International Journal of Molecular Sciences

Article Methylselenol Produced In Vivo from Methylseleninic Acid or Dimethyl Diselenide Induces Toxic Protein Aggregation in Saccharomyces cerevisiae

Marc Dauplais 1, Katarzyna Bierla 2, Coralie Maizeray 1, Roxane Lestini 3 , Ryszard Lobinski 2,4,5, Pierre Plateau 1, Joanna Szpunar 2 and Myriam Lazard 1,*

1 Laboratoire de Biologie Structurale de la Cellule, BIOC, École Polytechnique, CNRS-UMR7654, IP Paris, 91128 Palaiseau CEDEX, France; [email protected] (M.D.); [email protected] (C.M.); [email protected] (P.P.) 2 IPREM UMR5254, E2S UPPA, Institut des Sciences Analytiques et de Physico-Chimie Pour l’Environnement et les Matériaux, CNRS, Université de Pau et des Pays de l’Adour, Hélioparc, 64053 Pau, France; [email protected] (K.B.); [email protected] (R.L.); [email protected] (J.S.) 3 Laboratoire d’Optique et Biosciences, École Polytechnique, CNRS UMR7645—INSERM U1182, IP Paris, 91128 Palaiseau CEDEX, France; [email protected] 4 Laboratory of Molecular Dietetics, I.M. Sechenov First Moscow State Medical University, 19048 Moscow, Russia 5 Chair of Analytical Chemistry, Faculty of Chemistry, Warsaw University of Technology, Noakowskiego 3, 00-664 Warszawa, Poland * Correspondence: [email protected]



 Abstract: Methylselenol (MeSeH) has been suggested to be a critical metabolite for anticancer activity Citation: Dauplais, M.; Bierla, K.; of selenium, although the mechanisms underlying its activity remain to be fully established. The aim Maizeray, C.; Lestini, R.; Lobinski, R.; of this study was to identify metabolic pathways of MeSeH in Saccharomyces cerevisiae to decipher the Plateau, P.; Szpunar, J.; Lazard, M. mechanism of its toxicity. We first investigated in vitro the formation of MeSeH from methylseleninic Methylselenol Produced In Vivo from acid (MSeA) or dimethyldiselenide. Determination of the equilibrium and rate constants of the Methylseleninic Acid or Dimethyl Diselenide Induces Toxic Protein reactions between glutathione (GSH) and these MeSeH precursors indicates that in the conditions Aggregation in Saccharomyces that prevail in vivo, GSH can reduce the major part of MSeA or dimethyldiselenide into MeSeH. cerevisiae. Int. J. Mol. Sci. 2021, 22, MeSeH can also be enzymatically produced by glutathione reductase or thioredoxin/thioredoxin 2241. https://doi.org/10.3390/ reductase. Studies on the toxicity of MeSeH precursors (MSeA, dimethyldiselenide or a mixture of ijms22052241 MSeA and GSH) in S. cerevisiae revealed that cytotoxicity and selenomethionine content were severely reduced in a met17 mutant devoid of O-acetylhomoserine sulfhydrylase. This suggests conversion Academic Editor: Hideko Sone of MeSeH into selenomethionine by this enzyme. Protein aggregation was observed in wild-type but not in met17 cells. Altogether, our findings support the view that MeSeH is toxic in S. cerevisiae Received: 4 February 2021 because it is metabolized into selenomethionine which, in turn, induces toxic protein aggregation. Accepted: 19 February 2021 Published: 24 February 2021 Keywords: methylselenol; diselenide; methylseleninic acid; thiol/disulfide exchange; redox equilib- rium; Saccharomyces cerevisiae metabolism; toxicity; protein aggregation Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. 1. Introduction Selenium (Se) is an essential trace element for mammals. It is involved in redox functions as the amino acid selenocysteine, translationally inserted in the active site of a few proteins [1]. Se has attracted considerable interest in the last decades for its reported Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. beneficial effects on the prevention and/or treatment of several cancers and other diseases This article is an open access article but also from a toxicological perspective because of the narrow margin between intakes distributed under the terms and that result in efficacy and those that are toxic [2]. conditions of the Creative Commons Many studies have shown that Se compounds are selectively toxic to malignant cells Attribution (CC BY) license (https:// compared to normal cells, highlighting their potential for cancer therapy [3,4]. There is also creativecommons.org/licenses/by/ accumulating evidence that adverse health effects are associated with excess dietary Se sup- 4.0/). plementation [5,6]. Despite these studies, our understanding of the molecular mechanisms

Int. J. Mol. Sci. 2021, 22, 2241. https://doi.org/10.3390/ijms22052241 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 2241 2 of 20

underlying Se toxicity remains limited. Metabolization of selenocompounds in vivo gives rise to multiple metabolites [7–9], the biological activity of which depends on their trans- formation into different active products [10]. Therefore, defining the metabolic pathways used by selenocompounds and characterizing the toxicity of Se metabolic intermediates are important steps to better understand both the beneficial and toxic mechanisms of Se in human biology. Methylselenol (MeSeH, CH3SeH) has been suggested to be a critical Se-metabolite for anticancer activity [11]. Several studies have shown that Se-precursors metabolized into MeSeH are potent tumor inhibitors [12–15]. Methylseleninic acid (MSeA, CH3SeO2H) is an organoselenium derivative that can be converted into methylselenol by reducing agents. MSeA has shown encouraging anticancer activity in in vitro and in vivo models [16–19], although the underlying mechanism(s) of MSeA in anticancer activity remain elusive. Spallholz et al. proposed that MSeA triggered cell cycle arrest and apoptosis mediated by superoxide formation from redox cycling of MeSeH with oxygen and glutathione (GSH) [20,21]. However, MSeA was shown to induce apoptosis in prostate cancer cell lines without production of reactive oxygen species (ROS) [22]. Another possible mechanism accounting for MeSeH toxicity may be through its ability to reduce disulfide bonds of proteins and/or to form selenylsufide adducts with protein cysteine residues [23–25]. Because redox-sensitive cysteine residues play catalytic, structural, and regulatory roles in the biology of many proteins, reaction of MeSeH with these residues may impact many cellular processes including cell proliferation. For example, MSeA was shown to inactivate Protein kinase C, a receptor for tumor promoters, through a redox modification of critical cysteine sulfhydryls in the catalytic domain of the enzyme [26]. In vivo studies in the model Saccharomyces cerevisiae have significantly contributed to our understanding of the mechanisms that drive the mode of action and the toxicity of several Se compounds [27]. These studies have evidenced that the mode of action of Se is compound-specific. The inorganic Se compound sodium selenite (Na2SeO3) was − shown to be reduced by intracellular thiols to the key metabolite selenide (H2Se/HSe ) that can redox cycle with oxygen and GSH resulting in redox imbalance and generation of ROS that kill cells mainly through DNA damage. Thus, deletion of several genes involved in DNA repair and the GSH redox pathway resulted in hypersensitivity to selenite/selenide [28]. Considering the importance of redox balance in cancer cells, this mechanism might contribute to explain the high sensitivity of tumor cells to selenite. In contrast, the toxicity of selenomethionine (SeMet) involves its metabolic conversion to selenocysteine and misincorporation of the latter in proteins resulting in protein misfolding and aggregation [29]. In this paper, we applied a strategy that gave insights into selenite and SeMet toxicity in yeast to identify metabolic pathways affected by MeSeH. We first investigated in vitro the enzymatic and non-enzymatic formation of MeSeH starting from MSeA, methylse- lenoglutathione (MeSeSG) or dimethyl diselenide (DMDSe). Then, we analyzed the toxicity of these MeSeH precursors in S. cerevisiae. Analysis of various mutant strains supports the view that MeSeH is converted into SeMet, leading to toxic protein aggregation in yeast cells.

2. Result and Discussion 2.1. Thiol Reduction of MSeA, MeSeSG, and DMDSe It is generally accepted that MeSeH is produced spontaneously from MSeA by reaction with thiols such as glutathione (GSH) as schematized below (Equations (1)–(3)) [30].

MeSeA + 2 GSH → MSeOH + GSSG + H2O (1)

MeSeOH + GSH → MeSeSG + H2O (2)

MeSeSG + GSH ↔ MeSeH + GSSG (3) Int. J. Mol. Sci. 2021, 22, 2241 3 of 20

MSeA + 4 GSH → MeSeH + 2 GSSG + 2 H2O However, formation of MeSeH from a mixture of MSeA and GSH has never been precisely quantified. Because of its volatility and reactivity, MeSeH is difficult to detect and its identification usually relies on indirect methods. To study the above reactions, we first used a spectrophotometric method. UV-visible spectra were recorded after mixing 250 µM MSeA with increasing amounts of GSH. As shown in Figure1A, a peak at 260 nm appeared in the spectra immediately after addition of GSH. Then, spectra remained sta- ble over time. The intensity of this peak increased with increasing GSH concentrations until a molar GSH:MSeA ratio of 3:1 was reached. Further increase of this ratio did not result in significant modifications, apart from a slight decrease of the peak intensity. This stoichiometry suggests a reduction of MSeA by a three-fold amount of GSH yielding the selenylsulfide MeSeSG and GSSG, according to Equations (1) and (2). Significant pro- duction of MeSeH by Equation (3) would have consumed additional GSH molecules and would have resulted in a stoichiometry higher than 3. Analysis of the reaction products between MSeA and a 10-fold excess of GSH by electrospray mass spectrometry detected compounds at m/z = 307, 613 and 402, corresponding to GSH, GSSG and MeSeSG, respec- tively. Formation of MeSeSG is in accordance with a study by Kice and Lee [31] showing that benzeneseleninic acid reacts with excess thiols with a stoichiometry of three thiols per seleninic acid to give the selenenylsulfide. However, we cannot exclude at this stage that some MeSeH was formed and reacted with MeSeSG producing GSH and DMDSe (Equation (4)), which is not detectable by electrospray mass spectrometry.

MeSeH + MeSeSG ↔ DMDSe + GSH (4)

If such was the case, a stoichiometry of three thiols per seleninic acid would still be observed:

2 MSeA + 6 GSH ↔ DMDSe + 3 GSSG + 4 H2O

To test this possibility, we prepared MeSeSG and studied the exchange reactions (3) and (4) using spectrophotometry. Figure1B shows the absorption spectra of the different selenium compounds involved in these reactions. Methylselenol, obtained upon reduction of MSeA by TCEP, is characterized by a peak at 252 nm, typical of aliphatic selenolates. MeSeSG and DMDSe, the oxidized form of MeSeH, both display weak absorption above 240 nm with broad peaks centered around 275 and 310 nm, respectively. We first studied the reaction of MeSeH with MeSeSG. Figure1C shows the spectrum of MeSeH, produced by reduction of DMDSe with a stoichiometric amount of TCEP. Upon addition of an equimolar concentration of MeSeSG, the signal at 252 nm disappeared imme- diately (within the time necessary to record the spectrum) and was replaced by a spectrum characteristic of DMDSe, suggesting that MeSeH reacts rapidly with the selenylsulfide to form the diselenide and that the equilibrium of reaction (4) is in favor of the latter. Next, we studied the reaction of DMDSe with GSH. Because the equilibrium of reaction (4) is shifted towards the right, a large excess of GSH had to be used to obtain a detectable signal change at 252 nm. 100 or 200 µM DMDSe was mixed with 1 to 5 mM GSH and the absorbance at 252 nm was recorded as a function of time. Figure2 shows the kinetic traces for the reduction of 100 µM DMDSe by 1 mM GSH. The first experimental point was recorded 7 to 8 s after mixing. The change in absorbance during this dead time suggested that DMDSe rapidly reacted with GSH to form MeSeH. This burst was followed by a slow decrease of the absorbance at 252 nm, which we interpreted as the reaction of MeSeH with the GSSG contaminating the GSH solution. To confirm this hypothesis, we reproduced the experiment after the addition of 50 µM GSSG to the reaction mixture. In agreement with our interpretation, the initial bursts in absorbance were independent of the presence of additional GSSG whereas the initial velocity and the amplitude of the slow phase of the reaction were increased when GSSG concentration was increased (Figure2). The experimental curves were fitted according to a kinetic model Int. J. Mol. Sci. 2021, 22, 2241 4 of 20

describing Equations (5) and (6) (see Supplementary Materials). Because both forward and backward reactions of Equation (6) are rapid as shown above, we assumed that equilibrium of this equation was achieved at all times. In this model, changes in absorbance, therefore, Int. J. Int.Mol.Int.Int. J. J. J. Sci.Mol. Mol.Mol. 2021 Sci. Sci.Sci., 2021 22 20212021, 2241,, , ,22 2222,, , ,2241 2241 22412241 4 of 21444 of ofof 21 2121 Int. J. Mol. Sci. 2021, 22, 2241 depend on k1 and k−1, the rate constants for Equation (5), and K2, the equilibrium constant4 of 21

of Equation (6).

Figure 1. A FigureFigureFigure 1. UVUV 1.1. absorption absorptionUVUV absorptionabsorption spectra spectra spectraspectra of of various various ofof variousvarious Se Se-containing-containing SeSe--containing-containingcontaining species species speciesspeciesspecies and and effects andandand effects effectseffectseffects of ofreducing ofreducingofof reducingreducingreducing agents. agents. agents.agents.agents. (A) ( Reduction ((A()A) Reduction) ) ReductionReduction of m of e-ofof mme-e-e- Figure 1. UV absorption spectra of various Se-containing species and effects of reducing agents. (A) Reduction of me- methylseleninicthylseleninicthylseleninicthylseleninicthylseleninic acid acid (acid acidMSeA (MSeA) ( (MSeAMSeA) by glutathione by)) )by by glutathione glutathione glutathione (GSH (GSH). ( ()GSHGSH. UV) )). UVabsorption. .UV UVUV absorption absorption absorptionabsorption spectraspectra spectra spectraspectra in 100 inin inin mM 100 100100 potassium mM mMmM potassium potassiumpotassium phosphate, phosphate, phosphate,phosphate, pH 7.4 pH pH pH, of 7.4 7.47.4 7.4,MSeA,, ,of of of MSeA MSeA thylseleninic acid (MSeA) by glutathione (GSH). UV absorption spectra in 100 mM potassium phosphate, pH 7.4, of MSeA MSeA(250 (µM(250(250 (250) µMmixedµMµM))) ) mixedmixed mixed with withwithincreasing increasingincreasing increasing amounts amounts amountsamounts of GSH of ofof GSHGSHGSHas indicated asasas indicated indicatedindicatedindicated in the in ininin figure. the thethethefigure. figure.figure.figure. (B) (UV B ((B()B) UV) )absorption UVUV absorption absorptionabsorption spectra spectra spectraspectra of various of ofof various variousvarious Se- SeSe--- (250 µM) mixed with increasing amounts of GSH as indicated in the figure. (B) UV absorption spectra of various Se- Se-containingcontainingcontainingcontainingcontaining species. species. species. species.species. The The sampleThe TheThe sample sample samplesample cuvette cuvette cuvette cuvettecuvette contained contained contained containedcontained 100 µM 100 100 100100 µMSeA µM µMMµMMSeA MSeA MSeAMSeA in the inin inin absence thethe thethe absenceabsence absenceabsence ( ( )(( or presence))) )or oror presence presencepresence ( ( ( )( of 500)) )of of ofµM 500 500 500 TCEP, µM µMµM TCEP, TCEP, containing species. The sample cuvette contained 100 µM MSeA in the absence ( ) or presence ( ) of 500 µM TCEP, 100 µM100100 MeSeSG µMµMµ MeSeSGMeSeSG ( ()((, or 100)),), , or or µM 100100 DMDSe µMµµM DMDSeDMDSe ( ((( ) in 100)) ) in inin mM 100 100 potassiummMmM potassiumpotassium phosphate, phosphate,phosphate, pH 7.4 pHpH; spectra 7.47.4;; ; spectra spectraspectra in the in inin red the thethe box red redred are box boxbox are areare 100TCEP, µM 100 MeSeSGM MeSeSG ( ), ( or 100), orµM 100 DMDSeM DMDSe ( ( ) in 100) in mM 100 potassium mM potassium phosphate, phosphate, pH 7.4 pH; spectra 7.4; spectra in the in red the box red boxare extendedextextextendedendedended in the ininin inset thethethe .inset inset inset(C) .. Reaction. ((C(C)) ) ReactionReaction of MeSeSG ofof MeSeSGMeSeSG with withwithmethylselenol methylselenolmethylselenol (MeSeH ((MeSeH(MeSeH). MeSeH)).). . MeSeHMeSeHMeSeH was was producedwaswas produced produced produced by mixing bybyby mixingmixingmixing 25 µM 252525 µMµMµM extareended extended in the in the inset inset.. (C) (CReaction) Reaction of ofMeSeSG MeSeSG with with methylselenol methylselenol (MeSeH (MeSeH).). MeSeH MeSeH was was produced produced by by mixing mixing 25 25 µMµM DMDSeDMDSeDMDSe with with 25with µM 2525 TCEP µMµM TCEPTCEP in 100 inin mM 100100 potassiummMmM potassiumpotassium phosphate, phosphate,phosphate, pH 7.4. pHpH UV 7.4.7.4. absorption UVUV absorptionabsorption spectra spectraspectra were werewererecorded recordedrecorded before beforebefore ( ()(( )) ) DMDSeDMDSe with with 25 25 µMµM TCEP in 100 mM potassium phosphate, pH 7.4. UV absorption spectra were recorded before ()) and afterandand after theafter addition thethe additionaddition of 50 ofofµM 5050 MeSeSG µMµM MeSeSGMeSeSG (•••• ((•(•)••• •••to this)) ) to toto sample. this thisthis sample. sample.sample. andand after after the the addition addition of of 50 50 µMµM MeSeSG ( ••••)) toto thisthis sample.sample. Next,Next,Next, we studied wewe studiedstudied the reaction thethe reactionreaction of DMDSe ofof DMDSeDMDSe with with withGSH. GSH.GSH. Because BecauseBecause the equilibrium thethe equilibriumequilibrium of reac- ofof reac-reac- Next, we studied the reactionk of− DMDSe with GSH. Because the equilibrium of reac- tion tion(tiontion4) is ( ( (4shifted4))) is isis shifted shiftedshifted towards towards towardstowards the right, the thethe right, right,right,1 a large a aa large large largeexcess excess excessexcess of GSH of ofof GSH GSHGSHhad had tohadhad be to to toused be bebe used usedusedto obtain to toto obtain obtainobtain a de- a aa de- de-de- tion (4) is shiftedMeSeSG towards+ theGSH right, a largeMeSeH excess+ GSSGof GSH( K1had= tok be/ used k− ) to obtain a de-(5) tectabletectabletectabletectable signal signal signalsignal change change changechange at 252 at atat nm. 252 252252 nm. 100nm.nm. or 100 100100 200 or oror µM 200 200200 DMDSe µM µMµM DMDSe DMDSeDMDSe was was mixedwaswas1 mixed mixedmixed with1 with withwith1 to 51 11 mMto toto 5 55 mM GSHmMmM GSH GSHGSH tectable signal change at 252 nm. 100k or 200 µM DMDSe was mixed with 1 to 5 mM GSH and andtheand absorbance the the absorbance absorbance at 252 at at nm252 252 wasnm nm was recorded1was recorded recorded as a asfunction as a a function function of time. of of time. time. and theand absorbance the absorbance at 252 at nm 252 was nm recorded was recorded as a function as a function of time. of time. FigureFigureFigure 2 shows 2 2 shows shows the kinetic the the kinetic kinetic tracesk traces traces for the for for reduction the the reduction reduction of 100 of of µM100 100 DMDSeµM µM DMDSe DMDSe by 1 by mMby 1 1 mM GSH.mM GSH. GSH. FigureFigure 2 shows 2 shows the kinetic the kinetic traces −traces for2 the for reduction the reduction of 100 of µM 100 DMDSe µM DMDSe by 1 mM by 1 GSH. mM GSH. The ThefirstThe first experimentalfirst experimental experimentalMeSeH +pointMeSeSG point point was was recordedwas recorded recordedDMDSe 7 to 87 7 sto +to after 8 8GSH s s after aftermixing.(K2 mixing. mixing.= Thek / changeThe The k− change change) in absorbance in in absorbance absorbance(6) The firstThe experimental first experimental point pointwas recorded was recorded 7 to 8 7s toafter 8 s mixing.after mixing. The2 change The 2change in absorbance in absorbance duringduringduring this deadthis this dead deadtime time timesuggested suggested suggested thatk that DMDSethat DMDSe DMDSe rapidly rapidly rapidly reacted reacted reacted with with withGSH GSH GSHto form to to form formMeSeH. MeSeH. MeSeH. duringduring this dead this timedead suggestedtime suggested that 2DMDSe that DMDSe rapidly rapidly reacted reacted with GSHwith toGSH form to MeSeH.form MeSeH. This ThisburstThis burst burst was was followedwas followed followed by a byslowby a a slow slowdecrease decrease decrease of the of of absorbance the the absorbance absorbance at 252 at at nm,252 252 nm, whichnm, which which we inter- we we inter- inter- This burstTheThis best-fit burstwas followed was parameter followed by valuesa slow by afor decreaseslow the decrease reaction of the ofof absorbance 100the orabsorbance 200 µatM 252 DMDSe at nm, 252 which nm, in the which we presence inter- we inter- pretedpretedpreted as the as as reaction the the reaction reaction of MeSeH of of MeSeH MeSeH with with withthe GSSG the the GSSG GSSG contaminating contaminating contaminating the GSH the the GSH GSHsolution. solution. solution. To con- To To con- con- pretedof varyingpreted as the concentrationsas reaction the reaction of MeSeH of MeSeH GSH with and thewith GSSGGSSG the GSSG contaminating are given contaminating in Table the GSH1. the Mean solution.GSH values solution. To andcon- To con- firm firmfirmthisfirm hypothesis,this thisthis hypothesis, hypothesis,hypothesis, we reproduced we wewe reproduced reproducedreproduced the experiment the thethe experiment experimentexperiment after after afterafterthe addition the thethe addition additionaddition of 50 of of ofµM 50 5050 GSSG µM µMµM−1 GSSG GSSGGSSG− to1 to toto firmstatistical this hypothesis, errors calculated we reproduced from these the data experiment are K2 =1067 after ±the192, addition k−1 = of 325 50± µM84 MGSSGs to, the reactionthethethe reaction reactionreaction mixture. mixture. mixture.mixture. In− ag1 In InreementIn− 1 ag agagreementreementreement with with withwithour interpretation, our ourour interpretation, interpretation,interpretation, the initial the thethe initial initialinitial bursts bursts burstsbursts in absorbance in inin absorbance absorbanceabsorbance theand reaction k1 = 0.36 mixture.± 0.14 MIn agreements . with our interpretation, the initial bursts in absorbance werewere wereindependent independent independent of the of of presence the the presence presence of additional of of additional additional GSSG GSSG GSSG whereas whereas whereas the initial the the initial initial velocity velocity velocity and andthe and the the were independentwere independent of the ofpresence the presence of additional of additional GSSG GSSG whereas whereas the initial the initial velocity velocity and the and the amplitudeamplitudeamplitude of the of of slow the the slow slowphase phase phase of the of of reaction the the reaction reaction were were wereincreased increased increased when when when GSSG GSSG GSSG concentration concentration concentration was was was amplitudeamplitude of the ofslow the phaseslow phase of the ofreaction the reaction were increasedwere increased when whenGSSG GSSG concentration concentration was was increasedincreasedincreased (Fig ure (Fig(Fig 2ureure). The 22)). . TheexperimentalThe experimentalexperimental curves curvescurves were were werefitted fittedfitted according accordingaccording to a tokineticto aa kinetickinetic model modelmodel increasedincreased (Figure (Fig 2)ure. The 2) .experimental The experimental curves curves were werefitted fittedaccording according to a kinetic to a kinetic model model describingdescribingdescribing Equations Equations Equations (5) and ( (55)) (and 6and) (see ( (66)) Supplementary( (seesee Supplementary Supplementary Materials Materials Materials). Because)). .Because Because both both forwardboth forward forward and and and describingdescribing Equations Equations (5) and (5 ()6 and) (see (6 Supplementary) (see Supplementary Materials Materials). Because). Because both forward both forward and and backwardbackwardbackward reactions reactionsreactions of Equation ofof EquationEquation (6) are ((66) ) rapid areare rapidrapid as shown as as shown shown above, above,above, we assumed wewe assumed assumed that thatequilib-that equilib-equilib- backwardbackward reactions reactions of Equation of Equation (6) are (6 rapid) are rapidas shown as shown above, above, we assumed we assumed that equilib- that equilib- riumrium riumof th ofisof thequationthisis equationequation was wasachievedwas achievedachieved at all atat times. allall times.times. In this InIn thismodel,this model,model, changes changeschanges in absorbance, inin absorbance,absorbance, rium riumof th isof equation this equation was achieved was achieved at all attimes. all times. In this In model, this model, changes changes in absorbance, in absorbance,

Int. J. Mol. Sci. 2021, 22, 2241 5 of 21

therefore, depend on k1 and k-1, the rate constants for Equation (5), and K2, the equilibrium constant of Equation (6).

k−1 MeSeSG + GSH ⇆ MeSeH + GSSG (K1= k1/ k-1) (5) k1

k Int. J. Mol. Sci. 2021, 22, 2241 −2 5 of 20 MeSeH + MeSeSG ⇆ DMDSe + GSH (K2= k2/ k-2) (6) k2

FigureFigure 2.2. Reduction of DMDSe by by GSH. GSH. 100 100 µMµM DMDSe DMDSe was was mixed mixed with with 1 1mM mM GSH GSH (containing (containing 15 15µMµ MGSSG) GSSG) in inthe the absence absence (black (black trace) trace) or or presence presence (blue (blue trace) trace) of of 50 50 µMµM added GSSG inin 100100 mMmM potassiumpotassium phosphate,phosphate, pHpH 7.4.7.4. TheThe absorbanceabsorbance atat 252252 nmnm waswas recordedrecordedas asa afunction functionof oftime timestarting starting 77 to 8 s s after after mixing. mixing. The The absorbance absorbance of a of solution a solution of 100 of µM 100 DMDSeµM DMDSe alone, alone, measured measured with a withcontrol a controlsample, sample, was 0.016. was The 0.016. experimental The experimental data weredata fitted were to the fitted kinetic to themodel kinetic desc modelribed in described Supplemen- in Supplementarytary Materials (red Materials lines). (red lines).

TableThe 1. Equilibrium best-fit parameter and rate constants values for deduced the reaction from spectrophotometric of 100 or 200 µM experiments DMDSe in withthe presence mixtures ofof DMDSe,varying GSH,concentrations and GSSG. of GSH and GSSG are given in Table 1. Mean values and statis- tical errors calculated from these data are K2 =1067 ± 192, k-1 = 325 ± 84 M−1 s−1, and k1 = 0.36 ± 0.14 M−1Initial s−1. Concentrations (µM) k−1 k1 K2 −1 −1 −1 −1 DMDSe GSH GSSG (M .s ) (M .s ) Table 1. Equilibrium and rate constants deduced from spectrophotometric experiments with mix- 100 1000 15 * 1288 ± 7 431 ± 8 0.45 ± 0.02 tures of100 DMDSe, GSH 1000, and GSSG. 15 * + 50 1184 ± 10 311 ± 4 0.46 ± 0.02 100 2500 37.5 * 812 ± 4 232 ± 3 0.54 ± 0.01 Initial Concentrations (µM) k-1 k1 100 5000 75 *K2 1222 ± 5 226 ± 2 0.15 ± 0.01 −1 −1 −1 −1 DMDSe200 GSH 2500 GSSG 37.5 * 902 ± 5(M 388.s ±) 4 0.30(M ±.s0.01) 100200 1000 5000 15 * 75 *1288 994 ± 7± 10431 362 ± 8± 50.45 0.27 ± 0.00.012 * GSSG100 present as a contaminant1000 in the15 GSH* + 50 solution. 1184 ± 10 311 ± 4 0.46 ± 0.02 100 2500 37.5 * 812 ± 4 232 ± 3 0.54 ± 0.01 To determine more precisely the value of the rate constants, stopped-flow spectropho- 100 5000 75 * 1222 ± 5 226 ± 2 0.15 ± 0.01 tometry was employed. To obtain k−1, MeSeH was produced by reduction of DMDSe with a substoichiometric200 2500 amount of37.5 TCEP * and was902 immediately ± 5 mixed388 with± 4 various0.30 amounts ± 0.01 of GSSG in the stopped-flow instrument. To obtain k−2, DMDSe was mixed with GSH. The changes in absorbance at 252 nm were recorded. The kinetic traces from four (for k−2) or six different mixtures (for k−1) were fitted to the theoretical model. The resulting constants are −1 −1 given in Table2. The mean value determined for k −1 (371 ± 45 M .s ) was in excellent agreement with the value calculated above. A value of (3.7 ± 0.5) 104 M−1.s−1 was calcu- lated for k−2. We could not obtain an accurate value for k2 because the reaction between MeSeH and MeSeSG was too fast to be precisely quantified. However, an estimated value 7 −1 −1 of k2 close to 4 10 M .s could be calculated from the values of K2 and k−2. Int. J. Mol. Sci. 2021, 22, 2241 6 of 20

Table 2. Rate constants deduced from stopped-flow experiments performed with mixtures of MeSeH and GSSG (left) or GSH and DMDSe (right). Means and standard deviations were calculated from at least three replicate measurements.

MeSeH GSSG Rate Constant GSH DMDSe Rate Constant −1 −1 −1 −1 (µM) (µM) k−1 (M .s ) (µM) (µM) k−2 (M .s ) 25 100 418 ± 32 1250 50 2.9 ± 0.7 104 50 200 434 ± 43 1667 67 4.3 ± 1.3 104 50 300 365 ± 70 2500 100 3.6 ± 0.9 104 50 400 351 ± 140 1250 125 3.9 ± 1.3 104 100 400 356 ± 35 100 600 322± 54

The selenol/diselenide exchange reactions (5) and (6) were also studied starting from MeSeSG and GSH. 100 µM MeSeSG was mixed with 10 mM GSH and UV-spectra (200–400 nm) were recorded as a function of time (Figure3). The increase in absorbance at 252 nm indicates that methylselenol was slowly formed and that ~2.2 µM MeSeH was produced when equilibrium was reached. Changes in absorbance observed at 290 nm and 340 nm (Figure3, inset) indicate that DMDSe was also formed. The kinetic traces at 252 and 340 nm were simultaneously fitted to the kinetic equation system corresponding Int. J. Mol. Sci. 2021, 22, 2241 to Equations (5) and (6). The constants calculated from three independent experiments7 of 21 −1 −1 − 1 −1 (K2 = 692 ± 140, k−1 = 339 ± 195 M .s and k1 = 0.27 ± 0.05 M .s ) are in good agreement with those determined above.

FigureFigure 3.3.Reduction Reduction ofof MeSeSGMeSeSG byby GSH.GSH. 100100µ µMM MeSeSG MeSeSG was was mixed mixed with with 10 10 mM mM GSH GSH (containing (containing 150150µ µMM GSSG) GSSG) in in 100 100 mM mM potassium potassium phosphate, phosphate, pH pH 7.4, 7.4, and and UV UV absorption absorption spectra spectra were were recorded recorded after 20 s (blue line); 60 s (orange); 100 s (grey); 140 s (yellow); 180 s (light blue); 220 s (green); 260 s after 20 s (blue line); 60 s (orange); 100 s (grey); 140 s (yellow); 180 s (light blue); 220 s (green); 260 s (dark blue); 300 s (brown). The spectrum of a solution of 100 µM MeSeSG alone is displayed in black. (dark blue); 300 s (brown). The spectrum of a solution of 100 µM MeSeSG alone is displayed in black. Inset: The spectrum of MeSeSG (black solid line) was subtracted from that of the products of the Inset:reaction The after spectrum 5 min ( ofdotted MeSeSG line) (black to obtain solid the line) difference was subtracted spectrum from (red thatline). of the products of the reaction after 5 min (dotted line) to obtain the difference spectrum (red line). The equilibrium constants for thiol/diselenide and selenol/disulfide exchange reac- The equilibrium constants for thiol/diselenide and selenol/disulfide exchange re- tions determined in this study (K1 ~7.3 10–4; K2 ~ 1000) are comparable to those determined actions determined in this study (K1 ~7.3 10−4; K2 ~ 1000) are comparable to those de- for cysteine/selenocysteine in [32]. In addition, our results confirm that the reaction of se- lenols with selenylsulfides are several orders of magnitude faster than with disulfides [32]. To further validate our model, we performed a redox titration of DMDSe with DTT. DMDSe (approx. 50 µM) was mixed with increasing concentrations of DTT (0 to 2500 µM) and UV-spectra (200–400 nm) were recorded (Figure 4). The absorbance of the samples at 252 nm was plotted as a function of the DTT concentration added to the sample (Figure 4, inset). A value for the apparent equilibrium constant Keq ([DTTox][MeSeH]2)/([DTT][DMDSe]) of 1.15 ± 0.12 10−4 M was estimated by fitting experi- mental values from two independent experiments to the equation described in Supple- mentary Materials. From the known value of the equilibrium constant of the reaction be- tween GSH and DTT ([GSH]2·DTTox]/([GSSG]·[DTT]) = 200 M [33,34]), we calculated that the equilibrium constant between DMDSe and GSH (KDMDSe/GSH = K1/K2 = [GSSG]·[MeSeH]2/([DMDSe]·[GSH]2) was equal to 5.8 ± 0.6 10−7. This value agrees well with the value of K1/K2 = 7.3 10−7 calculated from the above kinetic experiments. From the value of KDMDSe/GSH and the redox potential of the GSSG/GSH couple E°’(GSSG/GSH) = −264 mV at pH 7.4 [35], a value of −446 ± 2 mV can be calculated for the redox potential of the DMDSe/MeSeH couple.

Int. J. Mol. Sci. 2021, 22, 2241 7 of 20

termined for cysteine/selenocysteine in [32]. In addition, our results confirm that the reaction of selenols with selenylsulfides are several orders of magnitude faster than with disulfides [32]. To further validate our model, we performed a redox titration of DMDSe with DTT. DMDSe (approx. 50 µM) was mixed with increasing concentrations of DTT (0 to 2500 µM) and UV-spectra (200–400 nm) were recorded (Figure4). The absorbance of the samples at 252 nm was plotted as a function of the DTT concentration added to the sample (Figure4, in- 2 set). A value for the apparent equilibrium constant Keq ([DTTox][MeSeH] )/([DTT][DMDSe]) of 1.15 ± 0.12 10−4 M was estimated by fitting experimental values from two independent experiments to the equation described in Supplementary Materials. From the known 2 value of the equilibrium constant of the reaction between GSH and DTT ([GSH] ·DTTox]/ ([GSSG]·[DTT]) = 200 M [33,34]), we calculated that the equilibrium constant between 2 2 DMDSe and GSH (KDMDSe/GSH = K1/K2 = [GSSG]·[MeSeH] /([DMDSe]·[GSH] ) was equal to 5.8 ± 0.6 10−7. This value agrees well with the value of K1/K2 = 7.3 10−7 calcu- lated from the above kinetic experiments. From the value of KDMDSe/GSH and the redox potential of the GSSG/GSH couple E◦’(GSSG/GSH) = −264 mV at pH 7.4 [35], a value of −446 ± 2 mV can be calculated for the redox potential of the DMDSe/MeSeH couple.

Figure 4. Redox titration of DMDSe with DTT. 50 µM DMDSe was mixed with increasing concentrations of DTT (0 to 2400 µM) in 100 mM potassium phosphate, pH 7.4 and UV absorption spectra were recorded. Inset: The absorbance at 252 nm was plotted against DTT concentration and fitted to the theoretical equation.

This value is lower than the redox potentials reported for other unconstrained dise- lenide bonds such as that of selenoglutathione (−407 mV [36]) or selenocystine (−386 mV at pH 7.0 [37]) and indicates that the reduction of DMDSe requires strong reducing con- ditions. However, glutathione is abundant in the eukaryotic cytosol with an estimated concentration of 10 mM in S. cerevisiae [38]. Moreover, cytosolic GSSG concentration is tightly regulated and the GSH/GSSG ratio was estimated to be as high as 50,000:1 in the yeast cytosol [39]. From the equilibrium constants calculated above, we can predict the equilibrium distribution of MeSeH, MeSeSG, and DMDSe concentrations (Figure5). For GSH/GSSG ratios > 5000, MeSeH is the major Se-species at total Se concentrations of Int. J. Mol. Sci. 2021, 22, 2241 8 of 20

1–10 µM. Therefore, it can be expected that, in eukaryotic cells, GSH is able to efficiently reduce both DMDSe and MeSeSG into MeSeH. In contrast, in the in vitro experiments shown in Figure1, reduction of MSeA by GSH generated GSSG. Therefore, the GSH/GSSG ratio was low and MeSeH is not expected to be significantly produced in these conditions. Int. J. Mol. Sci. 2021, 22, 2241 For example, according to our equilibrium constants values, reaction of 250 µM MSeA9 of with 21 a 10-fold excess of GSH is expected to produce 150 µM MeSeSG, 50 µM DMDSe, and only 0.58 µM MeSeH.

Figure 5. Equilibrium distribution of MeSeH, MeSeSG, and DMDSe as a function of the GSH/GSSG ratio for a GSH concentrationFigure of5. 10Equilibrium mM and a total distribution Se concentration of MeSeH, of 5 µM ([totalMeSeSG Se] =, and [MeSeH] DMDSe + [MeSeSG] as a function + 2 [DMDSe]). of the The GSH/GSSG curves were obtainedratio for using a GSH values concentration of 1350 for 1/K1 of and10 mM 1000 forand K2. a total Se concentration of 5 µM ([total Se] = [MeSeH] + [MeSeSG] + 2 [DMDSe]). The curves were obtained using values of 1350 for 1/K1 and 1000 for K2. 2.2. Enzymatic Reduction by Glutathione Reductase and Thioredoxin Reductase 2.2. Enzymatic ReductionTo determine by Glutathione how MeSeH Reductase could be, and alternatively, Thioredoxin produced Reductasein vivo , we tested whether MeSeSG is a substrate for the NADPH-dependent glutathione reductase (GR). Although To determineGR is how known MeSeH to be very could specific be, for alternatively,GSSG, Ganther [ 40 produced] has shown in that vivo, selenodiglutathione we tested whether MeSeSG(GSSeSG) is a substrate was a good for substrate the NADPH of GR from-dependent yeast. The reductionglutathione reaction reductase was followed (GR) by. Although GR is measuringknown to NADPH be very oxidation specific at 340for nm.GSSG, In Figure Ganther6A, the [40] reduction has shown rate of MeSeSG that sele- was nodiglutathionecompared (GSSeSG to) was that of a GSSG.good Insubstrate our experimental of GR from conditions yeast. (100 The mM reduction potassium reaction phosphate, pH 7.4, 22 ◦C), 1.5 nM of S. cerevisiae GR reduced 35 µM of GSSG in less than 15 min. The was followed bychange measuring in the absorbance NADPH atoxidation 340 nm was at weak 340 nm. when In MeSeSG Figure (50 6A,µM) the was reduction used as substrate rate of MeSeSG was incompared the same conditions, to that of but GSSG. became In significant our experimental when the concentration conditions of (100 GR inmM the assaypo- tassium phosphatewas, increasedpH 7.4, 22 to 73°C nM.), 1.5 Initial nM velocities of S. cerevisiae of NADPH GR oxidation, reduced deduced35 µM fromof GSSG the slope in less than 15 min.of The the absorbance change in variation the absorbance at 340 nm duringat 340 thenm first was min weak of the when reaction, MeSeSG were 7.5 ( and50 0.08 µmol NADPH oxidized min−1 nmol−1 for GSSG and MeSeSG, respectively. µM) was used as substrate in the same conditions, but became significant when the con- These results show that MeSeSG acts as a substrate for GR, albeit at a rate reduced centration of GRnearly in the 100-fold assay compared was increased to GSSG. to Upon 73 nM. addition Initial of enzyme velocities to MeSeSG, of NADPH there was oxida- a rapid tion, deduced fromphase the of slope NADPH of oxidationthe absorbance followed variation by a slower at continuous 340 nm during oxidation the phase first (Figure min 6ofB), the reaction, weresuggesting 7.5 and that 0.08 a product µmol of theNADPH reaction oxidized may redox-cycle min−1 withnmol oxygen.−1 for To GSSG confirm and this MeSeSG, respectivelhypothesis,y. experiments were performed in anaerobic conditions. The initial phases of the reaction were identical in aerobic and anaerobic conditions, whereas the slow phase of NADPH oxidation was oxygen-dependent (Figure6B). As shown in Figure6C, 0.52 mol of NADPH was oxidized per mol of MeSeSG at the end of the experiment in anaerobic conditions. This stoichiometry can be explained by the reaction between the MeSeH

Int. J. Mol. Sci. 2021, 22, 2241 9 of 20

produced by the GR-catalyzed reaction (7) with MeSeSG to produce DMDSe (reaction 4). Because the equilibrium constant of Equation (4) is high, the reverse equation of this Int. J. Mol. Sci. 2021, 22, 2241 10 of 21 equation is negligible until almost all the initial MeSeSG is consumed. In these conditions, Reactions (7) and (4) result in the overall reaction (8). Int. J. Mol. Sci. 2021, 22, 2241 10 of 21 Int. J. Mol. Sci. 2021, 22, 2241 10 of 21

Figure 6. Reduction of MeSeSG or DMDSe by S. cerevisiae glutathione reductase. (A) NADPH-de- Figure 6. Reduction ofFigure MeSeSGFigure 6. Reduction or DMDSe 6. Reduction of by MeSeSG S. cerevisiae of MeSeSG or DMDSe glutathione pendentor by DMDSeS. reductase. cerevisiae reduction by S.glutathione ( Aofcerevisiae) MeSeSGNADPH glutathione reductase. (-de-) or GSSG (A reductase. )( NADPH-dependent). The reaction (A) NADPH mixtures reduction-de- contained of 100 µM NADPH, pendent reduction of MeSeSG () or GSSG (). The reaction mixtures contained 100 µMµ NADPH, µ MeSeSGpendent () or GSSGreduction ( ). Theof MeSeSG reaction mixtures(and) or 50 GSSG µM contained of ( the) .substrate 100The reactionM NADPH,under mixtures study and in 50 100 containedM mM of thepotassium substrate100 µM phosphate, NADPH, under study pH 7.4. in The reactions were and 50 µM of the substrate under study in 100 mM potassium phosphate, pH 7.4. The reactions were 100 mMand potassium 50 µM of phosphate, the substrate pH 7.4.under Thestarted study reactions byin adding100 were mM started1.5 potassium nM byGR adding to thephosphate, GSSG 1.5 nM mixture GRpHto 7.4. or the The73 GSSG nM reactions GR mixture to the were orMeSeSG 73 nM mixture. (B) Aerobic started by adding 1.5 nMGR GR tothe to the MeSeSG GSSG mixture.mixture or (B 73) AerobicnM GR to (( the)) andand MeSeSG anaerobic mixture. ( , + +)()B consumption consumption) Aerobic of of NADPH NADPH in in the the presence presence of ofGR GR and and 100 µM DMDSe (+) or () and anaerobic (, +) consumptionstarted by of adding NADPH 1.5 in nM the GRpresence to the of GSSG GR and mixture 100 µM or DMDSe 73 nM (GR+) or to the MeSeSG mixture. (B) Aerobic 100 µM DMDSe (+) or 100 µM MeSeSG ( 100, ). µM The MeSe reactionSG ( mixtures, ). The contained reaction 200mixturesµM NADPH, contained 100 200 nM µM GR NADPH, in 100 mM 100 nM GR in 100 mM 100 µM MeSeSG (, ). The( reaction) and anaerobicmixtures contained (, +) consumption 200 µM NADPH, of NADPH 100 nM inGR the in 100presence mM of GR and 100 µM DMDSe (+) or potassium phosphate, pH 7.4, at 22 ◦C. Anaerobicpotassium reactions phosphate, were pH performed 7.4, at 22 in°C. a gloveAnaerobic box underreactions nitrogen were performed atmosphere. in a glove box under potassium phosphate, pH 7.4,100 at µM22 °C. MeSe AnaerobicSG ( ,reactions ). The werereaction performed mixtures in acontained glove box 200under µM NADPH, 100 nM GR in 100 mM (C) Stoichiometry of NADPH consumptionnitrogen as a function atmosphere. of MeSeSG (C) Stoichiometry concentration of NADPH in anaerobic consumption conditions. as a NADPHfunction of MeSeSG concen- nitrogen atmosphere. (C) Stoichiometrypotassium ofphosphate, NADPH consumption pH 7.4, attration 22 as °C.a function in Anaerobic anaerobic of MeSeSG conditions.reactions concen- wereNADPH performed consumption in a wasglove followed box under in anaerobic conditions with 0 consumption was followed in anaerobic conditions with 0 to 200 µM MeSeSG added to the reaction mixture as in B). The tration in anaerobic conditions.nitrogen NADPH atmosphere. consumption (C was) Stoichiometry followedto 200 in µM anaerobic of MeSeSG NADPH conditions added consumption to with the reaction 0 as a mixturefunction as of in MeSeSGB). The amount concen- of NADPH consumed after to 200 µM MeSeSG addedamount to the of NADPHreaction mixture consumed as in after B). 125The minamount was of calculated NADPHfrom consumed the ∆A after340 after subtraction of the value in the absence tration in anaerobic conditions. NADPH125 min was consumption calculated from was thefollowed ∆A340 after in anaerobic subtraction conditions of the value with in the 0 absence of substrate. 125 min was calculatedof from substrate.to the 200 ∆A µM340 after MeSeSG subtraction added of tothe the value reaction in the absencemixture of as substrate. in B). The amount of NADPH consumed after 125 min was calculated from the ∆A340These after resultssubtraction show of that the valueMeSeSG in theacts absence as a substrate of substrate. for GR, albeit at a rate reduced These results show that MeSeSG acts as a substrate fornearlyMeSeSG GR, 100 albeit+-foldNADPH at compareda rate+ reducedH +to →GSSG. MeSeH Upon+ additionGSH + ofNADP enzyme+ to MeSeSG,(7) there was a nearly 100-fold compared to GSSG. Upon addition of enzyme to MeSeSG, there was a These results show that rapidMeSeSG phase acts of NADPHas a substrate oxidation for followed GR, albeit by aat slower a rate continuous reduced oxidation phase (Fig- rapid phase of NADPH oxidation followed by a slower continuous oxidation phase (Fig-+ + nearly 100-fold compared to2ure MeSeSGGSSG. 6B), suggestingUpon+ NADPH addition that+ aH ofproduct enzyme→ DMDSe of the to reaction+MeSeSG,2 GSH may +there NADPredox was-cycle a with oxygen.(8) To con- ure 6B), suggesting that a product of the reaction may redoxfirm-cycle this hypothesis,with oxygen. experiments To con- were performed in anaerobic conditions. The initial rapid phase of NADPHThe oxidation sub-stoichiometric followed NADPH by a slower consumption continuous in the oxidation absence of phase dioxygen (Fig- implies that firm this hypothesis, experiments were performed in anaerobicphases of conditions. the reaction The were initial ide ntical in aerobic and anaerobic conditions, whereas the ure 6B), suggestingthe that DMDSe a product produced of the in this reaction reaction may is not redox reduced-cycle by with GR. Asoxygen. expected, To con- when 100 µM phases of the reaction were identical in aerobic and anaerobicslow phase conditions, of NADPH whereas oxidation the was oxygen-dependent (Figure 6B). As shown in Figure slow phase of NADPH oxidationfirm this was hypothesis, oxygenDMDSe-dependent experiments was (Figure mixed with were6B). 100As performed shown nM GR in in Figure anaerobic in anaerobic conditions, conditions. the consumption The initial of NADPH was lower than6C, 0.1 0.52µM/min, mol of NADPH confirming was that oxidized DMDSe per is mol not aof substrate MeSeSG ofat GRthe (Figureend of 6theB). experiment in 6C, 0.52 mol of NADPH wasphases oxidized of the per reaction mol of MeSeSG were anaerobicide at thentical end inconditions. of aerobicthe experiment Thisand stoicanaerobic in hiometry conditions, can be explained whereas by the the reaction between the anaerobic conditions. Thisslow stoic phasehiometry of NADPH can be Anexplained oxidation oxygen-dependent by was the oxygenreaction non-stoichiometric- dependentbetween the (Figure consumption 6B). As shown of NADPH in Figure was previously reported in theMeSeH reaction produced between by selenite the GR or-catalyzed selenodiglutathione reaction (7) with and theMeSeSG thioredoxin to produce sys- DMDSe (re- MeSeH produced by the GR6C,-catalyzed 0.52 mol reactionof NADPH (7) with was MeSeSG actionoxidized 4 )to. Because perproduce mol the DMDSeof equilibriumMeSeSG (re- at constant the end ofof Equation the experiment (4) is high, in the reverse equation of action 4). Because the equilibriumanaerobic constant conditions. of Equation This (4stoic)this is high,hiometry equation the reverse is can negligible be equation explained until of almost by theall thereaction initial MeSeSGbetween is theconsumed. In these condi- this equation is negligible MeSeHuntil almost produced all the initialby the MeSeSG GR-tions,catalyzed is consumed. Reactions reaction (In7) theseand (7) (with 4condi-) result MeSeSG in the overallto produce reaction DMDSe (8). (re- tions, Reactions (7) and (4) result in the overall reaction (8). action 4). Because the equilibrium constant of Equation (4) is high, the reverse equation of MeSeSG + NADPH + H+ → MeSeH + GSH + NADP+ (7) MeSeSGthis + equation NADPH +is H negligible+ → MeSeH u +ntil GSH almost + NADP all+ the initial MeSeSG(7) is consumed. In these condi- tions, Reactions (7) and (4) result in the overall2 reactionMeSeSG +(8 NADPH). + H+ → DMDSe + 2 GSH + NADP+ (8) 2 MeSeSG + NADPH + H+ → DMDSe + 2 GSH + NADP+ (8) MeSeSG + NADPH + H+ → MeSeH + GSH + NADP+ (7)

2 MeSeSG + NADPH + H+ → DMDSe + 2 GSH + NADP+ (8)

Int. J. Mol. Sci. 2021, 22, 2241 11 of 21

The sub-stoichiometric NADPH consumption in the absence of dioxygen implies that the DMDSe produced in this reaction is not reduced by GR. As expected, when 100 µM DMDSe was mixed with 100 nM GR in anaerobic conditions, the consumption of NADPH was lower than 0.1 µM/min, confirming that DMDSe is not a substrate of GR (Figure 6B). An oxygen-dependent non-stoichiometric consumption of NADPH was previously reported in the reaction between selenite or selenodiglutathione and the thioredoxin sys- tem [41] [42]. The authors proposed that the selenolates produced in the reaction continu- Int. J. Mol. Sci. 2021, 22, 2241 10 of 20 ously react with oxygen to catalyze the oxidation of TRX. In the present case, the non- stoichiometric consumption of NADPH may similarly be explained as follows: the reac- tion with oxygen of MeSeH formed in reaction (7) will produce DMDSe, which will react tem [41,42]. The authors proposed that the selenolates produced in the reaction con- withtinuously GSH react (reaction with oxygen 4), regenerating to catalyze the MeSeSG. oxidation MeSeSG of TRX. In will the again present be case, reduced the by GR con- sumingnon-stoichiometric NADPH consumptionand the reaction of NADPH will mayproceed similarly until be all explained the NADPH as follows: is exhausted. the reactionThe with main oxygen intracellular of MeSeH formed disulfide in reaction reductase (7) will responsible produce DMDSe, for maintaining which will proteins in theirreact withreduced GSH (reactionstate is 4),thioredoxin regenerating (TRX MeSeSG.), which MeSeSG is reduced will again by be reducedelectrons by from GR NADPH via consuming NADPH and the reaction will proceed until all the NADPH is exhausted. thioredoxin reductase (TRR) [43]. The TRX/TRR system acts as a general disulfide reduc- The main intracellular disulfide reductase responsible for maintaining proteins in tasetheir reduced with a state broad is thioredoxin substrate (TRX), specificity. which is Several reduced Se by- electronscontaining from compounds, NADPH including MSeA,via thioredoxin were shown reductase to (TRR)be directly [43]. The reduced TRX/TRR by system the human acts as selenoprotein a general disulfide TRR [44,45]. We studiedreductase the with reduction a broad substrate of MeSeH specificity. precursors Several Se-containing(MSeA, MeSeSG compounds,, and includingDMDSe) by a homolo- gousMSeA, system were shown composed to be directly of yeast reduced TRX by(2 theµM human) and TRR selenoprotein (0.5 µM TRR). We [44 first,45]. Wechecked NADPH studied the reduction of MeSeH precursors (MSeA, MeSeSG, and DMDSe) by a homologous consumptionsystem composed in the of yeast absence TRX of (2 µTRX.M) and Yeast TRR TRR (0.5 µwasM). unable We first to checked directly NADPH reduce MeSeH pre- cursors.consumption As shown in the absence in Figure of TRX. 7, TRX/TRR Yeast TRR-ca wastalyzed unable oxidation to directly reduceof NADPH MeSeH occurred in the presenceprecursors.Int. J. Mol. ofAs Sci. 50 shown2021 µM, 22 , inMSeA,2241 Figure MeSeSG7, TRX/TRR-catalyzed, or DMDSe. oxidation DMDSe of was NADPH the occurredpoorest insubstrate with a 10 of 21

reductionthe presence rate, of 50 µinM our MSeA, assay MeSeSG, conditions, or DMDSe. of DMDSe 4.6 µM/min, was the poorestcompared substrate to 12 with µM/min and 26 a reduction rate, in our assay conditions, of 4.6 µM/min, compared to 12 µM/min and µM/min for MeSeSG and MSeA, respectively. Similar rates were observed when the reac- 26 µM/minInt. J. Mol. forSci. MeSeSG2021, 22, 2241 and MSeA, respectively. Similar rates were observed when the 10 of 21

tionsreactions were were performed performed in in anaerobic anaerobic conditions conditions (4.8, 15, ( and4.8, 23 15µ,M/min and 23 for µM/min DMDSe, for DMDSe, MeSeSGMeSeSG, and, and MSeA, MSeA, respectively). respectively).

Figure 6. Reduction of MeSeSG or DMDSe by S. cerevisiae glutathione reductase. (A) NADPH-de- pendent reduction of MeSeSG () or GSSG (). The reaction mixtures contained 100 µM NADPH,

FigureFigure 7. 7.Reduction Reduction of MeSeH of MeSeH precursors precursorsand 50 by µM the TRX/TRRof by the the substrate TRX/TRR system. under The system. study reaction in 100 mixturesThe mM reaction potassium contained mixtures phosphate, contained pH 7.4. The reactions were µ µ µ S. cerevisiae 100100 µMM NADPH, NADPH, 2 M2 µM TRX TRX and 0.5 andstartedMFigure 0.5 TRR µM by from6. ReductionaddingTRR from 1.5 ofnM S. MeSeSGin GRcerevisiae 100 to mMthe or GSSG potassiumDMDSe in 100 mixture bymM phosphate, S. orpotassiumcerevisiae 73 nM pH GRglutathione phosphate,to the MeSeSG reductase. pH mixture. (A) NADPH (B) Aerobic-de- 7.4, 22 ◦C. The reactions were started(pendent by) and adding anaerobic reduction buffer ( of),, MeSeSG+ or) consumption 50 µM ( of) or DMDSe GSSG of NADPH ( ),). The MeSeSG( in thereaction presence), mixtures of GR contained and 100 µM 100 DMDSe µM NADPH, (+) or 7.4, 22 °C. The reactions were started by adding buffer (), or 50 µM of DMDSe (), MeSeSG(), or MSeA(∆). 100and µM 50 µM MeSe of SGthe (substrate, ). The under reaction study mixtures in 100 mM contained potassium 200 phosphate,µM NADPH, pH 100 7.4. nM The GR reactions in 100 weremM or MSeA(∆). potassiumstarted by addingphosphate, 1.5 nM pH GR 7.4, to at the 22 GSSG°C. Anaerobic mixture orreactions 73 nM GR were to theperformed MeSeSG in mixture. a glove (boxB) Aerobic under 2.3. Toxicity of MeSeH Precursors nitrogen() and anaerobicatmosphere. ( ,( C+)) consumptionStoichiometry of of NADPH NADPH in consumption the presence as of aGR function and 100 of µM MeSeSG DMDSe concen- (+) or 2.3. ToxicityThe above of experimentsMeSeH Precursors suggesttration100 µM that in MeSe anaerobic MSeA,SG ( MeSeSG,conditions., ). The reaction and NADPH DMDSe mixtures consumption are contained efficiently was 200 followed µM NADPH, in anaerobic 100 nM conditions GR in 100 with mM 0 converted into MeSeH inside the celltopotassium 200 and µM that MeSeSG phosphate, they are added good pH to7.4, candidates the at reaction 22 °C. toAnaerobic mixture studythe as reactions in toxicity B). The were amount performed of NADPH in a glove consumed box under after nitrogen atmosphere. (C) Stoichiometry340 of NADPH consumption as a function of MeSeSG concen- of MeSeH.The above The toxicity experiments of MeSeH precursors125suggest min was that was calculated investigatedMSeA, from MeSeSG the in ∆A a growth, after and subtraction inhibition DMDSe assay.of are the efficiently value in the absence con- of substrate. tration in anaerobic conditions. NADPH consumption was followed in anaerobic conditions with 0 vertedExponentially into MeSeH growing inside BY4742 the cells cell were and diluted that tothey a final are optical good density candidates of 0.04 to OD study at the toxicity to 200These µM MeSeSG results addedshow tothat the MeSeSG reaction mixtureacts as asa substratein B). The amountfor GR, of albeit NADPH at a consumed rate reduced after nearly125 min 100 was-fold calculated compared from theto GSSG.∆A340 after Upon subtraction addition of theof enzymevalue in theto MeSeSG,absence of theresubstrate. was a rapid phase of NADPH oxidation followed by a slower continuous oxidation phase (Fig- ure 6BThese), suggesting results show that athat product MeSeSG of the acts reaction as a substrate may redox for -GR,cycle albeit with atoxygen. a rate reducedTo con- firmnearly this 100 hypothesis,-fold compared experiments to GSSG. were Upon performed addition inof anaerobicenzyme to conditions. MeSeSG, there The initialwas a phasesrapid phase of the of reaction NADPH were oxidation identical followed in aerobic by a slowerand anaerobic continuous conditions, oxidation whereas phase (Fig- the slowure 6B phase), suggesting of NADPH that oxidation a product was of oxygenthe reaction-dependent may redox (Figure-cycle 6B )with. As shownoxygen. in To Figure con- 6firmC, 0.52 this mol hypothesis, of NADPH experiments was oxidized were per performedmol of MeSeSG in anaerobic at the end conditions. of the experiment The initial in anaerobicphases of conditions.the reaction This were stoic idehiometryntical in aerobiccan be explainedand anaerobic by the conditions, reaction betweenwhereas thethe MeSeHslow phase produced of NADPH by the oxidation GR-catalyzed was oxygen reaction-dependent (7) with MeSeSG (Figure to6B produce). As shown DMDSe in Figure (re- action6C, 0.52 4) .mol Because of NADPH the equilibrium was oxidized constant per molof Equation of MeSeSG (4) isat high, the end the of reverse the experiment equation ofin thisanaerobic equation conditions. is negligible This u ntilstoic almosthiometry all the can initial be explained MeSeSG isby consumed. the reaction In thesebetween condi- the tions,MeSeH Reactions produced (7 )by and the (4 GR) result-catalyzed in the overallreaction reaction (7) with ( 8MeSeSG). to produce DMDSe (re- action 4). Because the equilibrium constant of Equation (4) is high, the reverse equation of this equation is negligibleMeSeSG until + NADPH almost all + Hthe+ → initial MeSeH MeSeSG + GSH is + consumed. NADP+ In these condi-(7) tions, Reactions (7) and (4) result in the overall reaction (8). 2 MeSeSG + NADPH + H+ → DMDSe + 2 GSH + NADP+ (8) MeSeSG + NADPH + H+ → MeSeH + GSH + NADP+ (7)

2 MeSeSG + NADPH + H+ → DMDSe + 2 GSH + NADP+ (8)

Int. J. Mol. Sci. 2021, 22, 2241 12 of 21

of MeSeH. The toxicity of MeSeH precursors was investigated in a growth inhibition as- Int. J. Mol. Sci. 2021, 22, 2241 say. Exponentially growing BY4742 cells were diluted to a final optical density11 of 20 of 0.04 OD at 600 nm and incubated in SD medium containing various concentrations of MSeA, MSeA mixed with a three-fold excess of GSH (MSeA/GSH mix), or DMDSe. The cell density of the cultures600 nm andwas incubated measured inSD after medium 20–24 containing h. Inhibitory various concentration concentrations of of MSeA MSeA, MSeAwas in the mM range.mixed The withGI50 avalue three-fold (the excessconcentration of GSH (MSeA/GSH that reduces mix), by or DMDSe.50% the The cell cell growth density at of the end of the experimentthe cultures) was for measured MSeA was after 4 20–24 mM h. (Figure Inhibitory 8A concentration). The toxicity of MSeA of MSeA was in was the mM exacerbated range. The GI50 value (the concentration that reduces by 50% the cell growth at the end of whenthe it was experiment) mixed forwith MSeA a three was- 4fold mM excess (Figure8 ofA). GSH, The toxicity producing of MSeA MeSeSG was exacerbated extracellularly. In this case,when the it was GI mixed50 value with was a three-fold around excess2 µM of (Figure GSH, producing 8B). Likewise, MeSeSG the extracellularly. GI50 value In of DMDSe was 1this µM case, (Figure the GI 508Cvalue). Because was around of the 2 µM high (Figure concentration8B). Likewise, theof MSeA GI 50 value needed of DMDSe to inhibit cell growth,was we 1 µ Mused (Figure the8 MSeA/GSHC). Because of mix the high or DMDSe concentration in further of MSeA experiments. needed to inhibit We observed cell that growth, we used the MSeA/GSH mix or DMDSe in further experiments. We observed that additionaddition of methionine of methionine (100 (100 µMµM)) to to the growthgrowth medium medium markedly markedly reduced reduced the toxicity the toxicity of 50 the MSeA/of theGSH MSeA/GSH mix or mix of orDMDSe of DMDSe (Figure (Figure 8B,C8B,C),), the the GI GI 50 valuesvalues shifting to to 75 75 and and 30 µM, respectively.30 µM, respectively.

Int. J. Mol. Sci. 2021, 22, 2241 10 of 21

Int. J. Mol. Sci. 2021, 22, 2241 10 of 21

Figure 8. Cell growth inhibition by MeSeH precursors. BY4742 and mutant strains were grown overnight at 30 ◦C in SD Figure 8. Cell growth inhibition by MeSeH precursors. BY4742Figure and 6. Reductionmutant strainsof MeSeSG were or DMDSegrown by overnight S. cerevisiae at glutathione 30 °C in reductase.SD (A) NADPH-de- medium or SD medium supplemented as indicated. Exponentially growing cells were diluted to 0.04–0.08 OD in the pendent reduction of MeSeSG () or GSSG (). 600The reaction600 mixtures contained 100 µM NADPH, medium or SD medium supplemented as indicated.◦ Exponentially growing cells were diluted to 0.04–0.08 OD in the same same medium and grown for 20–24 h at 30 C in various concentrationsand 50 µM of of MeSeHthe substrate precursors. under study Growth in was100 mM monitored potassium phosphate, pH 7.4. The reactions were medium and grown for 20–24 h at 30°C in various concentrations of MeSeH precursors. Growth was monitored by change in by change in OD600 and plotted as % of the growth in the absencestarted of by toxic. adding (A) 1.5 BY4742 nM GR cells to the grown GSSG in mixture SD medium or 73 innM GR to the MeSeSG mixture. (B) Aerobic OD600 and plotted as % of the growth in the absence of toxic. (A) BY4742 cells grown in SD medium in the presence of increas- Figure 6. Reduction of MeSeSGthe or DMDSe presence by of S. increasing cerevisiae concentrationsglutathione reductase. of MSeA. (A (B) )NADPH BY4742-(de- cells) and grown anaerobic in SD ( ), + or) consumption SD supplemented of NADPH with 100 in theµM presence of GR and 100 µM DMDSe (+) or ing concentrations of MSeA. (B) BY4742 cells grown in SD () or SD supplemented with 100 µM methionine () in the pres- pendent reduction of MeSeSG (methionine) or GSSG ( ). inThe the reaction presence mixtures of increasing contained concentrations 100 µM NADPH, of100 a mixture µM MeSe containingSG (,  MSeA). The reaction with a three-fold mixtures contained excess of 200 µM NADPH, 100 nM GR in 100 mM and 50 µM of the substrateence under of increasingGSH. study (C in) BY4742100 concentrations mMcells potassium grown phosphate, inof SD a mixture ( ) orpH SD 7.4. containing supplemented The reactions MSeA withwerepotassium 100withµM aphosphate, methioninethree-fold pH ( excess 7.4,) in at the 22of presence °C. GSH. Anaerobic of(C increasing) BY4742 reactions cells were grown performed in in a glove box under started by adding 1.5 nM GR to the GSSG mixture or 73 nM GR to the MeSeSG mixture. (B) Aerobicnitrogen atmosphere. (C) Stoichiometry of NADPH consumption as a function of MeSeSG concen- SD ( ) orconcentrations SD supplemented of DMDSe. with (D )100 BY4742 µM (methionine) and ∆met17 ( ( ) ),in and the∆ presencecys3 () isogenic of increasing mutants cellsconcentrations grown in SD of medium DMDSe. (D) BY4742 () and anaerobic (, +) consumption of NADPH in the presence of GR and 100 µM DMDSe (+tration) or in anaerobic conditions. NADPH consumption was followed in anaerobic conditions with 0 () and supplemented∆met17 (), withand 100∆cys3µM cysteine() isogenic in the presencemutants of cells increasing grown concentrations in SD medium of DMDSe. supplemented with 100 µM cysteine in the 100 µM MeSeSG (, presence). The reaction of increasing mixtures contained concentrations 200 µM NADPH, of DMDSe. 100 nM GR in 100 mMto 200 µM MeSeSG added to the reaction mixture as in B). The amount of NADPH consumed after potassium phosphate, pH 7.4, at 22 °C. Anaerobic reactions were performed in a glove box under125 min was calculated from the ∆A340 after subtraction of the value in the absence of substrate. nitrogen atmosphere. (C) Stoichiometry of NADPH consumption as a function of MeSeSG concen- tration in anaerobic conditions. NADPH consumption was Thisfollowed protective in anaerobic effect conditions of methioninewith 0 These resultsprompted show usthat to MeSeSG study acts the as behavior a substrate of for various GR, albeit at a rate reduced to 200 µM MeSeSG added to the reaction mixture as mutantin B). The amountstrains. of ANADPH possible consumed explanation afternearly 100 for-fold the compared relief of to toxicityGSSG. Upon by methionineaddition of enzyme addition to MeSeSG, there was a 125 min was calculated from the ∆A340 after subtraction of the value in the absence of substrate. could be that the level of intracellularrapid phase NADPH of NADPH becomes oxidation growth followed-limiting by a slower because continuous reduc- oxidation phase (Fig- ure 6B), suggesting that a product of the reaction may redox-cycle with oxygen. To con- tion of MeSeSG or DMDSe results in continuous oxidation and depletion of NADPH. Be- These results show that MeSeSG acts as a substrate for GR, albeit at a rate reducedfirm this hypothesis, experiments were performed in anaerobic conditions. The initial nearly 100-fold compared to GSSG. Upon additioncause of the enzyme oxidation to MeSeSG, of four there molecules wasphases a of of NADPHthe reaction is requiredwere identical to produce in aerobic methionine and anaerobic from conditions, whereas the rapid phase of NADPH oxidation followed by a slower continuous oxidation phase (Fig-slow phase of NADPH oxidation was oxygen-dependent (Figure 6B). As shown in Figure ure 6B), suggesting that a product of the reaction may redox-cycle with oxygen. To con-6C, 0.52 mol of NADPH was oxidized per mol of MeSeSG at the end of the experiment in firm this hypothesis, experiments were performed in anaerobic conditions. The initialanaerobic conditions. This stoichiometry can be explained by the reaction between the phases of the reaction were identical in aerobic and anaerobic conditions, whereas the MeSeH produced by the GR-catalyzed reaction (7) with MeSeSG to produce DMDSe (re- slow phase of NADPH oxidation was oxygen-dependent (Figure 6B). As shown in Figureaction 4). Because the equilibrium constant of Equation (4) is high, the reverse equation of 6C, 0.52 mol of NADPH was oxidized per mol of MeSeSG at the end of the experimentthis in equation is negligible until almost all the initial MeSeSG is consumed. In these condi- anaerobic conditions. This stoichiometry can be explained by the reaction between tions,the Reactions (7) and (4) result in the overall reaction (8). MeSeH produced by the GR-catalyzed reaction (7) with MeSeSG to produce DMDSe (re- action 4). Because the equilibrium constant of Equation (4) is high, the reverse equation of MeSeSG + NADPH + H+ → MeSeH + GSH + NADP+ (7) this equation is negligible until almost all the initial MeSeSG is consumed. In these condi- tions, Reactions (7) and (4) result in the overall reaction (8). 2 MeSeSG + NADPH + H+ → DMDSe + 2 GSH + NADP+ (8)

MeSeSG + NADPH + H+ → MeSeH + GSH + NADP+ (7)

2 MeSeSG + NADPH + H+ → DMDSe + 2 GSH + NADP+ (8)

Int. J. Mol. Sci. 2021, 22, 2241 12 of 20

This protective effect of methionine prompted us to study the behavior of various mutant strains. A possible explanation for the relief of toxicity by methionine addition could be that the level of intracellular NADPH becomes growth-limiting because reduction of MeSeSG or DMDSe results in continuous oxidation and depletion of NADPH. Because the oxidation of four molecules of NADPH is required to produce methionine from sulfate, an external supply of methionine might help preserving cellular NADPH equivalents and alleviate toxicity. The main source of NADPH is the reaction catalyzed by glucose-6- phosphate dehydrogenase, encoded by ZWF1, the first and rate-limiting enzyme in the pentose phosphate pathway. The deletion of ZWF1 had no effect on the toxicity of the MSeA/GSH mix or DMDSe (result not shown), suggesting that improved growth in the presence of methionine is not related to the preservation of a high NADPH pool. Mutants defective in O-acetylhomoserine (OAH)-sulfhydrylase, which catalyzes the incorporation of sulfide into OAH to form homocysteine, were previously selected by resistance to DMDSe [46]. It was speculated that DMDSe was reduced to MeSeH and that the latter was converted into SeMet by OAH-sulfhydrylase, encoded by MET17. We have previously shown that SeMet toxicity was mediated by its metabolization to selenocysteine [29]. Therefore, we evaluated the sensitivity to DMDSe of strains deleted for MET17 or CYS3, which encodes cystathionine γ-lyase, an enzyme of the transsulfuration pathway, responsible for cysteine synthesis from methionine. In these experiments, we added 100 µM cysteine as sulfur source. In contrast to methionine, cysteine addition did not affect toxicity. As shown in Figure8D, both ∆cys3 and ∆met17 strains demonstrated a high level of resistance to DMDSe (GI50 = 70 µM and 40 µM, respectively). These results suggest that the toxicity of methylated Se compounds are the consequence of their reduction into MeSeH, followed by metabolization of this compound into SeMet by OAH-sulfhydrylase and conversion of SeMet into toxic selenocysteine (SeCys) by the transsulfuration pathway. Thus, the protective effect of methionine against MeSeH toxicity is likely to result from competition between the sulfur- and Se-compounds along the metabolic pathway leading to cysteine, as previously shown in the case of SeMet toxicity [47].

2.4. Selenium Content of Cells Exposed to MeSeH Precursors To determine whether the observed differences in toxicity were related to differences in intracellular Se accumulation, total Se-content was quantified in cells incubated for 30 min with different concentrations of MeSeH precursors. The results shown in Table3 indicate that exposure to increasing MSeA concentrations resulted in higher total Se-content in the BY4742 strain. When a three-fold excess of GSH was mixed with a non-toxic MSeA concentration (30 µM), cells accumulated 15-times more Se than in the presence of MSeA alone (2331 vs 162 µg/g dry weight). Thus, the observed toxicity of a mixture of MSeA and GSH is correlated to an increased Se accumulation, suggesting that MeSeSG is taken up more efficiently than MSeA by yeast cells. Exposure to a toxic concentration of DMDSe also led to a high accumulation of Se.

Table 3. Total Se content (µg/g dry weight) of BY4742 cells after 30 min incubation with MSeA, MSeA /GSH mix or DMDSe in SD medium. Means and standard deviations were calculated from three replicate measurements of at least two independent experiments.

Se Compound Total Se Strain Concentration Added (µg/g Dry Weight) BY4742 MSeA 30 µM 162 ± 32 MSeA 300 µM 314 ± 25 MSeA 10 mM 4053 ± 455 MSeA /GSH mix 30 µM + 90 µM 2331 ± 600 DMDSe 5 µM 1378 ± 290

Total Se- and SeMet-content in the water-soluble fraction of BY4742 or BY4742-∆met17 cells were compared (Table4). In the presence of MSeA, ∆met17 cells accumulated half as Int. J. Mol. Sci. 2021, 22, 2241 13 of 20

much total Se as the parental strain. When ∆met17 cells were exposed to the MSeA/GSH mix or DMDSe, water-soluble Se-content was reduced more than 25 times and the amount of SeMet was reduced more than 10 times compared to the wild-type. These results suggest that conversion of MeSeH to SeMet by OAH-sulfhydrylase is the major pathway by which MeSeH is converted into selenocompounds that accumulate in the cell.

Table 4. Total Se and SeMet content (µg/g dry weight) in the water-soluble fraction of BY4742 or BY4742-∆met17 cells grown in SD + 100 µM cysteine and incubated for 30 min in the presence of MSeA, MSeA/GSH mix, or DMDSe. Means and standard deviations were calculated from three replicate measurements of at least two independent experiments.

BY4742 BY4742-∆met17 Total Se in Water Total Se in Water Se Compound Added Concentration SeMet SeMet Fraction Fraction MSeA 300 µM 118 ± 16 47 ± 11 67 ± 17 12 ± 5 MSeA/GSH mix 30 µM + 90 µM 1462 ± 515 349 ± 159 33 ± 6 6 ± 0.5 DMDSe 5 µM 573 ± 49 81 ± 41 21 ± 5 7.5 ± 3

∆met17 cells are unable to synthesize methionine in the absence of an organic source of sulfur. However, a small amount of SeMet (around 10 µg/g of cells, which is 4 to 60 times lower than in BY4742) was observed when ∆met17 cells were incubated with MeSeH precursors. An incorporation of Se in SeMet had already been observed in ∆met17 cells incubated with selenite [48]. This points to a metabolic pathway resulting in SeMet synthesis independently of OAH-sulfhydrylase. This enzyme belongs to a family of closely related pyridoxal phosphate (PLP)-dependent enzymes that catalyze a wide variety of reactions in amino acid metabolism. In particular, cystathionine beta-synthase (Cys4p), which catalyzes cystathionine synthesis from serine and homocysteine, is structurally homologous to OAH-sulfhydrylase and was shown to possess serine sulfhydrylase activ- ity [49]. It is conceivable that this enzyme has low homoserine sulfhydrylase activity, which would directly generate SeMet from MeSeH. The identification of SeMet in MSeA-treated human lymphoma cells suggests that such a pathway might be relevant in mammalian cells, which cannot synthetize amino acids from inorganic sulfur [50,51].

2.5. Exposure to MeSeH Causes Protein Aggregation We recently showed that SeMet exposure induced Hsp104 expression and caused an accumulation of toxic protein aggregates in yeast cells resulting from the conversion of SeMet to SeCys by the transsulfuration pathway and the misincorporation of SeCys in the place of cysteine during protein biosynthesis [29]. Protein aggregation was visualized by fluorescence microscopy of live cells expressing an Hsp104-GFP construct. To confirm that MeSeH toxic effects were mediated by its metabolization to SeMet, we compared the effects of DMDSe on the expression (Figure9A) and localization (Figure9B) of Hsp104- GFP in BY4742 cells, expressing OAH-sulfhydrylase, to those of BY4741 cells, which are devoid of this activity (met17). Exposure to 20 µM SeMet for 2 h induced the expression of Hsp104-GFP in both strains, whereas induction of Hsp104-GFP by DMDSe (100 µM) was dependent on the presence of a functional MET17 gene (Figure9A). As shown in Figure9B, Hsp104-GFP distribution was diffuse in the cytoplasm of unexposed cells, whereas aggregates were visible in BY4742 cells exposed to toxic concentrations of SeMet or DMDSe. At 20 µM SeMet or 5 µM DMDSe, more than 70% of cells contained at least one distinct bright fluorescent focus. In contrast, Hsp104-GFP remained soluble in BY4741 cells exposed to 5 or 200 µM DMDSe. In a previous article, we showed that protein aggregation upon SeMet exposure was suppressed in a ∆cys3 mutant unable to synthesize SeCys from SeMet [29]. 5 µM DMDSe was unable to induce protein aggregation in a BY4742-∆cys3 mutant strain. Altogether, these results indicate that intracellular formation of MeSeH leads to toxic protein aggregation in wild-type yeast cells. Int. J. Mol. Sci. 2021, 22, 2241 14 of 20 Int. J. Mol. Sci. 2021, 22, 2241 15 of 21

Figure 9. DMDSe promotesFigure 9. DMDSe protein aggregationpromotes protein in a MET17 aggregation-dependent in a MET17 manner.-dependent (A) Induction manner. of Hsp104-GFP(A) Induction by of SeMet or DMDSe. ExponentiallyHsp104 growing-GFP by BY4742-Hsp104-GFP SeMet or DMDSe. (darkExponentially bars) or BY4741-Hsp104-GFP growing BY4742-Hsp104 (light-GFP bars) (dark cells bars were) or incubated in SC + 100 µM methionineBY4741-Hsp104 for 2 h-GFP at 30 (light◦C in bars the) absencecells were or incubated presence in of SC the + indicated100 µM methionine concentration for 2 ofh at SeMet 30°C orin DMDSe. the absence or presence of the indicated concentration of SeMet or DMDSe. The fluorescence in The fluorescence in whole cell extracts was recorded at 508 nm and normalized to the optical density of the extracts at whole cell extracts was recorded at 508 nm and normalized to the optical density of the extracts at 280 nm. The fluorescence280 nm. intensityThe fluorescence in the absence intensity of in toxic the absence (control) of wastoxic set (control as 1. () Bwas) Localization set as 1. (B) ofLocalization Hsp104-GFP of in cells exposed to SeMet orHsp104 DMDSe.-GFP Hsp104-GFP in cells exposed localization to SeMet was or DMDSe. monitored Hsp104 by fluorescence-GFP localization in living was cells monitored after 1 h by of flu- exposure to the indicated concentrationsorescence in of living SeMet cells or DMDSe.after 1 h BY4742of exposure and to BY4742- the indicated∆cys3 cellsconcentrations were grown of SeMet in SD supplementedor DMDSe. with 100 µM cysteine, BY4741BY4742 cells and wereBY4742 grown-∆cys3 in cells SD were supplemented grown in SD with supplemented 100 µM methionine. with 100 µM Representative cysteine, BY4 images741 cells obtained after maximum intensitywere grown z-projection, in SD supplemented bar equals 10 µwithm. The100 fractionµM methionine. of cells containingRepresentative at least images one Hsp104-GFPobtained after focus was determined by manual inspection of 100–200 cells in each condition.

Int. J. Mol. Sci. 2021, 22, 2241 15 of 20

3. Conclusions Our results show that MeSeH can be efficiently produced from MSeA, MeSeSG and DMDSe, provided that the cellular GSH/GSSG ratio is sufficiently high. Enzymatic reduc- tion of MeSeH precursors could also contribute to MeSeH formation in vivo. From toxicity experiments with MeSeH precursors, we conclude that MeSeH is toxic for yeast cells and that this toxicity results from (i) conversion of MeSeH into SeMet by OAH- sulfhydrylase, (ii) conversion of SeMet into SeCys by the transsulfuration pathway and (iii) SeCys misin- corporation during protein synthesis, which produces toxic protein aggregates. Because humans can synthetize only very low levels of SeMet from MSeA [50,51], this pathway is not likely to account for MSeA anti-cancer effects. However, at higher concentrations, MeSeH is toxic to met17 yeast cells which, like human cells, lack OAH-sulfhydrylase ac- tivity. We are currently studying the mechanism of toxicity of MeSeH precursors in a methionine-auxotroph yeast strain.

4. Materials and Methods 4.1. Reagents and Enzymes MSeA was purchased from Aldrich (Saint-Louis, MO, USA); GSH, GSSG, NADPH, DTT, DMDSe, and TCEP from Sigma (Saint-Louis, MO, USA). DMDSe was prepared as a 20 mM stock solution in 100% ethanol stored at −20 ◦C. Glutathione reductase from baker’s yeast (205 units/mg, 2.2 mg/mL) was from Sigma (Saint-Louis, MO, USA). Yeast thioredoxin (9.7 units/mg, 88.5 µM) was from Fisher Scientific (Waltham, MA, USA). Yeast thioredoxin reductase (25.6 units/mg) was purchased from Abnova (Taipei, Taiwan).

4.2. Spectrophotometric Measurements All spectrophotometric measurements were performed with a double beam Agilent Technologies (Santa Clara, CA, USA) Cary 100 spectrophotometer. All experiments were performed in 100 mM potassium phosphate, pH 7.4, at 22 ◦C. To minimize aerobic oxidation of MeSeH, the buffer was deoxygenized in a glove-box under nitrogen atmosphere until it was used aerobically. During the time necessary to perform the experiments, the rate of oxidation of MeSeH was reduced by 75% compared to that in fully oxygenated buffer. The molar extinction coefficients of MeSeH at 252 nm and 340 nm (4670 and 192 M−1 cm−1, respectively) were determined from the reactions of MSeA or DMDSe solutions of known concentration with a 10-fold excess of TCEP. Identical results were obtained with MSeA or DMDSe. Calculation of rate and equilibrium constants were made using values of 84, −1 −1 288, 226, 161, 35 and 107 M cm for the ε252nm of GSH, GSSG, MeSeSG, DMDSe, DTT, and DTTox respectively. The values used for ε340nm of GSH, GSSG, MeSeSG, MeSeH, and DMDSe were 3, 5, 15, 192, and 52 M−1 cm−1, respectively. When reduction reactions with GSH, DTT, or TCEP were analyzed by spectrophotometry, both cuvettes contained the reductant. The reactions were initiated by adding the Se compound in the sample cuvette. Stock solutions of GSH and DTT (100 mM), buffered at pH 7.4, were renewed every day. The concentrations of these stock solutions were determined by Ellman’s assay [52]. The amount of contaminant GSSG (1.2 to 1.5% (mol/mol)) in the GSH solution was measured by reduction with NADPH (200 µM) and glutathione reductase (0.1 units/mL). GSSG concentration was deduced from the change in absorbance at 340 nm, caused by the + conversion of NADPH to NADP . DTT solutions contained 0.5 to 0.6% (mol/mol) DTTox, −1 −1 as measured by UV absorbance at 285 nm (ε285 nm = 283 M cm ).

4.3. MeSeSG Purification 5 mM MSeA and 20 mM GSH were mixed in 100 mM potassium phosphate, pH 7.4, and left to react for 10 min. The mixture was then diluted twice with 20 % (v/v) acetic acid/water to bring the pH to 3.5 and applied on an Alltima C18 high-performance liquid chromatography column (0.32 × 15 cm, 5 µm), from Grace (Columbia, MD, USA) equilibrated in 0.05% (v/v) acetic acid/water at a flow rate of 0.4 mL/min. Elution was performed with the same solution for 5 min, followed by a linear gradient from 0.05% Int. J. Mol. Sci. 2021, 22, 2241 16 of 20

acetic acid to 100% methanol in 20 min. The retention times of GSH and GSSG were 3.1 and 11.2 min, respectively. Another peak, likely corresponding to MeSeSG, eluted at 14.8 min. Identity of this peak was confirmed by positive electrospray mass analysis. The result indicated the presence of a major ion species at m/z = 402.023 with an isotopic distribu- tion in agreement with the presence of a selenium atom. Fractions containing MeSeSG were collected, lyophilized, and aliquoted for storage at −20 ◦C. After resuspension in 100 mM potassium phosphate, pH 7.4, concentration of MeSeSG was determined from the absorbance of MeSeH at 252 nm after reduction with a ten-fold excess of TCEP.

4.4. Stopped-Flow Experiments An Applied Photophysics (Leatherhead, UK) SX20 and a BioLogic (Willow Hill, PA, USA) SFM-300 stopped-flow apparatus, with cell pathlength of 0.2 and 0.08 cm, respectively, were used to determine kinetic constants. Both instruments had a dead time of 1 ms. All experiments were performed in deoxygenized 100 mM potassium phosphate, pH 7.4, at 22 ◦C. To determine the reaction rate between MeSeH and GSSG, MeSeH was produced by reduction of DMDSe with a substoichiometric amount of TCEP. Reactions were initiated by mixing equal volumes of MeSeH (final concentrations: 50 or 100 µM) and GSSG (final concentrations: 100, 200, 300, 400, or 600 µM) or equal volumes of DMDSe (final concentrations, 50, 100, or 125 µM) and GSH (final concentrations, 1250, 1667, or 2500 µM). After mixing, the change of absorbance at 252 nm was recorded.

4.5. Determination of DMDSe Redox Potential 50 µM DMDSe was mixed with DTT (0 to 2500 µM) in 100 mM potassium phosphate, pH 7.4, at 22 ◦C. UV-spectra (200–400 nm) were recorded immediately after mixing and after 1 min. Identical results were obtained, showing that chemical equilibrium between reactants was reached. To obtain the equilibrium constant of the reaction, the increase in absorbance at 252 nm as a function of added DTT was fitted to the theoretical equation.

4.6. Data Analysis Experimental data were fitted to theoretical equations described in Supplementary Materials using OriginPro software (OriginLab Corporation, Northampton, MA, USA).

4.7. Enzymatic Assays The reduction reactions were performed at 22 ◦C in 100 mM potassium phosphate, pH 7.4. When glutathione reductase from S. cerevisiae was used, the reaction volume (600 µL) contained 50 or 100 µM of the substrate under study (GSSG, MeSeSG, or DMDSe), 100 to 200 µM NADPH, and glutathione reductase at the indicated concentration. Reduction by the S. cerevisiae thioredoxin/thioredoxin reductase system were performed in an assay mixture of 200 µL containing 2 µM thioredoxin and 0.5 µM thioredoxin reductase, 100 µM NADPH, and 50 µM substrates. The oxidation of NADPH was followed at 340 nm us- ing a millimolar extinction coefficient of 6.22 cm−1 for NADPH. Anaerobic assays were performed in a glove-box under nitrogen atmosphere.

4.8. Strains and Media The S. cerevisiae strains used in this study are derived from strain BY4742 (MATα his3∆1 leu2∆ lys2∆0 ura3∆0) or BY4741 (MATa his3∆1 leu2∆ met17∆0 ura3∆0). The parental strains and the single mutants were obtained from Euroscarf (Scientific Research and Development GmbH, Oberursel, Germany). The BY4741 strain expressing a GFP-tagged Hsp104 was purchased from ThermoFisher Scientific (Waltham, MA, USA). GFP tagging of Hsp104 in BY4742 was performed by PCR-mediated homologous recombination, and correct integrations were checked by PCR. Standard Synthetic Defined (SD) medium contained 0.67% (w/v) yeast nitrogen base (Difco, Becton, Dickinson and Company, Sparks, MD, USA), 2% (w/v) glucose and 50 mg/l of histidine, leucine, lysine, and uracil and was buffered at pH 6.0 by the addition of 50 mM MES-NaOH. Standard Synthetic Complete Int. J. Mol. Sci. 2021, 22, 2241 17 of 20

(SC) medium contained 0.67% (w/v) yeast nitrogen base (Difco), 2% (w/v) glucose and 80 mg/l of adenine, uracil and all amino acids (160 mg/mL leucine) except methionine and cysteine and was buffered at pH 6.0 by the addition of 50 mM MES-NaOH. Media were supplemented in methionine and cysteine as indicated in the legends to the figures.

4.9. Toxicity Assays Cells were pre-grown at 30 ◦C in the medium subsequently used. Exponentially growing cells were inoculated in the same medium to obtain an OD600 of 0.04–0.08 and left to grow at 30 ◦C for 1 h. After the addition of Se compounds, cells were grown at 30 ◦C and the OD600 was recorded after 20–24 h.

4.10. Determination of Selenium Content For the determination of total Se content, BY4742 cells were grown at 30 ◦C to an OD600 of 0.6–0.8 in SD medium, followed by 30 min exposure to the Se-compound under study. For the determination of SeMet and total water soluble Se contents, BY4742 and a ∆met17 mutant were grown in SD + 100 µM cysteine, followed by 30 min exposure to the Se-compound. Cells were harvested by centrifugation and washed 3 times in an equal volume of ultrapure water. Samples corresponding to 30 OD600 units were aliquoted in microfuge tubes and lyophilized. For each condition, at least three aliquots were prepared. The dry weight of each yeast sample was estimated by the difference of weight between the filled and empty microtubes. Total Se contents were determined in yeast samples after digestion of 4–8 mg (dry weight) of sample with 100 µL of nitric acid and 50 µL of H2O2. Se concentration in water extracts was determined after extraction of 4–8 mg samples with 200 µL of ultrapure water using ultrasonic bath (Sonorex, Bandelin, Berlin, Germany) for 1 h. After extraction, samples were centrifuged at 20,000× g for 15 min. 30 µL aliquots of water extract were used and digested with 100 µL of nitric acid. Digestion was carried out at 65 ◦C for 4h in a hot block. After digestion, samples were diluted and analyzed by inductively coupled plasma mass spectrometry (ICP-MS) (Agilent 7500cs, Tokyo, Japan) operating in hydrogen collision gas mode (H2) to remove possible interferences. Certified reference material (CRM) SELM1 (NRC Canada) was analyzed together with the samples to validate the method and to create a five-points calibration curve. SeMet concentration was determined after reverse phase HPLC separation on an Acquity UPLC BEH C18 column (150 × 2.1 mm, 1.7 µm), from Waters (Milford, MA, USA). Gradient elution, at a flow rate of 0.35 mL/min, was carried out using eluent A, 0.1% formic acid in water, and eluent B, 0.1% formic acid in acetonitrile. The gradient program was 0–0.5 min 0% B, 10.5–20.5 min up to 25% B, 20.5–27.5 min up to 50% B, 27.5–30 min at 50% B, 30–31 min down to 0% B, 31–35 min 0% B. Samples obtained from water extraction were filtrated through 3kDa cutoff filter and diluted with MilliQ water. A 5 µL aliquot of the supernatant was injected into the reverse phase column each time. SeMet standard (Sigma, Saint-Louis, MO, USA) was used in order to create a calibration curve. ICP-MS detection was achieved using a model 7700 instrument (Agilent) fitted with platinum cones, 1 mm internal diameter injector torch. 7.5% of oxygen was added to plasma gas to avoid carbon deposit on the cones. The RP ICP MS coupling was done via Scott spray chamber cooled to −5 ◦C[53].

4.11. Fluorescence Microscopy ◦ Yeast cells expressing Hsp104-GFP were grown at 30 C to an OD650 of 0.5 in SC medium supplemented with methionine and/or cysteine as indicated in the figure legends, followed by exposure to SeMet or to DMDSe for 1 h. Cells were mounted on glass slides covered with a thin layer of 1% agarose. Differential interference contrast (DIC; Nomarski interference contrast) and fluorescence images were obtained at room temperature using a Zeiss (Oberkochen, Germany) Axio Observer microscope equipped with a 40×, 1.4 NA oil immersion objective. 470 nm excitation at maximum available intensity (4 W cm−2) and Int. J. Mol. Sci. 2021, 22, 2241 18 of 20

a filter set 65 HE (EX BP 475/30, BS FT 495, EM BP550/100) were used for fluorescence imaging. The lateral resolution was estimated to be 180 nm. Images were captured with digital camera Zeiss AxioCam MRm. Z-stacks of 15 to 25 images with 260 nm spacing were recorded. Maximum intensity z-projections were performed with ImageJ and the images were analyzed manually.

4.12. Fluorescence Spectroscopy ◦ BY4742 or BY4741 cells expressing Hsp104-GFP were grown at 30 C to an OD600 of 0.5 in SC medium + 100 µM methionine, followed by 2 h exposure to 20 µM SeMet or 100 µM DMDSe. Whole cell extracts from 1–3 OD600 units were prepared in 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 250 mM NaCl, 5% (v/v) glycerol, 10 mM 2-mercaptoethanol, by vortexing cells at 4 ◦C for 10 × 30 s in the presence of an equal volume of glass beads (500–750 µm). After centrifugation at 10,000× g for 10 min, the supernatant was recovered and the optical density at 280 nm was determined. GFP fluorescence was recorded at 508 nm in a Jasco FP-8300 spectrofluorometer using an excitation wavelength of 487 nm (bandwidth 2.5 nm).

Supplementary Materials: The following are available online at https://www.mdpi.com/1422-006 7/22/5/2241/s1. Author Contributions: Formal analysis, M.D. and P.P.; Investigation, M.D., K.B., C.M. and M.L.; Resources, R.L. (Roxane Lestini), R.L. (Ryszard Lobinski) and J.S.; Supervision, J.S. and M.L.; Writing— original draft, M.L.; Writing—review & editing, M.D., P.P. and M.L. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data is contained within the article or supplementary material. Acknowledgments: We gratefully acknowledge Sophie Bourcier for mass spectrometric measure- ments and Marten Vos for his help using the stopped-flow apparatus. Conflicts of Interest: The authors declare no conflict of interest.

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