acylcarnitine carrier is regulated by hydrogen sulfide via interaction with C136 and C155

    The mitochondrial carnitine/acylcarnitine carrier is regulated by hydrogen sulfide via interaction with C136 and C155 Nicola Giangregorio, Annamaria Tonazzi, Lara Console, Imma Lorusso, Annalisa De Palma, Cesare Indiveri PII: DOI: Reference:

S0304-4165(15)00269-X doi: 10.1016/j.bbagen.2015.10.005 BBAGEN 28301

To appear in:

BBA - General Subjects

Received date: Revised date: Accepted date:

23 July 2015 24 September 2015 8 October 2015

Please cite this article as: Nicola Giangregorio, Annamaria Tonazzi, Lara Console, Imma Lorusso, Annalisa De Palma, Cesare Indiveri, The mitochondrial carnitine/acylcarnitine carrier is regulated by hydrogen sulfide via interaction with C136 and C155, BBA - General Subjects (2015), doi: 10.1016/j.bbagen.2015.10.005

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ACCEPTED MANUSCRIPT THE MITOCHONDRIAL CARNITINE/ACYLCARNITINE CARRIER IS REGULATED BY

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HYDROGEN SULFIDE VIA INTERACTION WITH C136 AND C155

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Nicola Giangregorio1a, Annamaria Tonazzi1a, Lara Console2, Imma Lorusso3, Annalisa De

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Palma3, Cesare Indiveri2,1

CNR Institute of Biomembranes and Bioenergetics, via Amendola 165/A, 70126 Bari, Italy

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Department DiBEST (Biologia, Ecologia, Scienze della Terra) Unit of Biochemistry and

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Molecular Biotechnology, Via Bucci 4C, University of Calabria, 87036 Arcavacata di Rende, Italy

Department of Bioscience, Biotechnology and Biopharmaceutics, University of Bari, Italy

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These authors contributed equally to this work.

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Corresponding author:

Cesare Indiveri, Department DiBEST(Biologia, Ecologia e Scienze della Terra) University of Calabria Via P. Bucci cubo 4C, 87036 Arcavacata di Rende (CS) Italy. Tel.:+39-0984-492939; Fax: +39-0984-492911. E-mail: [email protected].

Abbreviations DTE, dithioerythritol; NEM, N-ethylmaleimide; PLP, pyridoxal 5-phosphate;Pipes, 1,4-piperazinediethanesulfonic acid; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; CAC, Mitochondrial carnitine/acylcarnitine carrier; synonyms: CACT,

ACCEPTED MANUSCRIPT solute carrier family 25 member 20, symbol: SLC25A20 ; ORC ornithine carrier; ANC, adenine nucleotide carrier; WT, wild-type; N-acetylcysteine (NAC).

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Abstract

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Background: The Carnitine/acylcarnitine carrier (CAC or CACT) mediates transport of

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acylcarnitines into mitochondria for the β-oxidation. CAC possesses Cys residues which respond to redox changes undergoing to SH/disulfide interconversion. Methods: The effect of H2S has been investigated on the [3H]carnitine/carnitine antiport

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catalyzed by recombinant or native CAC reconstituted in proteoliposomes. Site-directed

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mutagenesis was employed for identifying Cys reacting with H2S. Results: H2S led to transport inhibition, which was dependent on concentration, pH and time of incubation. Best inhibition with IC50 of 0.70 µM was observed at physiological pH

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after 30-60 min incubation. At longer times of incubation, inhibition was reversed. After oxidation of the carrier by O2, transport activity was rescued by H2S indicating that the

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inhibition/activation depends on the initial redox state of the protein. The observed effects were more efficient on the native rat liver transporter than on the recombinant protein. Only

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the protein containing both C136 and C155 responded to the reagent as the WT. While reduced responses were observed in the mutants containing C136 or C155. Multialignment of known mitochondrial carriers, highlighted that only the CAC possesses both Cys residues. This correlates well with the absence of effects of H2S on carriers which does not contain the Cys couple. Conclusions: Altogether, these data demonstrate that H2S regulates the CAC by inhibiting or activating transport on the basis of the redox state of the protein. General Significance: CAC represents a specific target of H2S among mitochondrial carriers in agreement with the presence of a reactive Cys couple.

ACCEPTED MANUSCRIPT 1 Introduction Besides the known lethal effects of hydrogen sulfide at relatively high concentrations, it is

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now well assessed that this gas also plays important roles in human physiology [1-5]. The

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range of concentrations at which physiological effects are generated is still under debate.

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In general, it is believed that the cellular concentration of H2S ranges between nanomolar and three hundred micromolar depending on the measurement method and the tissues in which it is relieved [6-9]. The main known actions of this gas consist of relaxation of the

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cardiovascular systems (artery and veins), control of inflammation and neuroprotection,

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even though other roles have been postulated in liver, lung and other tissues, which however still need to be better defined [6, 10]. Among other effects, H2S has a documented effect in ameliorating reperfusion injury after ischemia [6, 11, 12]. The

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mechanism of action is, at least in some cases, mediated by interaction with sensitive protein residues such as Cys, which are reactive towards the gas. The molecular

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mechanisms of cellular action are poorly understood. Some proteins have already been described to interact with H2S with important consequences on modulation of physiological

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functions. It is demonstrated that H2S covalently modifies the ATP-sensitive K+ channels causing membrane hyper polarization and vasodilatation [13]. Cytochrome c oxidase is inhibited by H2S [9, 14]. This probably reflects the observed inhibition of mitochondrial respiration. However, other mitochondrial targets of H2S may exist which could better explain action of this gas on mitochondrial energy metabolism. Based on present knowledge, it is expected that the best targets of H2S would be those proteins whose Cys residues, on the one hand exhibit high affinity towards H2S, on the other hand modulate the protein function upon modification. A protein with these features might be the mitochondrial carnitine/acylcarnitine carrier. This transporter is essential for completion of the β-oxidation pathway as demonstrated by several studies performed with intact mitochondria and with native or recombinant purified

ACCEPTED MANUSCRIPT proteins [15] and confirmed by the lethality of human pathology caused by defects of this protein [15, 16]. CAC contains six Cys residues whose structure/function relationships

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have been well defined by site-directed mutagenesis, homology modeling and chemical

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modification. It has been demonstrated that two of these residues, C136 and C155, play a

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regulatory role on the protein function and, hence, to the fatty acid oxidation pathway. In particular C136 exhibits an high reactivity which is evident in terms of very low IC 50 towards a series of chemical reagents, such as mercurial compounds [17] but also towards

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physiological compounds involved in the cell redox homeostasis such as glutathione. C155

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is the counterpart for disulfide formation. The 2SH/S-S conversion acts as an on-off switch of the transporter, which is active only in the reduced (thiol) form [18-20]. In this work, the high affinity interaction of the CAC with H2S has been demonstrated to occur via the

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couple C136-C155 suggesting a gas sensing role for these residues. The expected

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consequences in the regulation of energy metabolism are discussed.

2 Materials and Methods

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2.1 Materials.

Sephadex G-75 was purchased from Pharmacia, L-[methyl-3H]carnitine and L-[2,33

H]ornithine from Scopus Research BV Costerweg, [ 2,5',8-3H]ATP from PerkinElmer, egg-

yolk phospholipids (L-α-phosphatidylcholine from fresh turkey egg yolk), PIPES, Triton X100, cardiolipin, L-carnitine, L-ornithine, ATP, N-dodecanoylsarcosine (sarkosyl), Sodium Hydrosulfide (NaHS), Sodium sulfide (Na2S), GSH, N-acetyl cysteine (NAC) and Cys from Sigma-Aldrich. All other reagents were of analytical grade.

2.2 Site-directed mutagenesis, overexpression and isolation of the CAC proteins. The previously constructed pMW7-WTratCAC recombinant plasmid was used as a template to introduce mutations in the CAC protein using complementary mutagenic

ACCEPTED MANUSCRIPT primers and the High Fidelity PCR System (Roche) [21]. The PCR products were purified by the QIAEX II Gel Extraction Kit (QIAGEN), digested with NdeI and HindIII and ligated

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into the pMW7 expression vector. All resulting pMW7-ratCAC constructs were verified by

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DNA sequencing. Except for the desired base changes, all the sequences corresponded to

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the CAC coding sequence. The human [22] and the Aspergillus nidulans (fungus) CAC, amplified from an A. nidulans cDNA library (kindly provided by Dr. JR De Lucas) were subcloned into pMW 7 between NdeI and BamHI restriction sites. Bacterial over-expression

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was obtained using E. coli CO214. Wild-type and mutants rat, human and A. nidulans

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CAC inclusion body fractions were isolated from E. coli, solubilized and purified as previously described [23].

2.3 Reconstitution of CAC, ANC and ORC in liposomes.

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Mitochondria has been isolated using a standard protocol that consists of: cell destruction by mechanical stress, centrifugation at low speed to remove debris and large cellular

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organelles, centrifugation at higher speed to isolate crude mitochondria [24].Mitochondrial Rat liver extract (0.3 mg proteins in 3% Triton X-100) containing native CAC or human, rat

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and A. nidulans CAC recombinant proteins (about 0.6 μg protein) were reconstituted into liposomes by removing the detergent from the mixed micelles through an hydrophobic ionexchange column containing the resin Amberlite XAD-4, as described previously [25]. The reconstitution mixture of mitochondrial or recombinant CAC proteins was composed of protein, 1% Triton X-100, 10 mg of egg yolk phospholipids in the form of sonicated liposomes, 10 mM Pipes at pH 7.2, 15 mM carnitine, in a final volume of 680 μl. This mixture was passed 15 times, at room temperature, through the same Amberlite column (pasteur pipette filled with 0.5 g resin) pre-equilibrated with a buffer containing 10 mM Pipes pH 7.2 and 15 mM carnitine. In the case of ANC and ORC, the reconstitution procedure of rat liver mitochondrial extract was the same described previously [25, 26].

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2.4 Transport measurements.

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The external substrate was removed by passing proteoliposomes through a Sephadex G-

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75 column (0.7 cm diameter; 15 cm height). The turbid eluate (500 µL) from the Sephadex column were collected and used for transport measurement by the inhibitor-stop method [27]. For uptake measurements, transport at 25°C was started by adding 0.1 mM

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[3H]carnitine or [3H]ornithine or [3H]ATP to proteoliposomes and stopped by the addition of

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0.1 mM NEM in the case of CAC or 20 mM PLP in the case of ORC or 5 µM carboxyatractyloside in the case of ANC as previously described [28, 29]. In controls, the inhibitor was added together with the labeled substrate at time zero. The experimental

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values were corrected by subtracting control values. Finally, the external substrate was removed by chromatography on Sephadex G-75 columns (0.7 cm diameter; 10 cm height)

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and intraliposomal radioactivity was measured. The measurement of CAC activity in isolated mitochondria has been performed as previously described [30].

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2.5 Other methods

Amount of reconstituted protein was estimated from Coomassie blue stained SDS-PAGE gels by using the Chemidoc imaging system equipped with Quantity One software (BioRad) as previously described [31]. The homology model of the human CAC was constructed using the crystallographic structure of the bovine ANC as template [32] and the computer application Swiss PDB Viewer as previously described for the CAC [33]. NaHS or Na2S solutions were prepared as described in ref [34]. Measurements of sulfide content in solutions was performed using the DTNB titration method. The Sulfide released by NaHS (0.90 ± 0.14 mol/mol NaHS) was very similar to that released by Na2S (1.1 ± 0.25 mol/mol) [33]. Each experiment dealing with effects of H2S was performed either

ACCEPTED MANUSCRIPT using NaHS or Na2S. Those perfomed with NaHS are reported in the figures. Very similar results were obtained also in those, not shown, performed with Na 2S.

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2.6 Statistical analysis

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Statistical analysis was performed by one-way analysis of variance (ANOVA) or Student's

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t-test, as indicated in figure legends. Values of p lower than 0.05 were considered statistically significant. Data points derived from means of three different experiments as

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specified in the figure legends.

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3 Results

3.1 Characterization of CAC inhibition by H2S Addition of 15 M NaHS to CAC proteoliposomes caused inhibition of transport function as

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described in Fig. 1. The [3H]carnitine/carnitine antiport measured as dependence on time, is indeed reduced up to 67% after 60 min of transport. Release of the reactive species of

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H2S is pH and time dependent [34]. The reaction was performed under different pH conditions ( Fig. 2). The results show that the optimal pH for inhibition is pH 7.2 while at

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more acidic pH a lower inhibition was observed. Nearly no inhibition was detected at pH 8.0 (Fig. 2). The time dependence of the reaction was then investigated. The results with proteoliposomes indicated that the incubation time is critical for the inhibition. The transport activity of CAC gradually decreases with increasing H2S concentration (fig. 3A). Indeed at 1µM NaHS a maximal inhibition of about 70% was found after 60 min, while at higher concentrations maximal inhibition was reached at shorter times, i.e., after 30 min at 30 µM or after 15 min at 100 µM (Fig. 3A). Surprisingly, by prolonging the incubation, after maximal inhibition, the activity was partially restored. This effect was more evident at 1 and 30 µM H2S concentrations. To investigate the effect of H2S on CAC in the context of the entire organelle, the same experiment has been carried out using isolated mitochondria. A very similar trend of the CAC response to H2S was observed (Fig. 3B). In this case, the

ACCEPTED MANUSCRIPT inhibition as well as the reactivation were faster than in proteoliposomes. To confirm the time/concentration relationships, the dose response, under different incubation times, was

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investigated with the recombinant protein. Reaction/inhibition was much more effective at

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60 min. The IC50 derived from the dose response curve at this incubation time was 0.70

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µM ± 0.10. At shorter times of incubation, the compound was less effective, indeed the curves were shifted towards higher NaHS concentrations and higher IC 50 were measured. The values were 21± 5.4 µM at 30 min or 58 ± 4.2 µM at 15 min incubation (Fig. 4).

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Addition of DTE led to recovery of transport activity at all concentrations of NaHS tested

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(not shown), indicating that the action of H2S is mediated by interaction with Cys residues of the protein. Interestingly, some physiological reducing agents such as GSH, Nacetylcysteine and Cys led to activity recovery (Fig. 5A). The inhibition was protected by

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the substrate, as shown in Fig. 5B. Protection was dependent on carnitine concentration indicating the implication of the substrate binding site in the interaction with H2S.

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3.2 Effect of H2S on the oxidized CAC Inactivation/recovery was studied for both recombinant and native CAC, extracted from rat

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liver mitochondria. Recovery of transport function was studied after oxidation of the reconstituted protein by oxygen exposure. The oxygen oxidation led to inhibition of the transport function, which was fully reversed by treatment with DTE. As control of oxygen induced oxidation of CAC, proteoliposomes were incubated in N2 atmosphere. Oxidation, i.e., loss of transport function was time-dependent (Fig. 6, inset) and fully prevented by N2 incubation. Treatment with DTE after O2 incubation fully recovered transport function confirming that oxidation caused formation of disulfide. As expected the DTE treatment on the N2 incubated protein did not exert any effect. Thus, effect of different concentrations of H2S was tested on the oxidized protein. Similar results were obtained with both the recombinant and the native protein (Fig. 7). In particular the native protein was reactivated

ACCEPTED MANUSCRIPT at a lower NaHS concentration; indeed recovery was almost complete at 0.5 µM, while the recombinant protein was fully reactivated at concentrations ten times higher.

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3.3 Identification of Cysteines involved in CAC inhibition.

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Given that the target of H2S must be Cys residues, site directed mutagenesis was

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performed to identify the residues of CAC responsible for H2S effects. An initial screening on single mutants of each of the six Cys CAC residues, i.e., C23S, C58S, C89S, C136S, C155S and C283S highlighted that only the mutants C136S and C155S exhibited

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significant impairment of inhibition (Fig. S1), while all the others behave as the WT, i.e.,

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were strongly inhibited (not shown). This finding suggested an involvement of C136 and C155 in the interaction with H2S. To confirm the critical role of the two Cys residues, the quadruple mutant (C23S/C58S/C89S/C283S) containing only C136 and C155, the five

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replacement mutants (C23V/C58V/C89S/C136V/C283S) containing the sole C155 and (C23V/C58V/C89S/C155V/C283S) containing the sole C136 were tested for H2S effects. It

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has to be stressed that in the five-replacement mutants several Cys residues have been replaced with Val instead of Ser since the substitution with five Ser led to strong

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impairment of transport function. Figure 8 shows the dose response analysis of WT and mutants. The quadruple mutant showed a behavior comparable to WT (see Fig. 4) with a derived IC50 of 1.0 ± 0.10. While mutants containing the sole C136 or C155 showed a clear shift towards higher concentrations of NaHS with IC50 of 52 µM ± 10 and 26 µM ± 6.3, respectively. The mutant in which all Cys are substituted (C-less protein) showed no inhibition, confirming the involvement of Cys residues in the mechanism. These data demonstrate that C136 and C155 are involved in the reaction. The higher affinity of C155 for H2S with respect to C136 indicates that this is the most critical residue for inhibition and, probably, the first in reacting with the gas. The dependence on H 2S incubation time was studied for the three mutants in comparison with the WT (Fig. 9). The mutant C23S/C58S/C89S/C283S containing both C136 and C155 behaved similarly to the WT

ACCEPTED MANUSCRIPT showing maximal inhibition after 30 min incubation and efficient recovery of transport function after longer incubation. Again a similar behavior was observed for the mutant

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containing only C155. In contrast, the mutant containing the sole C136 showed a slower

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decrease of transport function upon incubation with NaHS and no recovery up to 120 min

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of incubation. To gain further insights in the mechanism of interaction of H2S with the Cys residues involved in the regulation, additional mutants were analyzed. To test the influence of the vicinal Lys residue on the reactivity of C136, the mutant K135A has been tested for

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inhibition. This mutant was previously demonstrated to exhibit transport activity similar to

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that of WT [35]. Removal of K135 caused an increase of IC50 for NaHS from 0.70 to 40µM ± 0.50, confirming that the reaction occurs via formation of a thiolate on C136, which is facilitated by the vicinal Lys. An additional mutant has been tested in which the most

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critical residue C155 was shifted to position 156 (C155A/A156C). This mutant showed transport activity similar to the WT (not shown). The IC50 for H2S of this mutant was about

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one order of magnitude higher than that for WT, i.e. 9 µM ± 1.5, indicating that the position of C155 is indeed very critical.

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To investigate the occurrence of the H2S sensitive Cys couple within the carrier family, sequence alignment of mitochondrial carriers with known function was performed. Interestingly it was found that only the CAC possesses both Cys residues, while most of the other carriers have only one of the two homologous Cys. Several proteins have nor C136 neither C155 homologues. Few carriers have only the C136 homologous as depicted by alignment of several functionally characterized carriers (Fig. 10A) and by a complete alignment of all carriers (Fig. S2). It has to be noted that in the total alignment, larger gaps are present due to the longer insertions of SLC 25A18 and 47 (Fig. S2). Then, some of the most representative mitochondrial carriers were tested for inhibition. The IC 50 for H2S was measured for the ANC, which has only the C155 homologous, and the ORC, containing only the cysteine that is homologous of CAC’s C136. The IC50 derived from

ACCEPTED MANUSCRIPT dose response curves were 0.29 ± 0.080 mM and 1.1 ± 0.50 mM, respectively. In this experiment the ANC isoform 1 was assayed; however, the other two isoforms of the ANC

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also possess the sole C155 homologous (not shown). The experiments on ANC and ORC

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confirm the requirement of both Cys residues and, hence, that CAC is a specific

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mitochondrial target of H2S. The structures of the three carriers, CAC (Fig. 10 B), ORC (Fig. 10 C) built on the basis of the 3D structure of ANC (Fig. 10 D), highlight the position of two Cys residues which are close enough to form a disulfide in the CAC as previously

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demonstrated [33]. Moreover the C136 or C155 homologues of ORC or ANC, respectively,

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are not located exactly in the same position of CAC. This is in agreement with the lower affinity of ANC and ORC for H2S compared to CAC mutant containing only one of the two Cys, i.e., C23V/C58V/C89S/C136V/C283S containing the sole C155 and

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C23V/C58V/C89S/C155V/C283S containing the sole C136. A similar bioinformatic analysis was performed on members of the CAC sub-family (Fig. 11). Very interestingly,

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the Cys couple was present only in higher species. To confirm the link between H 2S effect and presence of the Cys couple, the human and the A. nidulans transporters, were tested.

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Indeed, while the human CAC showed high affinity for NaHS (IC50 1.5 ± 0.40 µM) comparable to that of the rat protein, no inhibition could be detected for the A. nidulans transporter (Fig. S1). The activity of this transporter in proteoliposomes was 2.0 ± 0.5 nmol/mg protein/15 min similar to that of the recombinant rat protein and remained higher than 90% after exposure to NaHS concentration from 1 to 100 µM. 4 Discussion and conclusion Physiological concentration of H2S ranges from 30 to 300 µM [7, 13], even though it was also reported that its actual concentration could be much lower, i.e. in the nanomolar range. This was attributable to the effect of removing H2S from tissues when its release is detected under aerobic conditions [8]. The most recent reports assert that the peculiar chemical properties of H2S make it difficult to exactly measure its concentrations leaving

ACCEPTED MANUSCRIPT huge discrepancies both in this issue and in its physiological role [36]. It can be however hypothesized that owing to the enzymatic production of H2S, its concentration could vary

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depending on the activity and localization of the producing enzymes [9]. Therefore, its

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concentration could be higher in the sites of production and lower in the sites of removal

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from tissues by interaction with proteins. Indeed this reagent has been used at 100 µM for discovering effects on K+ channels [13]. Also the chemical behavior of H2S is not as well understood as for CO or NO [37]. Concerning the targets, it is acknowledged that H2S can

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interact with thiol groups of protein and on the basis of proteins features, some proteins

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could "sense" H2S responding to its concentration with alterations of their function, other proteins could just buffer/remove H2S interacting with lower affinity [9]. It is expected that proteins with higher affinity for H2S react specifically at selected Cys residues. CAC,

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indeed, shows high affinity. The interaction occurs via C155 and C136. The final proofs of the crucial role played by the C136-C155 couple is given by analysis of Cys mutants.

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Indeed only the mutant containing the couple behaves exactly as the WT. C136 is located in the third transmembrane segment, facing the water filled cavity (Fig. 10 A and B). C155

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is located on a matrix loop and can come in close vicinity of C136 during the transport cycle and the two sensitive Cys residues are reachable by reagents from the cytosolic side of the transporter as previously demonstrated [33, 38]. The higher affinity of H2S for C155 compared to C136 is in contrast with the higher affinity of C136 towards several specific thiol chemical reagents such as NEM and MTS [38, 39]. This discrepancy can be explained by the different solubility, reactivity and size of H2S (or HS-) with respect to NEM, MTS and other SH reagents. Therefore, the smaller H2S or HS- can reach easily C155. Reactivity with H2S is also expected to be influenced by vicinal residues which may change the pK of the Cys-SH. This correlates well with the finding that the mutant K135A is much less reactive than WT towards the reagent (IC50 = 40 µM). This confirms that the K135-NH2, adjacent to the C136-SH, lowers the C136-SH pK leading to the formation of

ACCEPTED MANUSCRIPT C136-S-, responsible of the disulfide formation. A mechanism could be proposed in which H2S first reacts with C155 forming -SSH. The free -SH of the close residue (C136) reacts

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with the -SSH forming the disulfide C136-S-S-C155, which is responsible of the

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inactivation of CAC [38]. In this reaction H2S is released. It is well known that, at longer

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times, H2S in solution can form polysulfides [40]. These molecular species have capacity to reduce protein disulfides [41-43] and, hence, to reduce C136-S-S-C155 adduct forming again free C136-SH and C155-SH. Such reaction well explains the reactivation of the

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inhibited protein observed at longer times (Figs. 3 A, B and 7). Indeed, formation of

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polysulfides follows a time scale similar [40] to that describing the inhibition/activation response of CAC. The initial state of the CAC also determines the effect of H2S (or of its products). It has been demonstrated in this work that molecular oxygen oxidizes CAC

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leading to inhibition of transport activity. Thus, while exposure of fully active CAC to H2S lead to inhibition, the oxygen-oxidized protein is activated by incubation with H2S. The

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different responses under different time, concentration and oxygen level is somewhat similar to the behavior observed by CAC in response to H2O2 [44], therefore confirming the

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role of the Cys couple as sensing molecular relay. The other carriers, such as the ANC and ORC, contain only one or no of the two residues and, if one is present it is not located in the critical position as in the CAC. Therefore, other carriers show low or no sensitivity to the gas. Concerning the possible physiological role of the modulation by H2S, this is linked to the unique property of the CAC of containing the Cys couple which sense redox changes. This carrier can be predicted to modulate mitochondrial bioenergetics in response to H2S and, possibly, to other gasses and/or reactive compounds, which will be dealt with in future work. In fact, CAC constitutes an essential step for the mitochondrial β-oxidation pathway as demonstrated by the severe life-threatening pathology caused by its defect [15]. The response to H2S is expected to finely tune CAC activity, and hence, mitochondrial

ACCEPTED MANUSCRIPT bioenergetics, which could vary on the basis of the actual energy state and oxidative stress. In this respect, inhibition of CAC by H2S may favor cardiovascular protection,

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playing a positive effect on ischemia/reperfusion, i.e., under condition of oxidative stress.

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Indeed, suppression of β-oxidation following CAC inhibition, will favor a switch of

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metabolism to glycolysis with consequent cardio-protection [6, 11, 12]. However, we have observed after spontaneous oxygen-dependent oxidation which causes CAC inhibition, that H2S reactivate the protein instead of inhibiting. Thus, H2S exerts a double effect

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depending on its concentration, incubation time and initial state of the protein, suggesting

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a more complex pathway of modulation (Fig. 3 A, B and 7). It can be hypothesized that when H2S is produced by activation of the enzyme pathway [9, 45], it acts on the mitochondrial site, i.e., CAC, decreasing its activity in case of lower oxygen concentration.

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In contrast to this, at higher oxygen level which causes CAC activity depression, H2S acts as a reducing agent recovering the carrier activity. Formation of polysulfides would also

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play important roles in these differential actions of H2S. To our knowledge, this is the first experimental evidence demonstrating capacity of H2S to specifically regulate the function

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of a mitochondrial carrier interacting with Cys residues. Interestingly CAC is the sole carrier harboring the Cys couple and hence responding to H2S with a high affinity. The Cys couple represents a molecular sensor of oxygen level and responds to the gas-transmitter as well as to other redox regulatory compounds such as GSH [18] or to toxic compounds such as Hg2+ and methylmercury [17]. Very interestingly this regulatory role is typical of higher organisms since the Cys couple is not present in lower organisms.

Aknowledgements This work was supported by funds from MIUR (Ministery of Instruction, University and Research): Programma Operativo Nazionale [01_00937] - "Modelli sperimentali

ACCEPTED MANUSCRIPT biotecnologici integrati per lo sviluppo e la selezione di molecole di interesse per la salute

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dell’uomo" to CI.

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Legends to Figures

Fig. 1. Effect of H2S on carnitine antiport activity in proteoliposomes. Transport activity of CAC was started adding 0.1 mM [3H]carnitine to proteoliposomes containing 15 mM

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carnitine and terminated at the indicated times as described in “Materials and Methods”.

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The uptake was measured in presence of 15 μM NaHS (●) or buffer (○).The values were

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the means ± SD from three experiments.

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Fig. 2. Influence of pH on the H2S inhibition on CAC. CAC, reconstituted in the liposomes at the indicated pH, was incubated for 30 min in presence of 30µM NaHS or buffer

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(control). After this incubation time proteoliposomes were passed through a Sephadex G75 column to eliminate unreacted NaHS. Transport activity was started by adding 0.1 mM

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[3 H]carnitine to proteoliposomes and stopped after 30 min as described in “Materials and Methods”. The percent of activity was calculated with respect to the control. The data represent means ± S.D. of three independent experiments. Fig. 3. Influence of time on the H2S inhibition on CAC. (A) CAC proteoliposomes or (B) isolated mitochondria were incubated with different NaHS concentrations (white 1µM, light gray 30µM and dark gray 100μM) for the indicated time. After each incubation time, proteoliposomes were passed through Sephadex G-75 column to eliminate unreacted NaHS. Transport activity was started by adding 0.1 mM [3 H]carnitine to proteoliposomes and stopped after 30 min as described in “Materials and Methods”. The values were the means ± SD from three experiments. *P < 0.05 **P < 0.01 versus control was estimated by Student's t-test.

ACCEPTED MANUSCRIPT Fig. 4. Dose−response curves for the inhibition of the WT by H2S at different times of incubation. Dose−response curves was measured after incubation with H2S at the

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indicated concentrations for 60 min (□), 30 min (●) or 15 min (○). Transport activity was

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measured by adding 0.1mM [3H]carnitine and stopped after 15 min as described in

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“Materials and Methods”. Percent residual activity with respect to the control was reported. The X axis has a semi-logarithmic scale. The values were the means ± SD from three

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experiments.

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Fig. 5. Influence of substrate and -SH reducing compounds on CAC inhibition by H2S. (A) Effect of DTE, GSH, and N-acetylcysteine on the CAC inhibition. Proteoliposomes were incubated in presence of 1µM NaHS; 1µM NaHS plus 5 mM DTE or 5 mM GSH, 5 mM

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NAC or 5 mM Cys. After 60 min of incubation unreacted compounds were removed by passing the proteoliposomes through Sephadex G-75 column and transport was started

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adding 0.1 mM [3H]carnitine. (B) Effect of substrate on CAC inhibition. Carnitine at the indicated concentrations was added to proteoliposomes and then the mixture was

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incubated for 60 min with H2S. After each incubation time, unreacted compound was removed by passing the proteoliposomes through Sephadex G-75 column. Transport was started adding 0.1 mM [3H]carnitine to the proteoliposomes and stopped after 30 min. Percent residual activity is reported with respect to the control without the addition of H2S. Reported values were the means ± SD from three different experiments. *P < 0.05 **P < 0.01 versus control was estimated by Student's t-test.

Figure 6. Effect of O2 and N2 on redox state of CAC. Native (black columns) and recombinant CAC (gray columns) were exposed to O2 or N2 and incubated with DTE where indicated. Transport was started adding 0.1 mM [3H]carnitine to the proteoliposomes and stopped after 30 min. Percent of residual activity is reported with

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time. The uptake was measured after exposure to O2 or N2.

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Reported values were means ± SD from three different experiment. *P < 0.05 **P < 0.01

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versus control was estimated by Student's t-test.

Figure 7. Effect of NaHS on oxidized CAC protein. In order to obtain the oxidized CAC,

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liposomes reconstituted with the native (dark gray columns) or the recombinant (light gray

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columns) protein were exposed to the oxygen for 6 h. After this time, proteoliposomes were incubated with NaHS at indicated concentrations for 1h.Transport was measured as described in “Materials and Methods”. Percent residual activity was reported with respect

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to the control without O2 exposure. Reported values were the means ± SD from three different experiments. *P < 0.05 **P < 0.01 versus control was estimated by Student's t-

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test.

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Figure 8. Dose−response curves for the inhibition by NaHS of different mutants of CAC. (●) C23S/C58S/C89S/C283S, (□) C23V/C58V/C89S/C136V/C283S, (■) C23V/C58V/C89S/C155V/C283S and (C23V/C58V/C89S/C136V/C155V/C283S (). After 60 min of incubation with NaHS at the indicated concentrations, transport activity was measured by adding 0.1 mM [3H]carnitine and stopped after 15 min as described in “Materials and Methods”. Percent of residual activity with respect to the control, without NaHS treatment, was reported. The X axis has a semi-logarithmic scale. The values were the means ± SD from three experiments.

Figure. 9. Time-dependence of inhibition of different CAC mutants. Proteoliposomes were incubated with 30µM NaHS for indicate times. After each incubation time,

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and stopped after 30 min as described in “Materials and Methods”. WT was depicted with

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white columns, C23S/C58S/C89S/C283S was depicted with light gray columns,

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C23V/C58V/C89S/C155V/C283S was depicted with dark gray columns and C23V/C58V/C89S/C136V/C283S was depicted with black columns. The values were the means ± SD from three experiments. *Significantly different from WT

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as determined from ANOVA one way test for p < 0.05.

Fig. 10. Structure comparison among members of the mitochondrial carriers family. (A), sequences were aligned using the Clustal W software. The amino acid position

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corresponding to Cys 136 and Cys 155 of CAC were highlighted gray and Cys were written in bold type. (B) ribbon diagram from a lateral view of the CAC structural model; (C)

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ribbon diagram of ANC structure [28]; (D) ribbon diagram of the ORC structural model. The reactive Cys were depicted in gray. The homology model has been represented using

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the molecular visualization program VMD.

Fig. 11. Alignment of proteins of the mitochondrial carnitine carrier subfamily. The amino acid sequence of the CAC proteins from the different species were aligned using the Clustal W software. The Cys corresponding to Cys136 and Cys 155 of rat CAC were highlighted gray and written in bold type.

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Figure 11

ACCEPTED MANUSCRIPT Highlights The carnitine/acylcarnitine carrier (CAC) possesses Cys residues sensitive to H2S. H2S lead to concentration and time dependent transport inhibition.

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After 60 min incubation of CAC with H2S the inhibition is reversed by the same reagent.

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The regulation effect of H2S occurs via reaction with C136 and C155 of CAC.