Tài liệu Báo cáo khoa học: ATP-dependent modulation and autophosphorylation of rapeseed 2-Cys peroxiredoxin docx

ATP-dependent modulation and autophosphorylation of rapeseed 2-Cys peroxiredoxin Martin Aran 1 , Daniel Caporaletti 1 , Alejandro M. Senn 1 , Marı ´a T. Tellez de In ˜ on 2 , Marı ´a R. Girotti 1 , Andrea S. Llera 1 and Ricardo A. Wolosiuk 1 1 Instituto Leloir, IIBBA-CONICET, Universidad de Buenos Aires, Argentina 2 INGEBI-CONICET, Buenos Aires, Argentina Rather than viewing reactive oxygen species (ROS) as toxic by-products of aerobic metabolism we now know them to be members of signaling networks that modulate important physiological processes [1,2]. Germane to the homeostatic regulation of ROS con- centrations, a large group of peroxidases devoid of selenium- and heme-prosthetic groups, the peroxi- redoxins (Prx) (EC 1.11.1.15), catalyze the reduction of hydroperoxides and peroxinitrite [3–6]. The number of subfamilies in this ubiquitous family of proteins varies depending on the classification criteria used but, in all cases, one subfamily encompasses polypep- tides in which there is strict conservation of two cyste- ine residues – the 2-Cys Prx [7–9]. The typical 2-Cys Prx, found in prokaryotes and eukaryotes, is a head- to-tail arranged homodimer in which one of the con- served cysteines of the polypeptide is linked via an intercatenary disulfide bond to the complementary cysteine of the paired subunit. Crucial to the peroxi- dase activity is the cysteine residue located at the N-terminal region, ‘the peroxidatic cysteine’, whose sulfhydryl group (-Cys-SH) turns into sulfenic acid (-Cys-SOH) after reacting with a hydroperoxide (ROOH). The sulfenate subsequently reacts with the cysteine located in the C-terminal region of the paired polypeptide, ‘the resolving cysteine’, forming a second intermolecular disulfide linkage (-Cys-S-S-Cys-). Com- pleting the peroxidatic cycle, one of the disulfides is Keywords 2-Cys peroxiredoxin; ATP binding; autophosphorylation; sulfinic-phosphoryl anhydride; sulfonic-phosphoryl anhydride Correspondence R. A. Wolosiuk, Instituto Leloir, Patricias Argentinas 435, C1405BWE Buenos Aires, Argentina Fax: +54 11 5238 7501 Tel: +54 11 5238 7500 E-mail: rwolosiuk@leloir.org.ar (Received 12 November 2007, revised 14 January 2008, accepted 16 January 2008) doi:10.1111/j.1742-4658.2008.06299.x 2-Cys peroxiredoxins (2-Cys Prx) are ubiquitous thiol-containing peroxidas- es that have been implicated in antioxidant defense and signal transduction. Although their biochemical features have been extensively studied, little is known about the mechanisms that link the redox activity and non-redox processes. Here we report that the concerted action of a nucleoside triphos- phate and Mg 2+ on rapeseed 2-Cys Prx reversibly impairs the peroxidase activity and promotes the formation of high molecular mass species. Using protein intrinsic fluorescence in the analysis of site-directed mutants, we demonstrate that ATP quenches the emission intensity of Trp179, a residue close to the conserved Cys175. More importantly, we found that ATP facilitates the autophosphorylation of 2-Cys Prx when the protein is succes- sively reduced with thiol-bearing compounds and oxidized with hydroper- oxides or quinones. MS analyses reveal that 2-Cys Prx incorporates the phosphoryl group into the Cys175 residue yielding the sulfinic-phosphoryl [Prx-(Cys175)-SO 2 PO 3 2) ] and the sulfonic-phosphoryl [Prx-(Cys175)-SO 3 PO 3 2) ] anhydrides. Hence, the functional coupling between ATP and 2-Cys Prx gives novel insights into not only the removal of reactive oxygen species, but also mechanisms that link the energy status of the cell and the oxidation of cysteine residues. Abbreviations 2-Cys Prx, 2-Cys peroxiredoxin; ANS, 8-anilinonaphthalene-1-sulfonate; ROS, reactive oxygen species. 1450 FEBS Journal 275 (2008) 1450–1463 ª 2008 The Authors Journal compilation ª 2008 FEBS cleaved back to thiols by the concerted action of cel- lular reductants and protein-disulfide oxido-reductases. Often regarded as a peroxidase, the additional func- tion as a molecular chaperone, identified in human and yeast 2-Cys Prx [10,11], exhibits a marked sensi- tivity to a variety of compounds and conditions, such as reductants and ROS. An imbalance between these dual functions is probably associated with many human pathologies, such as thyroid tumors, breast and lung cancer, Alzheimer’s disease and neurodegen- erative disorders [12]. By contrast to the extensive literature on the redox modulation of different enzyme activities in chloro- plasts and non-photosynthetic systems, mainly via thiol–disulfide exchange [13–16], systematic efforts to examine the opposite action of non-redox processes on redox reactions are scarce. Most biochemical research on 2-Cys Prx has studied the interplay between the stimuli and the abundance of reductants and oxidants (thermodynamic control), whereas the catalytic fea- tures (kinetic control) have been less clear. Therefore, the reactions leading to ROS generation and detoxifi- cation have been elucidated, but little is known about how oxidative stress is linked to non-redox processes in the signaling networks that modulate cellular func- tions. Studies addressing this issue have found signi- ficant changes in the quaternary structure and dual functions when human 2-Cys Prx is phosphorylated on Thr90 by cyclin-dependent protein kinases, preferably CDK1 (formerly Cdc2) [17,18]. A putative intermedi- ate at the peroxidatic cysteine (-Cys-S(=O)-O-PO 3 2) ) has recently been suggested in the multiple-step pro- cess underlying sulfiredoxin-mediated reduction of 2-Cys Prx-SO 2 H, however, experimental evidence is lack- ing [19–21]. As with many proteins, phosphorylation of 2-Cys Prx via these two mechanisms requires the participation of additional catalysts, i.e. protein kinase and sulfiredoxin. Despite numerous studies showing the close association between ATP and chaperone activity [22], with the exception of serving as the phos- phoryl donor for CDK1 and sulfiredoxin, the direct interaction of a nucleotide with 2-Cys Prx has not been previously addressed. Here, we report that the con- certed action of a nucleoside triphosphate and Mg 2+ on rapeseed 2-Cys Prx impairs the peroxidase activity. More importantly, MS studies show that the successive action of a reductant and an oxidant makes the pro- tein a recipient of the phosphoryl moiety in sulfonic and sulfinic acid forms of Cys175. Hence, ATP has emerged as both a novel regulator of 2-Cys Prx func- tions and the phosphoryl donor for the autophospho- rylation at the resolving cysteine. Results The concerted action of nucleotides and Mg 2+ modulates the peroxidase activity of 2-Cys Prx A thorough inspection of the available X-ray structure of human 2-Cys Prx (PDB entry: 1QMV) revealed that the covalently linked homodimer creates a large inter- nal cavity comprising segments of two polypeptides (-Leu42–Pro46, Arg129–Ile133, Gln141–Asn146, Gly151–Arg159-) and (-Pro54–Ile57, Cys175–Gly178-). Because the size of the cavity (0.515 nm 3 ) in silico eas- ily docks a molecule of ATP (0.33 nm 3 ), it was impor- tant to know whether the functions of 2-Cys Prx were sensitive to insertion of the nucleotide. As shown in Fig. 1A, a nucleoside triphosphate in concert with Mg 2+ lowered the peroxidase activity in a dose-depen- dent manner, purine nucleotides being more potent than pyrimidine derivatives. In particular, the response of the peroxidase activity to increasing concentrations of ATP exhibited three well-defined stages: (a) monot- onous decay (I 0.5 = 0.25 mm), (b) stabilization at half of the maximal activity from 0.9 to 1.2 mm, and (c) a sharp decrease to undetectable levels beyond 1.5 mm (I 0.5 = 1.40 mm). Interestingly, inhibition mediated by the other purine nucleotide, GTP, was significantly similar in the first two stages, but lacked the third. Following these initial experiments, we investigated whether other phosphorylated compounds and bivalent cations exhibited similar capacity. In the presence of 2mm Mg 2+ , the rate of H 2 O 2 removal was inhibited by 60, 5 and 5% when it was assayed with 2 mm ADP, AMP or orthophosphate, respectively (not shown). By contrast, Mg 2+ was the most efficient cation in assisting nucleotide-dependent inhibition (100%), whereas Ca 2+ (92%), Mn 2+ (85%) and Zn 2+ (65%) exhibited a varying capacity when the peroxi- dase activity was assayed in the presence of fixed con- centrations of both ATP (3 mm) and bivalent cations (3 mm), (Fig. 1B). At this stage, the lack of peroxidase activity might be attributed to an irreversible change in 2-Cys Prx triggered by the binding of ligands. Against this possibility, totally inactive 2-Cys Prx, caused by incubation with 3 mm ATP and 3 mm Mg 2+ , immedi- ately recovered 75% of its original activity upon chela- tion of Mg 2+ by the addition of 5 mm EGTA (Fig. 1C). Clearly, the capacity of ATP to lower the peroxidase activity in the absence of exogenous com- ponents revealed a mechanism that is substantially different from the inhibition brought about by the phosphorylation of human 2-Cys Prx mediated by the CDK1–cyclin B complex [17]. M. Aran et al. ATP modulates 2-Cys peroxiredoxin FEBS Journal 275 (2008) 1450–1463 ª 2008 The Authors Journal compilation ª 2008 FEBS 1451 In addition to the peroxidase activity that functions in the cellular defense against ROS, yeast and human 2-Cys Prx are molecular chaperones [10,11]. Therefore, it was important to examine 2-Cys Prx beyond a single activity and establish whether the regulation described above previously had wider implications. We found that the rapeseed orthologue efficiently prevents the thermal aggregation of citrate synthase indicating that the chaperone activity is likely to be a general function of typical 2-Cys Prx (Fig. 1D). Remarkably, incorpo- ration of increasing amounts of Mg 2+ into the incuba- tion milieu led to a concomitant reduction in the chaperone capacity which, at variance with the peroxi- dase activity, was not affected by the presence of 2.5 mm ATP. These data provide the first evidence that the dual functions of 2-Cys Prx can be differen- tially regulated by ATP and Mg 2+ . The interaction with ATP modifies structural features of 2-Cys Prx Given the essential role of ATP in the peroxidase activity, we evaluated changes in the structure of 2-Cys Prx brought about by the concerted action of the nucleotide and the bivalent cation. To accurately determine the molecular mass of our 2-Cys Prx prepa- rations, static light-scattering measurements were per- formed, because this spectroscopic technique allows direct estimation of the species in solution [23]. In the absence of perturbants, the predominant form of A C B D Fig. 1. Effect of nucleotides ⁄ Me 2+ on the functions of 2-Cys Prx. (A) Concerted action of nucleotides ⁄ Mg 2+ on the peroxidase activity. The reaction, carried out in a solution supplemented with 3 l M 2-Cys Prx, 2 mM MgCl 2 and the indicated nucleoside triphosphate, was started by the addition of 0.13 m M H 2 O 2 and the remnant of reduced dithiothreitol was estimated with Ellman’s reagent after 15 min. Data from seven independent experiments were averaged and standard deviations were calculated. Control activity: 4.3 nmol H 2 O 2 reduced per min. (B) Effect of ATP ⁄ Me 2+ on peroxidase activity. The assay was essentially similar to (A), except that the concentrations of ATP and the Me 2+ (Mg 2+ ,Ca 2+ ,Mn 2+ ,Zn 2+ ) were both fixed at 3 mM. (C) EGTA-mediated reversal of (ATP ⁄ Mg 2+ )-dependent inhibition of peroxidase activity. 2-Cys Prx (3 l M) was incubated for 3 min with 2 mM ATP and 2 mM MgCl 2 . After the addition of EGTA to a final concentration of 5 mM, the protein solution was further incubated for 5 min and the peroxidase activity was assayed as in (A). (D) Effect of ATP and Mg 2+ on the chap- erone activity. 2-Cys Prx (5 l M) was incubated in 25 mM Tris ⁄ HCl (pH 8) containing, as indicated, different concentrations of MgCl 2 and 2.5 m M ATP. After 10 min at 25 °C followed by 10 min at 45 °C, the assay was started by the addition of citrate synthase and measured as described in Experimental procedures [25]. ATP modulates 2-Cys peroxiredoxin M. Aran et al. 1452 FEBS Journal 275 (2008) 1450–1463 ª 2008 The Authors Journal compilation ª 2008 FEBS rapeseed 2-Cys Prx (polypeptide: 22 316 kDa) had a molecular mass of 260 kDa indicating that it was essentially similar to counterparts from other sources, wherein covalently linked dimers (a 2 ) associate non- covalently, forming doughnut-shaped decamers (a 2 ) 5 (not shown) [24,25]. By contrast, size-exclusion chro- matography revealed that protein dissolved in solu- tions containing 3 mm ATP and 3 mm Mg 2+ exhibited a 2550 kDa higher-order assembly that returned imme- diately to the decameric state upon the removal of ATP or Mg 2+ . It is noteworthy that low concentra- tions of two well-known intracellular components converted the rather stable decamer to higher-order assemblies whose molecular mass approached that of the dodecahedron [(a 2 ) 5 ] 12 observed recently in electron microscopy preparations of the erythrocyte orthologue treated with polyethylene glycol [26]. Data on the inhibition of peroxidase activity along with the reversible oligomerization of 2-Cys Prx were consistent with a specific binding of ATP ⁄ Mg to the protein. In line with this prediction, positive and nega- tive differences in absorbance appeared following incu- bation of 2-Cys Prx with ATP in the absence and presence of Mg 2+ , respectively (supplementary Fig. S1). Although these experiments confirmed an interaction between the nucleotide ⁄ Me 2+ couple and the protein, the differential response could not be attributed specifically to any of the interacting species. Therefore, we turned our attention to fluorescence emission spectroscopy which provides information about the polarity of local environments surrounding either extrinsic probes that bind to proteins or intrinsic fluorophores buried in the protein interior. In a first set of experiments, we relied on a biophysical probe commonly used to study the characteristics of protein surfaces, 8-anilinonaphthalene-1-sulfonate (ANS), which, as expected, exhibited an emission maximum wavelength at 512 nm that was not modified by the presence of 3 mm ATP or 3 mm Mg 2+ (Fig. 2A). At variance, reflecting the affinity of this extrinsic probe towards exposed protein hydrophobic surfaces, 2-Cys Prx led to a marked enhancement of the emis- sion intensity with a concurrent displacement of the A C B Fig. 2. Effect of the binding of ATP ⁄ Mg 2+ to 2-Cys Prx on the fluo- rescence emission of extrinsic and intrinsic chromophores. (A) Sen- sitivity of the extrinsic probe ANS. Binding of ANS to 2-Cys Prx was performed for 10 min at 25 °C in solutions of 20 m M Tris ⁄ HCl (pH 7.8) containing 75 l M ANS (e 350nm : 5000 M )1 Æcm )1 ), and, as indicated, 10 l M 2-Cys Prx, 3 mM ATP and 3 mM Mg 2+ . Protein solutions were excited at 370 nm and emission spectra were scanned from 410 to 600 nm. The spectral bandwidths were 5 nm. (B) Quenching of tryptophan fluorescence. Equilibrium fluorescence measurements were conducted increasing the concentrations of ATP or ADP, as indicated, while keeping constant the concentration of 2-Cys Prx (2 l M) and Mg 2+ (2 mM). After correction for the inner filter effect, data were fitted to the saturation curve equation using nonlinear least-squares regression analyses. The difference in fluo- rescence (DF) between 2-Cys Prx (F o ) and 2-Cys Prx-ATP-Mg 2+ complex (F) at 340 nm was plotted according to Lehrer [28] (inset). (C) Quenching of emission intensity in W88F and W179F 2-Cys Prx. Fluorescence measurements were performed as described in (B), except that W88F and W179F mutants replaced for the wild-type 2-Cys Prx. M. Aran et al. ATP modulates 2-Cys peroxiredoxin FEBS Journal 275 (2008) 1450–1463 ª 2008 The Authors Journal compilation ª 2008 FEBS 1453 spectrum to a maximum at 480 nm. At this stage, the addition of 3 mm ATP and 3 mm Mg 2+ did not shift the maximum emission wavelength, but progressively increased the emission intensity, indicating that the nucleotide and the bivalent cation significantly enhanced the proportion of protein hydrophobic patches. Although these experiments were informative regard- ing the ability of 2-Cys Prx to interact with ATP, it was imperative to determine the nucleotide binding site. This information could be gained from the intrin- sic fluorescence because the constituent polypeptide held two conserved tryptophan residues that exhibited a maximum emission wavelength centered at 343 nm, suggesting a rather polar environment around the in- dol side chains (supplementary Fig. S2A) [27]. Unfor- tunately, the concentration of nucleotides in these experiments never exceeded 0.2 mm because the intense inner filter effect caused by the purine ring impaired the excitation of tryptophan residues. Despite this limi- tation, if ATP perturbs the environment of Trp88 or Trp179 to some extent, the fluorescence emission should show a shift in maximum wavelength or a decrease in intensity when the nucleotide changes the conformation of the protein or collides with the fluoro- phore, respectively. Incorporation of ATP ⁄ Mg did not shift the maximum wavelength but caused a marked quenching of the emission intensity that was much less pronounced with ADP ⁄ Mg (Fig. 2B). Stern–Volmer analyses showed a pronounced downward curvature as result of a heterogeneous response of intrinsic fluoro- phores towards the quencher. In this context, if the deviation from linearity reflected a fluorophore inac- cessible to the nucleotide, the Stern–Volmer relationship should become linear using the expression developed by Lehrer [F o ⁄ (F o )F)=1⁄ f a +(1⁄ {f a ÆK SV Æ[Q]})] [28,29]. As shown in Fig. 2B (inset), the straight line was con- gruent with a unique tryptophan residue of 2-Cys Prx accessible to ATP⁄ Mg (f a = 0.26; K SV = 9.7 · 10 )3 Æ m )1 ). To unambiguously define the indol ring sensitive to ATP ⁄ Mg, we examined the intrinsic emission fluo- rescence in variants of 2-Cys Prx where Trp88 and Trp179 were replaced conservatively by phenylalanine via site-directed mutagenesis. The results in Fig. 2C clearly illustrate that the marked reduction in emission intensity caused by the quencher in W88F 2-Cys Prx was similar to its wild-type counterpart, whereas the W179F variant was insensitive to ATP ⁄ Mg. These findings demonstrated that the ATP binds to 2-Cys Prx close to Trp179 and, as a consequence, to the resolving Cys175. In this study, two complementary experiments indicated that the conservative replace- ment of tryptophan residues did not lead to gross alterations in the structure of 2-Cys Prx. First, the emission spectrum of W88F 2-Cys Prx was similar to its wild-type counterpart (k max = 343 nm), whereas that of the W179F variant was slightly blue-shifted (k max = 338 nm) (supplementary Fig. S2A). Second, the catalytic capacity was not affected because neither the basal nor the ATP-inhibited peroxidase activities were appreciably different from wild-type 2-Cys Prx (Fig. S2B). The sequential action of reductants and oxidants predisposes 2-Cys Prx to autophosphorylation In considering whether the interaction with ATP ⁄ Mg proceeded further to the specific phosphorylation of 2-Cys Prx, we noted that ten serine, one threonine and two tyrosine residues appeared as putative sites for phosphorylation (program netphos 2.0, Expassy). Therefore, we conducted a phosphorylation assay in which our preparation of rapeseed 2-Cys Prx was first treated with reductants and oxidants, then sub- sequently incubated with [c 32 P]ATP ⁄ Mg 2+ and finally subjected to non-reducing SDS ⁄ PAGE (Fig. 3A). In this successive in vitro reduction fi oxidation of 2-Cys Prx, we were compelled to use high concentra- tions of cumene hydroperoxide in the oxidation step because high concentrations of dithiothreitol, required for the complete and fast cleavage of disulfide bonds, remained in the solution. To our surprise, a 23 kDa- labeled band appeared when the recombinant protein was (a) incubated successively with 10 mm dithiothrei- tol, 10 mm cumene hydroperoxide and [ c 32 P]ATP ⁄ Mg 2+ , (b) subjected to non-reducing SDS ⁄ PAGE, and (c) characterized by Ponceau Red staining and autora- diography (Fig. 3B). Although not shown, four control experiments carried out under comparable conditions were consistent with the specific covalent binding of the phosphoryl moiety to 2-Cys Prx. First, 32 P-labeled bands did not appear in the autoradiography when chloroplast thioredoxin-m, chloroplast fructose-1,6- bisphosphatase or a-lactalbumin were used in place of 2-Cys Prx. Second, the autophosphorylation of 2-Cys Prx could not be attributed to artifacts linked to the unspecific binding of the nucleotide, as neither the presence of ADP, AMP or GTP, nor pulse and chase experiments with 3 mm nonradioactive ATP affected the incorporation of the 32 P-label into the protein. Third, supporting the formation of a covalent link as opposed to a protein highly resistant to SDS denatur- ation [30], the radioactive label remained linked to 2-Cys Prx after boiling or digestion with trypsin but was completely stripped from the protein by incubation with alkaline phosphatase. Fourth, the requirement for ATP modulates 2-Cys peroxiredoxin M. Aran et al. 1454 FEBS Journal 275 (2008) 1450–1463 ª 2008 The Authors Journal compilation ª 2008 FEBS MgCl 2 was neither replaced nor affected by CaCl 2 or MnCl 2 . Moreover, the requirement for the sequence reduction fi oxidation was further supported by experiments in which a compound generally used for cleaving disulfide bonds (2-mercaptoethanol) partially substituted for dithiothreitol and hydroperoxides (H 2 O 2 , t-butyl hydroperoxide) and two structurally dif- ferent quinones (2-hydroxy-1,4-naphthoquinone and 1,4-dihydroxyanthraquinone) were as efficient as cum- ene hydroperoxide (supplementary Fig. S3). The above quenching of Trp179 fluorescence by ATP was of particular interest in characterizing the autophosphorylation because the evolutionary conser- vation of this residue in the 2-Cys Prx subfamily is unknown. We therefore examined the ability of W88F and W179F 2-Cys Prx to incorporate the 32 P-label after successive incubations with dithiothreitol and cumene hydroperoxide. As shown in Fig. 3C, the for- mer variant was indistinguishable from wild-type 2-Cys Prx, whereas the latter was not functional. Near Trp179, the resolving cysteine is an additional con- served residue that can be predicted to interact with ATP. Supporting this view, we estimated in modeling work on 2-Cys Prx that the nitrogen atom in the indol ring of Trp179 is located 1.571 and 0.401 nm from the sulfur atoms of the peroxidatic and resolving cysteines, respectively [31]. Taken together, the close proximity to Trp179 and the requirement for sequential reduc- tion fi oxidation raised the possibility that Cys175 was actively involved in incorporation of the phos- phoryl moiety. Consistent with this, Fig. 3C shows that a serine in place of Cys53 and Cys175 retained and abrogated, respectively, the ability to incorporate the 32 P-label into 2-Cys Prx. Notably, this active par- ticipation of the resolving cysteine in the autophospho- rylation uncovered a new function that departed markedly from the known role in the peroxidase activity. Surprisingly, autophosphorylation of C53S 2-Cys Prx did not require successive incubation with dithiothreitol and the hydroperoxide but it was extre- mely sensitive to the addition of dithiothreitol (Fig. 3D). Given that the sulfur atom in the cysteine residues of proteins can adopt various oxidation numbers, our preparations of C53S 2-Cys Prx may have contained some proportion of spontaneously oxidized Cys175. To evaluate this possibility, C53S 2-Cys Prx was digested with trypsin and the peptides were examined by MS. A peak at m ⁄ z 2800.36 exhibited the expected mass of the intrapeptide span- ning from residue 160 to residue 184 [-RflT 160 LQAL- QYVQENPDEVCPAGWKPGEK 184 flS-], wherein the sulfur atom of Cys175 was totally reduced (Fig. 4). Of note, the presence of the sulfhydryl group at Cys175 was confirmed in parallel experiments in which MS studies were conducted with the adduct formed between C53S 2-Cys Prx and iodoacetate (not shown). Because the less intense peak at m ⁄ z 2832.36 was consistent with the addition of two A CD B Fig. 3. Autophosphorylation of 2-Cys Prx. (A) Experimental outline. (B) Requirement of reductants and oxidants. 2-Cys Prx was (a) incubated successively with 10 m M dithiothreitol (DTT), 10 mM cumene hydroperoxide (CuOOH) and [c 32 P]ATP, (b) subjected to non-reducing SDS ⁄ PAGE, and (c) transferred to nitrocellulose membranes for protein estimation and autoradiography, as described in Experimental procedures. (C) Role of conserved tryptophan and cysteine residues. W88F, W179F, C53S and C175S 2-Cys Prx were incubated, as indicated, with 10 m M dithiothreitol, 10 mM cumene hydroperoxide and [c 32 P]ATP prior to non-reducing SDS ⁄ PAGE and autoradiography, as outlined in (A). (D) Autophosphorylation of C53S 2-Cys Prx. C53S 2-Cys Prx was incubated for 10 min only in the presence and absence of 10 m M dithiothreitol prior to the addition of [c 32 P]ATP, non-reducing SDS ⁄ PAGE and autoradiography. M. Aran et al. ATP modulates 2-Cys peroxiredoxin FEBS Journal 275 (2008) 1450–1463 ª 2008 The Authors Journal compilation ª 2008 FEBS 1455 oxygen atoms to the respective 160–184 tryptic pep- tide, we further analyzed the sequence of informative ions to confirm the presence of a sulfinic group at Cys175. Accordingly, fragment ions from y1 to y9 showed the expected mass for residues spanning from Lys184 to Pro176, whereas trapped ions beyond y10 exhibited a mass shift of 32. The unequivocal assignment of two oxygen atoms to the Cys175 residue of C53S 2-Cys Prx revealed the unsuspected formation of oxyacid groups at sulfur atoms of the resolving cysteine. ATP phosphorylates the sulfinic and sulfonic forms of the Cys175 residue Although the Tyr166 residue in the 160–184 peptide appeared in silico to be one site for the incorporation of a phosphoryl group (netphos 2.0, Expassy), we found in separate experiments that autophosphoryla- tion of the Y166F mutant was similar to wild-type 2-Cys Prx. Given the absence of another putative site, we approached the localization of the phosphoryl moi- ety by examining the mass spectra of proteolytic digests obtained from 2-Cys Prx treated successively with dithiothreitol fi cumene hydroperoxide fi ATP. To locate any modification in the sequence of the 160–184 peptide, we relied on not only the difference in masses (m ⁄ z value), but also the y-series, the complementary b-series and the coincidence of stretches assigned to identical signals from different experiments. In these analyses, the peaks at m ⁄ z 2800.36 and 2832.36 reflected the expected mass of the 160–184 peptide holding at Cys175 a sulfhydryl group and two addi- tional oxygen atoms, respectively (Fig. 5A). As illus- trated for the former signal and in consonance with above results (see Fig. 4), the y- and b-ion series obtained for selected trapped ions confirmed that the sulfur atom of the resolving Cys175 bore a sulfhydryl group. From the repertoire of less intense signals but with low noise levels, two novel species at m ⁄ z 2934.36 and 2950.35 were particularly attractive because the masses matched the monosodium adducts [M + Na] + of the phosphorylated 160–184 peptide bearing sulfinic and sulfonic groups, respectively (Fig. 5B) [32–34]. As illustrated for the latter signal, sequence informative y-ions from m ⁄ z 0 to 970 were identical to those obtained in the spectra of m ⁄ z 2800.36 (Fig. 5A) and 2832.36 (see Fig. 4), thus proving that they originated from the 160–184 peptide. But more importantly, the absence of ions from y10 to y19 and the presence of Fig. 4. MS ⁄ MS spectra of the 160–184 tryptic peptide from C53S 2-Cys Prx. Expanded view of peaks at m ⁄ z 2800.36 and 2832.35 and the fragmentation of the peak at m ⁄ z 2832.35. 2-Cys Prx was digested with trypsin and prepared for MALDI-TOF MS as described in Experi- mental procedures. Data were first collected, smoothed and calculated the centroid using the software FLEXANALYSIS, and then plotted in GRAPHPAD PRISM. All labeled peaks were at least three times above background. The amino acid sequence of the 160–184 tryptic peptide bearing the sulfinic group is displayed above the spectrum. The fragmentation patterns that generate ions y and b are illustrated along the peptide sequence wherein (*) are fragment ions bearing –SO 2 H. ATP modulates 2-Cys peroxiredoxin M. Aran et al. 1456 FEBS Journal 275 (2008) 1450–1463 ª 2008 The Authors Journal compilation ª 2008 FEBS shifted ions from y10* to y17* revealed that the sul- fonic form of Cys175 held the monosodium adduct of one phosphoryl group (-SO 3 PO 3 2) ) thereby providing the first direct evidence for the phosphorylation of an oxyacid group at a cysteine residue. The diagnostic value of MS profiles regarding mainly the selected peaks was confirmed in a total of 17 independent spectra obtained with different instruments and samples. Discussion Over the last decade it has become apparent that 2-Cys Prx is a key component of signal transduction A B Fig. 5. MS ⁄ MS spectra of the 160–184 tryptic peptide from 2-Cys Prx. Phosphorylated 2-Cys Prx was digested with trypsin and prepared for MALDI-TOF MS as described in Experimental Procedures. Data were examined as described in Fig. 4. The amino acid sequence of the 160–184 tryptic peptide bearing unphosphorylated and phosphorylated cysteines are displayed above the spectrum. The fragmentation pat- terns that generate ions y and b are illustrated along the peptide sequence wherein (*) are fragment ions bearing –SO 3 PO 3 HNa. (A) Expanded view of peaks at m ⁄ z 2800.36 and 2832.35 and the fragmentation of the peak at m ⁄ z 2800.36. (B) Expanded view of peaks at m ⁄ z 2934.36 and 2950.35 and the fragmentation of the peak at m ⁄ z 2950.35. M. Aran et al. ATP modulates 2-Cys peroxiredoxin FEBS Journal 275 (2008) 1450–1463 ª 2008 The Authors Journal compilation ª 2008 FEBS 1457 pathways, ultimately controlling proteins involved in diverse cellular processes, such as cell proliferation, differentiation, apoptosis and photosynthesis [25,35– 38]. This study is the first to demonstrate that the activities associated with 2-Cys Prx are regulated directly by mechanisms sensitive to nucleotides and bivalent cations in which the concerted action of both compounds reversibly impairs the peroxidase activity, whereas only Mg 2+ lowers the chaperone capacity [10,11]. In addition to the differential regula- tion of the dual functions, inhibition of the peroxi- dase activity is highly specific because, of the nucleotides presented here, purine derivatives are markedly more effective than pyrimidine bases. Given that nucleotides do not participate directly in the reduction of hydroperoxides, it follows that the observed loss of activity is almost certainly due to a local effect on the structure of the protein (see below). These findings are important for understand- ing the fundamental question of how 2-Cys Prx uti- lizes non-redox compounds to regulate the associated functions and, in so doing, to cope with situations of oxidative stress. This extremely rapid and reversible association with low molecular mass compounds devoid of redox capacity may have wide applicability because we recently reported that 2-Cys Prx in con- certed action with fructose-1,6-bisphosphate and Ca 2+ stimulates the activity of chloroplast fructose- 1,6-bisphosphatase [25]. 2-Cys Prx is an obligate homodimer (a 2 ) whose con- version to doughnut-shaped (a 2 ) 5 decamer is redox sensitive [24]. Apropos, oxidants drive the human and yeast orthologues from lower molecular mass forms to higher molecular mass complexes and, in so doing, impair the peroxidase activity and enhance the chaper- one capacity [10,11]. Although the transition of 2-Cys Prx among oligomers with different molecular masses may be conceptually adequate for the regula- tion of associated functions [24,27], the unprecedented ATP-mediated oligomerization is beyond the scope of this study and it will be reported elsewhere. However, spectroscopic studies of 2-Cys Prx variants clearly dis- cerned the role of ATP. UV-differential spectropho- tometry and fluorescence emission of the extrinsic probe ANS initially revealed that the protein interacts directly with ATP, and further exploration of the intrinsic fluorescence emission in site-directed mutants unambiguously assigned the binding site close to Trp179. In the crystal structure of human 2-Cys Prx, this region encloses a cavity large enough to hold nu- cleotides in which the tryptophan residue homologous to rapeseed Trp179 is located far from the peroxidatic Cys53 and close to the resolving Cys175 [31]. Given that (a) the mechanism of peroxidase activity includes the formation of an intercatenary disulfide bond link- ing the peroxidatic cysteine with the resolving counter- part [3] and (b) ATP locates near the latter (this study), it is reasonable to suggest that the reversible binding of ATP ⁄ Mg 2+ halts the catalytic cycle via ste- ric perturbation of the resolving cysteine. However, we can not exclude the possibility that the reduction of hydroperoxides is inhibited by an allosteric effect of ATP ⁄ Mg 2+ on the peroxidatic cysteine. Although fur- ther studies are required to clarify this issue, our data definitively identify the region surrounding the resolv- ing cysteine of typical 2-Cys Prx as the target for nucleotides. The main outcome of our study is, however, the importance of oxyacid groups at the resolving Cys175 for the in vitro autophosphorylation of 2-Cys Prx. A combination of evidence from the lack of a similar capacity in other proteins to the behavior of site-direc- ted mutants clearly dismiss the possibility that trace quantities of contaminating bacterial kinases may co- purify with the recombinant protein [38]. The finding that the successive addition of a reductant and an oxi- dant promotes incorporation of the c-phosphoryl moi- ety of ATP indicates that, like other events mediated by 2-Cys Prx, autophosphorylation depends on a spe- cific redox state. The 23 kDa subunit contains two cysteines conserved throughout evolution, and analyses of site-directed mutants show that Cys175 holds the unique reactive thiol involved in autophosphorylation. Moreover, MS detection of over-oxidized sulfur atoms at the resolving cysteine led us to conclude that the sulfonic and sulfinic forms are necessary for linking the phosphoryl moiety to the protein. Further exami- nation of the oxidative step reveals that the autophos- phorylation proceeds in redox environments milder than those induced by harsh oxidants. Indeed, the midpoint redox potentials of 2-hydroxy-1,4-naphtho- quinone (E m7 = )0.15 V) and 1,4-dihydroxy-9,10- anthraquinone (E m7 = )0.18 V) are much lower than H 2 O 2 (E m7 = )1.76 V) which is usually used in studies of ROS [1,2,4]. These data uncover the capacity of the rapeseed resolving Cys175 for the oxidation to sulfinic acid, a process that clearly departs from similar sulfur chemistry at the peroxidatic cysteine [19,20,39,40]. Two lines of research have examined the phos- phorylation of 2-Cys Prx. First, it has been shown that several cyclin-dependent protein kinases promote in vitro the specific phosphorylation of human 2-Cys Prx at a threonine residue homologous to Thr91 in the rapeseed orthologue [17]. Second, the finding that the thiol of mammalian sulfiredoxin [Srx-SH] recruits the c-phosphoryl moiety of ATP ATP modulates 2-Cys peroxiredoxin M. Aran et al. 1458 FEBS Journal 275 (2008) 1450–1463 ª 2008 The Authors Journal compilation ª 2008 FEBS yielding a thiophosphate [Srx-S-PO 3 2) ] led to the proposal that sulfiredoxin subsequently transfers the phosphoryl group to the sulfinic form of the peroxid- atic cysteine in human PrxI [-Cys-S(=O)-OH] [19–21]. At this stage, the sulfinic–phosphoric mixed anhydride [-Cys-S(=O)-O-PO 3 2) ] would be cleaved by a thiol reductant [R-S-H] yielding a disulfide-S-monoxide [-Cys-S(=O)-S-R] that would be finally reduced back to thiol [-Cys-SH]. In this context, the strategy of our phosphorylation of 2-Cys Prx diverges markedly from previous studies in two important aspects: neither requires a complementary catalyst, like cyclin-depen- dent kinases or sulfiredoxin, nor proceeds via Thr91 or the peroxidatic cysteine. Indeed, our data provide entry into a previously unsuspected mechanism by which the successive reductionfioxidation of 2-Cys Prx generates oxyacid groups at Cys175 for the subse- quent formation of the sulfinic-phosphoryl [-(Cys175)- SO 2 PO 3 2) ] and sulfonic-phosphoryl [-(Cys175)-SO 3 PO 3 2) ] anhydrides (Scheme 1). Related to this, the mechanism by which dithiothreitol alone dramatically abrogates the autophosphorylation remains unknown. Does the reductant impair the process (a) before incorporation of the phosphoryl moiety by remov- ing the oxyacid groups or (b) after formation of -(Cys175)-S(=O) 1–2 -O-PO 3 2) by cleaving the mixed anhydride? The answer to these questions will reveal whether the oxyacid group itself at Cys175 or the sulfi(o)nic-phosphoric anhydride are endowed with an unusual reactivity to reductants. Although many studies have concentrated on the events underlying phosphorylation of the peroxidatic cysteine of 2-Cys Prx [19–21], we know of none that addressed the resolving cysteine. In almost all typical 2-Cys Prx, the function of the latter residue was hith- ertho confined to participating in the formation of an intercatenary disulfide bond with the sulfenic acid of the peroxidatic cysteine. Moreover, in line with the current paradigm on the mechanism for the reduction of hydroperoxides [8], the resolving cysteines of try- paredoxin peroxidase and AhpC from Trypanosoma brucei brucei and Salmonella typhymurium have been identified as targets in the reduction of the disulfide bond for the reactivation of peroxidase activity [41,42]. Against this background, we put forward a new scenario wherein ATP interacts actively with 2-Cys Prx and, in so doing, modifies the quaternary structure and associated functions. Moreover, the unusual phosphorylation of Cys175 oxyacid groups brings together the redox chemistry of the sulfur atom and the phosphorylating capacity of ATP, thereby providing a versatile mechanism wherein Cys175 appears as dual sensor able to perceive changes in the redox and energy status of the cell. By virtue of the flexibility of using redox and nonredox chemistries at a single cysteine residue, the possibilities to process a wide spectrum of stimuli into different cellular responses greatly extend the prevalent view circum- scribed to redox transformations of sulfhydryl groups [43]. Experimental procedures Materials Recombinant rapeseed 2-Cys Prx was prepared as described previously [25]. Biochemicals were purchased from Sigma- Aldrich (St Louis, MO, USA). Construction of 2-Cys Prx mutants C-terminal hexahistidine-tagged variants of 2-Cys Prx were generated by the PCR megaprimer method using, in the Scheme 1. Reaction scheme for the autophosphorylation of 2-Cys Prx. A reductant cleaves the intercatenary disulfide bond yielding the reduced form of the sulfur atom at Cys175 (reaction 1). The subsequent oxidation transforms the sulfhydryl group into the sulfinic and sulfonic species (reaction 2). The reactivities of these groups and the close proximity to the ATP binding site facilitate the incorporation of the c–phosphoryl moiety (reaction 3) linking in con- sequence redox and nonredox chemistries. Eventually, a phospha- tase facilitates the hydrolysis of the phosphoryl group and returns Cys175 to the oxidized state. M. Aran et al. ATP modulates 2-Cys peroxiredoxin FEBS Journal 275 (2008) 1450–1463 ª 2008 The Authors Journal compilation ª 2008 FEBS 1459 [...]...ATP modulates 2-Cys peroxiredoxin M Aran et al first round of amplification, the cDNA of rapeseed 2-Cys Prx cloned in pET-22b(+) vector as template, 5¢TAATACGACTCACTATAGG-3¢ [for the T7 promoter of pET-22b(+) vector] as 5¢-primer and 5¢-TCTCCGTAGG GGAGACAAAAGT-3¢, 5¢-ATCCCGCGGGGGAAACCT CATC-3¢ and 5¢-CTGTTTGGACGAACGCAAGATG-3¢ as 3¢-primers for C53S, C175S and W88F variants, respectively... struction of the human erythrocyte peroxiredoxin- 2 dodecahedral higher-order assembly Micron 38, 29–39 Konig J, Lotte K, Plessow R, Brockhinke A, Baier M & Dietz KJ (2003) Reaction mechanism of plant 2-Cys peroxiredoxin Role of the C terminus and the quaternary structure J Biol Chem 278, 24409–24420 Lehrer SS (1971) Solute perturbation of protein fluorescence The quenching of the tryptophyl fluorescence of. .. modulates 2-Cys peroxiredoxin 47 Studer S, Obrist M, Lentze N & Naberhaus F (2002) A critical motif for oligomerization and chaperone activity of bacterial a-heat shock proteins Eur J Biochem 269, 3578–3586 Supplementary material The following supplementary material is available online: Fig S1 Effect ATP-Mg2+ on UV differential spectra of rapeseed 2-Cys Prx Fig S2 W88F and W179F 2-Cys Prx Fig S3 Autophosphorylation. .. After addition of ammonium bicarbonate (pH 8.5) and urea to a final concentration of 0.1 and 2 m, respectively, 2-Cys Prx was digested at 37 °C for 2 h with trypsin (2-Cys Prx ⁄ trypsin 100 : 1, w ⁄ w) A matrix of a-cyano-4-hydroxy-cinnamic acid (5 mgÆmL)1 in 50% acetonitrile containing 0.1% trifluoroacetic acid) was mixed with the sample in a ratio of 8 : 2, spotted onto the Anchor-Chip plate and air dried... 0.2 and 1 cm, respectively) Static light scattering measurements The average molecular mass of 2-Cys Prx was determined at 25 °C on a Precision Detectors PD2010 light-scattering instrument connected in tandem to a Sephadex G-50 column and a LKB 2142 differential refractometer 2-Cys Prx (43 lm) was incubated for 5 min in 50 mm Tris ⁄ HCl (pH 7.8) in the presence and absence of 3 mm ATP ⁄ 3 mm Mg2+ and. .. 2-Cys peroxiredoxin in different environments Nonlinear least-squares regression analyses were performed with the program graphpad prism ´ cas y Tecnicas (MA, DC, AS and MRG) MTI, ASL and RAW are Established Investigators of the latter institution Autophosphorylation of 2-Cys Prx References Protein phosphorylation was carried out at 25 °C in 0.04 mL of 50 mm Tris ⁄ HCl (pH 7.9) containing 10 lm 2-Cys. .. solutions Intrinsic fluorescence measurements The steady-state fluorescence of 2 lm 2-Cys Prx in 20 mm Tris ⁄ HCl (pH 7.8) containing 2 mm MgCl2 was measured at 25 °C in a Jasco FP 770 spectrofluorometer Tryptophan emission spectra were taken from 305 to 370 nm using an excitation wavelength of 295 nm and excitation and emission bandwidths of 4 and 5 nm, respectively To minimize the inner filter effect, aliquots... mass range of m ⁄ z 500–3500 and MS ⁄ MS spectra of select ions were collected on an Ultraflex II MALDI-TOF ⁄ TOF (Bruker Daltonik, Bremen, Germany) Peak identification and monoisotopic peptide mass assignation were automatically performed using the software flexanalysis (Bruker Daltonik) The spectra obtained were interpreted using the FindPept tool (http:// ca.expasy.org/tools/findpept.html) and the MS-Product... redox-sensitive oligomerization of 2-cysteine peroxiredoxins Biochemistry 41, 5493–5504 25 Caporaletti D, D’Alessio AC, Rodriguez-Suarez RJ, Senn AM, Duek PD & Wolosiuk RA (2007) Nonreductive modulation of chloroplast fructose-1,6bisphosphatase by 2-Cys peroxiredoxin Biochem Biophys Res Commun 355, 722–727 26 Meissner U, Schroder E, Scheffler D, Martin AG & Harris JR (2007) Formation, TEM study and 3D recon- 1462... beforehand with the incubation solution The 90° light scattering and refractive index signals of the eluting material were transferred to a PC and analyzed with the discovery32 software supplied by the manufacturer The 90° light scattering detector was calibrated using bovine serum albumin (66.5 kDa) as a standard Molecular masses were determined from the ratio of the two detectors, light-scattering and . ATP-dependent modulation and autophosphorylation of rapeseed 2-Cys peroxiredoxin Martin Aran 1 , Daniel Caporaletti 1 , Alejandro M. Senn 1 ,. spectra of rapeseed 2-Cys Prx. Fig. S2. W88F and W179F 2-Cys Prx. Fig. S3. Autophosphorylation of 2-Cys Prx. This material is available as part of the online