found to depend on the resolving cysteine of Orp1 and to operate on the basis of conventional thiol-disulfide exchange. We then addressed the question whether mammalian peroxidases also have the ability to transfer oxidizing equivalents to roGFP. We found that the glutathione peroxidase Gpx4, but not the peroxiredoxin Prx6, promotes roGFP oxidation in living cells, suggesting that only certain peroxidases harbor the capacity to play pro-oxidative roles in mammalian redox signaling.DISCUSSIONPeroxiredoxins and glutathione peroxidases have exceptionally high reaction rates with H2O2 (22.Fourquet S. Huang M.E. D'Autreaux B. Toledano M.B. Antioxid. Redox Signal. 2008; 10: 1565-1576Crossref PubMed Scopus (130) Google Scholar) and are likely to trap most of the H2O2 generated under physiological conditions. Assuming that typical oxidant target proteins are in kinetic competition with “professional” H2O2 scavengers, it is not obvious how they become oxidized (3.Winterbourn C.C. Hampton M.B. Free Radic. Biol. Med. 2008; 45: 549-561Crossref PubMed Scopus (929) Google Scholar). One possible resolution of the paradox is that certain peroxidases serve as primary oxidant receptors to convey oxidation to secondary oxidant target proteins. The best studied example for such a peroxidase redox relay is the interaction of the oxidant receptor Orp1 with the oxidant target Yap1. It has not been known whether the Orp1-Yap1 relay represents a unique adaptation, whether Orp1 is more generally able to pass on oxidative equivalents to other proteins, or whether GPx-like peroxidases commonly fulfill relay functions.We have found that Orp1 efficiently mediates electron flow between H2O2 and roGFP in intact cells. Therefore, Orp1 is not mechanistically restricted to use Yap1 as the recipient of oxidative equivalents and thus harbors a more general ability to act as a thiol oxidase for other proteins. This observation suggests the further reaching possibility that other peroxidases, especially those of the GPx family, have a similar intrinsic capacity to promote the oxidation of other proteins. In fact, the human selenocysteine-based glutathione peroxidase Gpx4 was already implicated in the disulfide cross-linking of protamines (23.Conrad M. Moreno S.G. Sinowatz F. Ursini F. Kölle S. Roveri A. Brielmeier M. Wurst W. Maiorino M. Bornkamm G.W. Mol. Cell. Biol. 2005; 25: 7637-7644Crossref PubMed Scopus (199) Google Scholar) and, as shown here, is also capable of mediating the oxidation of roGFP2 in living cells.Coupling peroxidase redox activity to a fluorescent real-time probe offered the opportunity to characterize the conditions required to establish and maintain a functional peroxidase-based redox relay. As expected, it turned out that proximity between the peroxidase and the target protein is a key factor for directing the flow of oxidative equivalents toward the target protein. The efficiency by which wild type Orp1 oxidized roGFP2 was dramatically improved by the co-localization of both proteins within the same fusion protein. Likewise, the natural Orp1-Yap1 redox relay depends on a dedicated adapter protein (9.Veal E.A. Ross S.J. Malakasi P. Peacock E. Morgan B.A. J. Biol. Chem. 2003; 278: 30896-30904Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). It is therefore conceivable that repositioning of peroxidases relative to other proteins, by adapters or otherwise, allows them to switch between oxidant scavenging (i.e. the transfer of oxidative equivalents to Trx or GSH) and oxidant signaling (i.e. the diversion of oxidative equivalents toward redox-regulated target proteins).By comparing roGFP2 fusion proteins based on wild type and mutant Orp1 in dynamic redox measurements, we addressed the reaction mechanism of the Orp1-roGFP2 redox relay. Generally, there seem to exist two possibilities for a target protein to pick up an oxidizing equivalent from a peroxidase. Either it is able to tap the Cys-SOH before the same is converted into a disulfide bridge, or it engages with the disulfide bridge before the same is eliminated by the responsible reducing system. The potential advantage of the first mechanism is that Cys-SOH are highly reactive and that their condensation with free thiols is thermodynamically favored and not significantly impeded by an activation barrier (24.Claiborne A. Yeh J.I. Mallett T.C. Luba J. Crane 3rd, E.J. Charrier V. Parsonage D. Biochemistry. 1999; 38: 15407-15416Crossref PubMed Scopus (457) Google Scholar). However, due to the high driving force of the reaction, the target thiol would have to be prepositioned very close to the nascent Cys-SOH to preempt its reaction with other thiols.We used an Orp1 mutant lacking the resolving cysteine (Cys82) to explore the consequences of stabilizing Cys36-SOH within the fusion protein. In the absence of other thiols, roGFP2-Orp1(CS) responded to H2O2 more rapidly than roGFP2-Orp1(wt). However, the stabilization of Cys36-SOH made the fusion protein prone to inactivation by overoxidation (Fig. 3B), and the whole redox relay became highly susceptible to quenching by glutathione (Fig. 4B). Accordingly, we confirmed that in living cells, Orp1(CS) does not form a working redox relay with roGFP2. The reason appears to be the competition by millimolar concentrations of GSH in the cytosolic environment.Wild type Orp1 was found to mediate roGFP2 oxidation exclusively by thiol-disulfide exchange, in agreement with the previous observation that newly formed Cys36-SOH is rapidly converted into the intramolecular Cys36–Cys82 disulfide bond (16.Ma L.H. Takanishi C.L. Wood M.J. J. Biol. Chem. 2007; 282: 31429-31436Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Although thiol-disulfide exchange is slower than Cys-SOH-triggered roGFP oxidation, the degree of oxidation reached after equilibration was almost identical for both kinds of fusion proteins.The Orp1-Yap1 redox relay is presumed to depend on the direct condensation between Cys36-SOH and Cys598 of Yap1 (7.Toledano M.B. Delaunay A. Monceau L. Tacnet F. Trends Biochem. Sci. 2004; 29: 351-357Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). The fact that in yeast cells the Orp1-Yap1 relay is fully operational in the absence of Cys82 demonstrates that the Orp1 intramolecular disulfide is dispensable for Yap1 oxidation (14.Delaunay A. Pflieger D. Barrault M.B. Vinh J. Toledano M.B. Cell. 2002; 111: 471-481Abstract Full Text Full Text PDF PubMed Scopus (709) Google Scholar). However, this observation does not strictly exclude the possibility that Yap1/Ybp1-associated wild type Orp1 forms the intramolecular disulfide and also uses thiol-disulfide exchange to oxidize Yap1. Nevertheless, it is possible that the Orp1-Yap1 relay exclusively uses the Cys-SOH pathway and, thus, actually differs from the Orp1-roGFP2 relay. As Orp1, Yap1, and Ybp1 co-evolved to interact with one another, they may create a special microenvironment for the optimal alignment between Cys-SOH and the target thiol. The same microenvironment may also protect Cys-SOH against competing thiol-based reductants, including GSH. It is furthermore possible that Ybp1, rather than being a simple adapter, actively suppresses formation of the Orp1 intramolecular disulfide to enforce the Cys-SOH pathway of target oxidation. In contrast, the flexible peptide linker that brings together Orp1 and roGFP2 in the fusion protein, although effective in creating proximity and facilitating oxidant transfer, apparently does not create a microenvironment suitably shielded to support Cys-SOH-mediated oxidant transfer. Thus, the interaction of Orp1 with Yap1 and roGFP2, respectively, may be representative of the two principal pathways by which peroxidases can oxidize target protein thiols. Of note, it has been reported that oxidation of the Saccharomyces pombe Yap1-homologue Pap1 by the peroxiredoxin Tpx1 depends on both the peroxidatic and the resolving cysteine (25.Bozonet S.M. Findlay V.J. Day A.M. Cameron J. Veal E.A. Morgan B.A. J. Biol. Chem. 2005; 280: 23319-23327Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar), suggesting an underlying thiol-disulfide exchange mechanism similar to the Orp1-roGFP2 redox relay. In any case, the in vivo responsiveness of roGFP2-Orp1 demonstrates that a functional peroxidase redox relay does not have to be based on the Cys-SOH mechanism and does not necessarily require highly specialized adapter proteins.Our observations support the idea that at least some peroxidases in the GPx family possess a general propensity to act as protein thiol oxidases when specifically recruited to oxidizable target proteins. Indirect protein thiol oxidation by scaffold-based redox relays potentially is more target-specific than direct protein oxidation by H2O2, thus suggesting a mechanistic basis for H2O2-based redox signaling.Finally, our work establishes peroxidase-roGFP relays as a novel design principle for genetically encoded redox probes. roGFP-based fusion proteins now offer the possibility to study the intracellular behavior of individual peroxidases. We compared roGFP2-Orp1 with the recently described OxyR-based H2O2 probe HyPer. In live cell imaging, both probes showed a similar response and sensitivity. The somewhat slower response of roGFP2-Orp1, relative to HyPer (Fig. 5B), is not unexpected as it is based on the redox equilibration between thiol-disulfide pairs of two protein domains, whereas OxyR transmits a conformational change through the formation of a single intramolecular disulfide bond (26.Lee C. Lee S.M. Mukhopadhyay P. Kim S.J. Lee S.C. Ahn W.S. Yu M.H. Storz G. Ryu S.E. Nat. Struct. Mol. Biol. 2004; 11: 1179-1185Crossref PubMed Scopus (202) Google Scholar). Nevertheless, as we have made no attempts to further optimize the roGFP2-Orp1 construct, in terms of linker length or otherwise, the generation of kinetically enhanced versions is well conceivable. IntroductionBy now, it is well accepted that hydrogen peroxide (H2O2) acts as a signaling molecule. In a variety of physiological situations, it is generated in a controlled manner and leads to the selective posttranslational modification of cysteine residues on target proteins (1.D'Autréaux B. Toledano M.B. Nat. Rev. Mol. Cell Biol. 2007; 8: 813-824Crossref PubMed Scopus (2399) Google Scholar). Reversible thiol oxidation, in particular disulfide bond formation, changes the functional properties of affected proteins. One prominent example is the transient inactivation of protein tyrosine phosphatases in receptor tyrosine kinase signaling (2.Tonks N.K. Nat. Rev. Mol. Cell Biol. 2006; 7: 833-846Crossref PubMed Scopus (1231) Google Scholar).It is less clear how H2O2 actually oxidizes its target proteins. It is frequently assumed that a low pKa is sufficient to make protein thiols directly reactive toward H2O2. However, it has been pointed out that a low pKa by itself will not lead to a high reaction rate as the rate-limiting step is still impeded by a high activation barrier (3.Winterbourn C.C. Hampton M.B. Free Radic. Biol. Med. 2008; 45: 549-561Crossref PubMed Scopus (929) Google Scholar). To overcome kinetic inhibition, thiol-based peroxidases are equipped with highly evolved catalytic mechanisms (triads or tetrads (4.Tosatto S.C. Bosello V. Fogolari F. Mauri P. Roveri A. Toppo S. Flohé L. Ursini F. Maiorino M. Antioxid. Redox Signal. 2008; 10: 1515-1526Crossref PubMed Scopus (130) Google Scholar)) to bring about the reaction between the peroxidatic thiol and H2O2, thus achieving high rate constants (e.g. 2 × 107 m−1s−1 for Prx2 2The abbreviations and trivial names used are: PrxperoxiredoxinTrxthioredoxinTrxSthioredoxin systemroGFPredox-sensitive green fluorescent proteinGpxglutathione peroxidaseCys-SOHcysteine sulfenic acidH2DCFDA2′,7′-dichlorodihydrofluorescein diacetateDCFdichlorofluoresceinDTTdithiothreitolPBSphosphate-buffered salinedimedone5,5-dimethyl-1,3-cyclohexadionewtwild type. (5.Peskin A.V. Low F.M. Paton L.N. Maghzal G.J. Hampton M.B. Winterbourn C.C. J. Biol. Chem. 2007; 282: 11885-11892Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar)). Specialized catalytic mechanisms for “self-oxidation” are unlikely to exist in redox-regulated proteins, thus explaining their slow reaction with H2O2 when probed in vitro (e.g. 20 m−1s−1 for PTP1B) (3.Winterbourn C.C. Hampton M.B. Free Radic. Biol. Med. 2008; 45: 549-561Crossref PubMed Scopus (929) Google Scholar). It has been argued recently that under the intracellular conditions of kinetic competition, given the high reactivity and abundance of peroxiredoxins, oxidant-sensitive redox-regulated proteins like PTP1B are not likely to be oxidized directly by H2O2 (6.Winterbourn C.C. Nat. Chem. Biol. 2008; 4: 278-286Crossref PubMed Scopus (1687) Google Scholar). A plausible alternative to direct oxidation is a mechanism whereby a peroxidase acts as a primary oxidant acceptor and then passes on the oxidation to a target protein. Oxidative redox relays may also explain the observed selectivity of H2O2 in redox signaling. In support of this idea, a few specific observations of peroxidase-based redox relays have been made in recent years.The best known example is the Orp1-Yap1 redox relay in yeast (7.Toledano M.B. Delaunay A. Monceau L. Tacnet F. Trends Biochem. Sci. 2004; 29: 351-357Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Orp1 (also known as Gpx3) is a seleno-independent member of the glutathione peroxidase (GPx) family. Orp1 is thioredoxin (Trx)-dependent and therefore functionally classified as a peroxiredoxin (8.Maiorino M. Ursini F. Bosello V. Toppo S. Tosatto S.C. Mauri P. Becker K. Roveri A. Bulato C. Benazzi L. De Palma A. Flohé L. J. Mol. Biol. 2007; 365: 1033-1046Crossref PubMed Scopus (98) Google Scholar). Upon encountering H2O2, Orp1 forms a sulfenic acid (Cys-SOH) at its peroxidatic cysteine (Cys36). In the conventional catalytic cycle, Cys36-SOH rapidly condenses with the resolving cysteine of Orp1 (Cys82) to generate an intramolecular disulfide bond that is subject to reduction by Trx. In its redox relay mode, however, Orp1 mediates oxidation of the transcription factor Yap1 rather than oxidation of Trx. In a first step, Orp1 forms an intermolecular mixed disulfide bond with Yap1. It is thought that Cys36-SOH, instead of condensing with Cys82, directly reacts with the target thiol of Yap1 (Cys598). In a second step, the Orp1-Yap1 intermolecular disulfide is attacked by Cys303 of Yap1 and thus exchanged into a Yap1 intramolecular disulfide bond. The disulfide form of Yap1 accumulates in the nucleus and initiates a transcriptional response.The case of Orp1-Yap1 clearly demonstrates the possibility of peroxidase-mediated protein oxidation. However, the Orp1 redox relay may be a unique adaptation, restricted to a specific pairing of proteins in the context of the yeast cell. Therefore, in this study, we asked whether Orp1, as well as other GPx-type peroxidases, may harbor a general intrinsic ability to promote the oxidation of other proteins.Overall, two mechanisms of peroxidase-mediated protein oxidation are conceivable. Firstly, as suggested for Yap1, a target protein thiol may react directly with Cys-SOH (or Sec-SeOH in selenocysteine-based peroxidases), creating an intermolecular disulfide, which is then rearranged into a target intramolecular disulfide by virtue of a second target thiol. Secondly, following the conversion of Cys-SOH into a disulfide bond (either within or between peroxidase subunits or between peroxidase and glutathione), the target protein may be oxidized by a conventional thiol-disulfide exchange reaction. Either scenario implies that proximity and spatial orientation are important. Only if peroxidase and target protein are close to each other can the target protein thiol be expected to intercept either the Cys-SOH or the disulfide state of the peroxidase cycle, both of which are highly transient. Inside the cell, proximity and alignment may be established in a specific manner by adaptor or scaffolding proteins. In fact, the redox relay between Orp1 and Yap1 is known to depend on an additional protein, Ybp1 (9.Veal E.A. Ross S.J. Malakasi P. Peacock E. Morgan B.A. J. Biol. Chem. 2003; 278: 30896-30904Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar), which appears to act as a scaffold.We first addressed the question whether Orp1 is generally able to promote oxidation of thiol proteins when they come into close proximity. As a model target protein, we used redox-sensitive green fluorescent protein (roGFP), which is equipped with an oxidizable dithiol pair on its surface and allows fluorometric real-time measurements of its redox state (10.Dooley C.T. Dore T.M. Hanson G.T. Jackson W.C. Remington S.J. Tsien R.Y. J. Biol. Chem. 2004; 279: 22284-22293Abstract Full Text Full Text PDF PubMed Scopus (597) Google Scholar, 11.Gutscher M. Pauleau A.L. Marty L. Brach T. Wabnitz G.H. Samstag Y. Meyer A.J. Dick T.P. Nat. Methods. 2008; 5: 553-559Crossref PubMed Scopus (601) Google Scholar). We demonstrate that Orp1 does indeed promote the oxidation of roGFP2 in a proximity-dependent manner, both in vitro and inside living cells. In vitro, close proximity of the two proteins in a fusion protein leads to near quantitative conversion of H2O2 molecules into roGFP disulfide bridges. Inside living cells, expression of a roGFP2-Orp1 fusion protein enables specific and sensitive real-time measurements of intracellular H2O2 under physiologically relevant conditions, thus demonstrating the extraordinary efficiency of proximity-based peroxidase redox relays. The mechanism of the Orp1-roGFP2 redox relay in living cells was found to depend on the resolving cysteine of Orp1 and to operate on the basis of conventional thiol-disulfide exchange. We then addressed the question whether mammalian peroxidases also have the ability to transfer oxidizing equivalents to roGFP. We found that the glutathione peroxidase Gpx4, but not the peroxiredoxin Prx6, promotes roGFP oxidation in living cells, suggesting that only certain peroxidases harbor the capacity to play pro-oxidative roles in mammalian redox signaling.
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