acylcarnitine carrier through reversible S-nitrosylation of cysteine 136

    Nitric Oxide Inhibits the Mitochondrial Carnitine/Acylcarnitine Carrier through Reversible S-Nitrosylation of Cysteine 136 Annamaria Tonazzi, Nicola Giangregorio, Lara Console, Annalisa De Palma, Cesare Indiveri PII: DOI: Reference:

S0005-2728(17)30058-0 doi:10.1016/j.bbabio.2017.04.002 BBABIO 47802

To appear in:

BBA - Bioenergetics

Received date: Revised date: Accepted date:

7 January 2017 29 March 2017 20 April 2017

Please cite this article as: Annamaria Tonazzi, Nicola Giangregorio, Lara Console, Annalisa De Palma, Cesare Indiveri, Nitric Oxide Inhibits the Mitochondrial Carnitine/Acylcarnitine Carrier through Reversible S-Nitrosylation of Cysteine 136, BBA Bioenergetics (2017), doi:10.1016/j.bbabio.2017.04.002

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ACCEPTED MANUSCRIPT Nitric Oxide Inhibits the Mitochondrial Carnitine/Acylcarnitine Carrier through Reversible S-Nitrosylation of Cysteine 136.

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Annamaria Tonazzi1a, Nicola Giangregorio1a, Lara Console2, Annalisa De Palma3, Cesare Indiveri1,2 1

CNR Institute of Biomembranes and Bioenergetics, via Amendola 165/A, 70126 Bari, Italy Department DiBEST (Biologia, Ecologia, Scienze della Terra) Unit of Biochemistry and Molecular Biotechnology, Via Bucci 4C, University of Calabria, 87036 Arcavacata di Rende, Italy 3 Department of Bioscience, Biotechnology and Biopharmaceutics, University of Bari, Italy a These authors contributed equally to this work.

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Correspondence to:

Cesare Indiveri, Department DiBEST (Biologia, Ecologia e Scienze della Terra) University of Calabria Via P. Bucci cubo 4C, 87036 Arcavacata di Rende (CS) Italy.

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Tel.:+39-0984-492939; Fax: +39-0984-492911. E-mail: [email protected].

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Key words: Membrane transport, liposome, ß-oxidation, carnitine, mitochondria.

Abbreviations used: DTE, dithioerythritol; NEM, N-ethylmaleimide; Pipes, 1,4-piperazinediethanesulfonic acid; CACT, carnitine/acylcarnitine translocase; WT, wild-type, VLCAD, long chain acyl CoA dehydrogenase; GSH, L-Glutathione reduced; GSNO, SNitrosoglutathione.

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ACCEPTED MANUSCRIPT Abstract S-nitrosylation of the mitochondrial Carnitine/Acylcarnitine transporter (CACT) has been

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investigated on the native and the recombinant proteins reconstituted in proteoliposomes, and on

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intact mitochondria. The widely-used NO-releasing compound, GSNO, strongly inhibited the

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antiport measured in proteoliposomes reconstituted with the native CACT from rat liver mitochondria or the recombinant rat CACT over-expressed in E. coli. Inhibition was reversed

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by the reducing agent dithioerithritol, indicating a reaction mechanism based on nitrosylation of Cys residues of the CACT. The half inhibition constant (IC50) was very similar for the native

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and recombinant proteins, i.e., 74 and 71 μM, respectively. The inhibition resulted to be competitive with respect the substrate, carnitine. NO competed also with NEM, correlating well

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with previous data showing interference of NEM with the substrate transport path. Using a site-

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directed mutagenesis approach on Cys residues of the recombinant CACT, the target of NO was identified. C136 plays a major role in the reaction mechanism. The occurrence of S-

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nitrosylation was demonstrated in intact mitochondria after tretment with GSNO, immunoprecipitation and immunostaining of CACT with a specific anti NO-Cys antibody. In

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parallel samples, transport activity of CACT measured in intact mitochondria, was strongly inhibited after GSNO treatment. The possible physiological and pathological implications of the post-translational modification of CACT are discussed.

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ACCEPTED MANUSCRIPT 1. Introduction. Nitric Oxide (NO) is one of the gaseous compounds which control the function of many proteins

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[1]. The action mechanisms of this signaling molecule occurs by post-translational modification

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(PTM), essentially by Cys S-nitrosylation or binding to heme groups. Therefore, proteins harboring

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reactive Cys residues are prone to this modification. A previously described proteomic analysis highlighted the presence of many S-nitrosylated mitochondrial proteins. In these analyses,

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membrane nitrosylated proteins are under-represented because of difficulty in extraction from membranes [2]. However, a nitrosylated peptide belonging to the CACT was previously

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described[2]. Indeed, the CACT is a potential candidate for nitrosylation, since it possesses six Cys residues, some of which are exposed towards the aqueous environment and are particularly prone to

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be targeted by reactive SH compounds [3-5]. This transporter is essential for completion of the

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mitochondrial fatty acid oxidation since it allows fatty acyl units to enter the mitochondrial matrix where the enzymes of the β-oxidation pathway are located. Demonstration of the essential role of

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this transporter is given by the occurrence of a severe rare inherited pathology, belonging to the secondary carnitine deficiencies, caused by defects of the SLC25A20 gene, coding for CACT [6].

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The transport process catalyzed by CACT occurs by an antiport reaction in which acyl moieties are translocated as acyl-carnitines in exchange with free carnitine. That the transporter could be a target of NO is suggested by the presence of Cys residues, namely C136 and C155, that are very sensitive to both physiological and chemical SH reactive compounds. Among the physiological ones, there are the GSH and the gaseous compound H2S, which have modulatory effects on the transport function of CACT [3, 4]. Chemical SH reagents, such as N-ethylmaleimide (NEM), were previously found to react with one or both the Cys residues leading to inactivation of transport. Moreover, the redox state of the two Cys, C136 and C155, regulates the transporter that switches from an active to an inactive form, upon conversion of the Cys residues from a thiol, i.e., reduced, to a disulfide, i.e., oxidized state [3, 5, 7, 8]. In the frame of these regulatory aspects, this 3

ACCEPTED MANUSCRIPT transporter has been described as a potential modulator of the mitochondrial β-oxidation in studies performed with intact mitochondria as well as with the native and recombinant transporters [4]. In

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the present work, the action of NO on the transporter has been characterized at the molecular level.

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By using site-directed mutagenesis, the Cys residue responsible for S-nitrosylation and, hence, for

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the functional effect has been identified.

1. Materials and methods.

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1.1. Materials

l-[methyl-3H]carnitine from Scopus Research BV Costerweg , Sephadex G-75, egg-yolk

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phospholipids (l- α-phosphatidylcholine from fresh turkey egg yolk), Pipes, Triton X-100, cardiolipin, l-carnitine, antibodies anti-NO-cysteine (N5411) were purchased from Sigma-Aldrich,

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Milano Italy, protein G agarose beads was purchase from Invitrogen Carlsbad, CA 92008 USA. All

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other reagents were of analytical grade.

2.2 Overexpression of the CACT WT and mutant proteins.

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The previously constructed pMW7-WTratCACT recombinant plasmid was used for producing the rat CAC. This carrier exists as a single isoform that is expressed in virtually all rat tissues [6]. To introduce the mutations in the CACT protein as previously described [9]. The amino acid replacements were performed with complementary mutagenic primers using the overlap extension method [10] and the High Fidelity PCR System (Roche). The PCR products were purified by the QIAEX II Gel Extraction Kit (QIAGEN), digested with NdeI and HindIII (restriction sites added at the 5' end of forward and reverse primers, respectively) and ligated into the pMW7 expression vector. All mutations had been verified by DNA sequencing, and, except for the desired base changes, all the sequences were identical to that of rat CACT cDNA [11]. The resulting plasmids were transformed into E. coli C0214. Bacterial overexpression, isolation of the inclusion body

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ACCEPTED MANUSCRIPT fraction, solubilization and purification of the wild-type CACT and mutant CACT proteins were performed as described previously [9, 12].

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2.3 Reconstitution of CACT in liposomes.

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The recombinant protein, after purification to homogeneity, was reconstituted into liposomes as described previously [9, 12]. In the case of the native protein, rat liver mitochondria were first purified using a standard procedure consisting in cell destruction and differential centrifugation

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[13]. Then, the native protein was extracted from rat liver mitochondria by solubilisation with 3% Triton X-100 and reconstituted as previously described [4], in absence of reducing agents. In all the

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preparations, the concentration of intraliposomal carnitine was 15 mM.

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2.4 Transport measurements in proteoliposomes or intact mitochondria.

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For transport assay in proteoliposomes, the external substrate was removed from reconstituted proteoliposomes (see section 2.3) by passing 550μl of proteoliposomes through a Sephadex G-75

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column. The first 600 μl of the turbid eluate from the Sephadex column were collected, transferred to reaction vessels (100 μl each), and readily used for transport measurement by the inhibitor-stop

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method [14].

For uptake measurements, transport at 25°C was started by adding 0.1 mM [3H]carnitine to proteoliposomes and at the required time interval the reaction was terminated by the addition of 1.5 mM NEM. In controls, the inhibitor was added together with the labelled substrate at time zero. Finally, the external substrate was removed by chromatography on Sephadex G-75 columns, and the radioactivity in the liposomes was measured [14]. The experimental values were corrected by subtracting control values. For efflux measurements, the proteoliposomes were loaded with radioactivity before starting the transport assay. This was achieved by incubating the proteoliposomes (600 ml), passed through Sephadex G-75 with 5 M [3H] carnitine with a specific radioactivity of 10 nCi/nmol, for 60 min at 25 °C. Then, the external radioactivity was removed by 5

ACCEPTED MANUSCRIPT passing again the proteoliposomes through Sephadex G-75 as described above except that this chromatography was performed at 4°C to minimize the loss of internal substrate during the

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chromatography. Transport (efflux) was started by adding external buffer or substrate and stopped

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at the appropriate time interval. For rate measurements, transport was stopped at 5 min, i.e., within

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the initial linear range of the time course. The transport assay temperature was 25 C°. Finally, each sample of proteoliposomes (100 l) was passed through a Sephadex G-50 column to separate the external from the internal radioactivity. Efflux activity was expressed as percentage of

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intraliposomal radioactivity compared to control at time zero.

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Transport was measured in intact mitochondria as previously described [4], after loading intact mitochondrial with 20 mM carnitine for 30 min, in order to measure antiport function.

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Specific transport activities were expressed as nmol/g protein in intact mitochondria, as µmol/g

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protein in the case of the native protein extracted from rat live mitochondria and in mmol/g proteins

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in the case of the purified recombinant protein. Differences of orders of magnitudes in specific activities among the different protein sources were due to the great differences in total protein among intact mitochondria, mitochondrial extract and the homogeneously purified recombinant

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protein. In particular, the relative amount of CACT with respect to total mitochondrial proteins (from rat liver) is about 0.1 % [15]. This is in good agreement with the differences in specific transport activities between the mitochondrial extract and the purified recombinant protein (see as example Fig. 1A and 1B). 2.5 Immunoprecipitation with anti- CACT antibody. Five μg of in house produced anti-CACT antibody [16, 17] in PBS buffer were incubated 2 hours with protein G agarose beads (4°C). Then, antibodies (conjugated) were incubated with extract of mitochondria obtained by solubilizing 1 mg protein with 1.5 % Triton X-100 in PBS, centrifuging at 16000 g for 15 min and collecting the supernatant. After overnight incubation with the 6

ACCEPTED MANUSCRIPT conjugated antibody, beads were washed with PBS, collected by centrifugation and resuspended in 3X Laemmli buffer (with or without DTE). After centrifugation, proteins from the supernatant were

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used for western blot analysis.

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2.6 SDS-PAGE and western blot analysis

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SDS-PAGE electrophoresis was performed in the presence of 0.1 SDS according to Laemmli [18]. Minigel system, sizes 8 cm x 10 cm x 0.75 mm, was used. Stacking gel and separation gel 5% and

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17.5% acrylamide respectively (acrylamide/bisacrylamide ratio 30 : 0.2). Proteins were transferred to nitrocellulose membrane (Schleicher and Schuell, PROTRAN BA 85

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cellulosenitrat). Residual binding sites on the membrane were blocked by incubation with 3% BSA in buffer composed of NaCl 150mM; 50mM Tris-HCl, Tween20 0.05 % pH 7, for 10 min and then

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incubated with a rabbit polyclonal anti-CACT (dilution 1:1.000) or anti-NOcysteine (dilution

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1:1.000) in 0.5% BSA solution for 1 h at room temperature (RT). The anti-CACT antibody was produced in our laboratory injecting the purified recombinant over expressed CACT in rabbit and

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1. Results

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resulted specific for the CACT of rat and human [16, 18].

3.1 Reversible inhibition of carnitine transport by NO [3H]carnitine/carnitine antiport catalyzed by rat liver CACT reconstituted in proteoliposomes, was strongly inhibited by GSNO, the most widely used NO-releasing compound [2, 19, 20]. Inhibition was mostly reversed by the reducing agent DTE, added after incubation of proteoliposomes with GSNO. Treatment with DTE caused a slight and not statistically significant increase of transport also in control proteoliposomes (not treated with GSNO). This is due to the reducing action of DTE and hence re-activation of a partially oxidized (disulfide containing) protein fraction, as previously reported [21]. The GSNO inhibitory effect reached its maximal efficiency after about 2 min incubation (inset to Fig. 1 A), indicating a relatively slow reaction rate. The effect of 0.2 mM GSH, 7

ACCEPTED MANUSCRIPT which remains as free molecule after releasing of NO, was tested on the [3H]carnitine/carnitine antiport, as control. Under the same conditions used for GSNO, nearly no effect was observed (not

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shown), according to previous data [3]. A similar experiment was performed with the recombinant

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WT rat CACT reconstituted in proteoliposomes (Fig. 1 B). Overlapping results were found. Indeed,

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GSNO strongly inhibited the [3H]carnitine/carnitine antiport; addition of DTE reversed this effect in a time dependent mode. After 20 min about 80% activity was recovered. A complete recovery was expected at longer times as previously found [21]. Therefore, the recombinant protein could be used

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in further experiments for defining molecular determinants of the NO effect. It was previously shown that the CACT catalyzes, besides an antiport reaction, also a slower uniport

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that can be better resolved following [3H]carnitine efflux from proteoliposomes instead of uptake. It has to be stressed that the unidirectional transport is symmetrical, i.e., it can be inwardly or

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outwardly directed, as well [14, 22, 23]. Figure 2 describes the effect of the GSNO on the [3H]carnitine efflux from preloaded proteoliposomes in the absence of external substrate (carnitine

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unidirectional transport). As shown by the figure, the unidirectional transport was virtually abolished by the addition of GSNO. As control the inhibition was also tested on the antiport

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reaction measured as efflux of [3H]carnitine. As expected the antiport was much faster than the uniport [22]. Addition of GSNO led to strong inhibition of the antiport according to the same data obtained measuring uptake of [3H]carnitine (see Fig. 1). Percentages of uniport and antiport inhibition at 20 min were very similar, i.e, 68 % and 69 %, respectively, as expected from previous results on inhibition by NEM [22].

3.2 Mechanism of action of NO The dependence of the inhibition on the concentration of GSNO was studied on both the native and the recombinant proteins. The antiport rate, measured as uptake of 0.1 mM [3H]carnitine into proteoliposomes containing 15 mM carnitine, was assayed in the presence of increasing concentration of GSNO. The dose response curves obtained are shown in Fig. 3 A. Virtually 8

ACCEPTED MANUSCRIPT complete inhibition of the transport was observed, with both the proteins, at concentrations of GSNO of about 1 mM. The calculated IC50 from 3 independent experiments was respectively 74.0 ±

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3.0 µM for the native protein or 71.0 ± 4.9 for the recombinant CACT. Inhibition kinetics were

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further performed on GSNO to obtain data on the localization of the NO binding site with respect to

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the substrate binding site. The data obtained on the WT recombinant CACT, analyzed in double reciprocal (Lineweaver-Burk) plot are shown in Fig. 3 B. The experimental data were interpolated by straight lines, which showed a common intersection point on the ordinate axis. This behavior is

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typical of competitive inhibition. The half saturation constant (Ki) for the inhibitor, derived from

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the plot, was 180 ± 25 µM (from three experiments).

It was known from previous studies [8, 9, 24] that NEM strongly inhibits the CACT function by binding to SH residues of the protein. Taking advantage by the difference in reactivity of NEM

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(irreversible) and NO (reversible), we have investigated whether the reagents binds to the same SH group(s) of NEM on the native CACT protein. As shown in Fig. 4, proteoliposomes incubated with

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GSNO showed strong inhibition of carnitine transport which was mostly recovered after addition of DTE, according to the data above described. NEM also strongly inhibited transport, but DTE, in

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this case, was not able to recover the transport function due to the irreversible NEM-protein bond, according to previous data [8, 9, 24]. If the reconstituted CACT was preincubated with GSNO before NEM, transport was still strongly inhibited, even though at a slightly lower but significant extent with respect to NEM alone. However, after addition of DTE, the activity was recovered indicating that GSNO protected the inhibition by NEM, i.e., the released NO interacted with the same SH group(s) of NEM. This was also corroborated by comparing previous results on NEM inhibition [9] with the results described in this work (see below). As control, the same experiment was performed by adding NEM before GSNO. In this case, inhibition could not be reversed by DTE, due to the irreversible NEM-protein bond.

3.3 Identification of Cys residues responsible for nitrosylation. 9

ACCEPTED MANUSCRIPT To identify the Cys residues involved in the nitrosylation of the CACT, experiments were performed with Cys mutants of the CACT lacking one or more Cys residues (Fig. 5). It has to be

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evidenced that substitution of more than four Cys residues with Ser led to inactive proteins [9, 21].

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Thus, multiple replacement mutants carry some substitutions of Cys to Val instead of Ser. Most of

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the single Cys mutants, i.e, C23S, C58S, C89S and C283S, did not show any variation of inhibition by GSNO with respect to the WT (not shown). While, it is evident that the mutant C136S mostly lost sensitivity to NO with a residual activity higher than 75% which corresponds to an inhibition

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lower than 25 %. A very small, apparently not significant, loss of inhibition was also observed in

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the mutant C155S. On the other hand, the mutant carrying the double mutation, C136/155S was completely insensitive to GSNO treatment confirming the major role of C136 and a possibly marginal role of C155, which may have a much lower affinity for NO. This correlates well with

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virtual absence of S-nitrosylation of the C136/155S after treatment with GSNO (Supplementary Fig. 1). The mutant containing only C136, i.e., C23V/C58V/C89S/C155V/C283S showed a slightly

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lower inhibition with respect to the WT; the difference with respect to the WT was statistically not significant. While the mutant containing only C155, i.e., C23V/C58V/C89S/C136V/C283S, lost the

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sensitivity to GSNO as the C136S single mutant. The mutant containing both C136 and C155 but not the other Cys residues (C23S/C58S/C89S/C283S) was inhibited by GSNO at the same level of WT, confirming the major role of C136 and a possible marginal role of C155. As expected and according to all previous data, data, the C-less was insensitive to the reagent. Interestingly, C136 is conserved in several members of the CACT subfamily which correspond to higher animals or plants (Fig. 6). While it is not conserved within the entire mitochondrial carrier family of rat members, except that in the ornithine transporter (SLC25A15) and its isoform (SLC25A2) (Supplemetary Fig. 2). However, the ornithine transporter is not or very poorly inhibited by NO (Supplemetary Fig. 3) indicating that the regulation by this post-translational modification is specific for CACT.

3.4 CACT nitrosylation in intact mitochondria. 10

ACCEPTED MANUSCRIPT The existence of a nitrosylated form of CACT upon reaction of mitochondria with NO was tested by immunoprecipitating the CACT after treatment of intact mitochondria with GSNO. Fig. 7A

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shows the results of western blot reaction performed on CACT from rat liver mitochondria

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immunoprecipitated with a specific CACT antibody and immunoassayed with the anti-NO

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antibody. Upon treatment of mitochondria with the NO releasing compound a clear anti-NO immunostaining of the immunoprecipitated CACT was observed which was absent in the untreated control, i.e., CACT immunoprecipitated from rat liver mitochondrial but not treated with GSNO.

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Interestingly, after incubation of the protein with DTE the anti-NO reaction disappeared being

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similar to the untreated sample. These results correlated well with the data on transport assay (Fig. 1). All samples were stained with Ponceau red before incubation with the antibody (lower panel), confirming the presence of the protein. In parallel samples, the transport activity in GSNO treated

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or not treated mitochondria was measured as [3H]carnitine uptake (Fig. 7 B). The carnitine transport activity after GSNO treatment was strongly inhibited with respect to the untreated control. This

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2. Discussion

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correlates well with data in proteoliposomes.

The described inhibition by the NO releasing compound GSNO suggests that S-nitrosylation of the CACT may have a role in the regulation of the β-oxidation pathway, in which the transporter is a central player. The response of CACT to S-nitrosylation is similar to that previously described for the Complex I of the mitochondrial respiratory chain (NADH dehydrogenase) [25, 26]. Thanks to availability of the recombinant CACT which shows the same properties of the native transporter, including its sensitivity to NO, the molecular basis of the NO regulation could be defined. The major target of NO is C136, while C155 may contribute only at a much lower extent to the action of NO. Different mass spectrometry analyses previously reported nitrosylated C136 in peptides of mouse CACT 11

ACCEPTED MANUSCRIPT which shares 94% sequence identity with the rat protein (see supplementary Fig. 4) [2, 27], corroborates our finding. As shown in section 3, this Cys residue is strictly conserved in CACT of

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higher animals and plants (Fig 6). The target of NO is similar to that of GSH, which, however,

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activates the CACT by a mechanism which is completely different from that of NO interaction.

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GSH reacts with the oxidized inactive form of the protein in which C136 and C155 are linked by a disulfide. GSH first gluthathionylates C136, then leading to Cys reduction with rescue of transport activity [3]. In the case of H2S, the major target is different from that of NO. Indeed, C155 was the

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major residue for reaction of H2S; the mechanism and the effect of the reaction was, also in this

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case, quite different from that of NO [4]. The effect of NO strictly depends on the presence of C136 and its reaction with CACT results in inhibition of transport. This is related to the mechanism of inhibition based on steric hindrance. A further support to this finding is that the effect of NO is very

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similar to that of NEM, which was previously demonstrated to have an obstructive action on the transport pathway by interacting with C136. Indeed, NO protects the inhibition by NEM confirming

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that the same Cys residue represents the target (see Fig. 4). Moreover, the competitive inhibition observed for NO correlates well with the inhibition mechanism of both the compounds [9, 23].

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When the concentration of carnitine rises, inhibition by NO is prevented since binding of NO is slower than binding of carnitine (see also inset to Fig. 1 A). Reversible binding of NO to CACT was revealed by western blot (see Fig. 7A). It is evident in this figure that the apparent molecular mass of the CACT is lower when SDS-PAGE is performed in the absence of DTE, i.e. under nonreducing conditions. This is due to formation of disulphide(s) in the denatured protein, that cause a shift towards lower apparent molecular mass. This phenomenon is prevented by the presence of DTE, correlating well with previous observations [7, 24]. Concerning the possible role of nitrosylation of CACT, some considerations have to be made. Regulation of CACT differs from that of VLCAD whose nitrosylation caused a decrease of the enzyme Km for acyl-CoA substrates thus increasing the catalytic efficiency with respect to the non-nitrosylated enzyme [2]. However, non-nitrosylated form of VLCAD was only described in ob/ob mouse or in mouse carrying a 12

ACCEPTED MANUSCRIPT VLCAD mutation. Thus, it seems that VLCAD is constitutively nitrosylated in WT animals. Later on, administration of GSNO was proposed for correcting VLCAD deficiencies [28]. Very

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interestingly the VLCAD residue identified as target of NO, i.e., C238, has a vicinal K (K236) [2]

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as the C136 of the CACT which has the vicinal K135. Indeed, it is known that vicinal K residues

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decrease the pKa of Cys-SH facilitating its reaction with reactive nitrogen species. Differently from VLCAD, CACT seems not nitrosylated under physiological conditions (see Fig. 7). Under the experimental conditions used in this work, i.e., absence of other reactants (ROS) or enzymes,

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nitrosylation likely occurs by direct reaction of released NO with a Cys residue, as it was previously

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reported for Complex I [26]. However, it cannot be excluded that in vivo, also other mechanisms may concur to nitrosylation of CACT. As it was previously well described for Complex I, the inhibition of CACT is observed at GSNO concentrations higher than those corresponding to the

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basal levels of NO, that are lower than 1 µM in liver and in some other tissues, even though methodological difficulties do not guarantee exact and reproducible measurements [29, 30].

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Moreover, the actual concentration of NO released from GSNO after 2-10 minutes is about 2 orders of magnitude lower than the concentration of GSNO itself [31]. Accordingly, the actual Ki of

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CACT for NO should be about two orders of magnitude lower than that measured for GSNO (180 µM) thus fitting with a level little higher than the basal concentration of NO in liver [30]. Therefore, it is very likely that, similarly to Complex I [26, 32], CACT will be inhibited in vivo when NO level rises above the basal threshold. On this basis, S-nitrosylation of CACT may have importance under specific conditions in which mitochondrial oxidation of fatty acids must be slowed down to avoid CoA trapping by acetyl-CoA. Such conditions may occur under impairment of respiratory chain activity which can be caused, among other conditions, by increased intramitochondrial NO level and inhibition of Complex I [25, 33]. Interestingly, inhibition of CACT by NO exhibits similar properties of Complex I, i.e., sensitivity to relatively high concentration of NO and slow reaction rate; CACT seems more sensitive to reaction with NO since maximal inhibition is reached at incubation time and GSNO concentration slightly lower than those required by Complex I [34]. 13

ACCEPTED MANUSCRIPT Inhibition of CACT by NO may play the important role of controlling the fatty acyl flux into βoxidation during altered mitochondrial metabolism such as ischemia and reperfusion. Similarly, to

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the well-assessed role of nitrosylation in Complex I, inhibition of CACT may act in preventing

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accumulation of reducing equivalents and decreasing ROS formation following re-oxygenation [26,

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32]. As a further support to the physiological relevance of the CACT nitrosylation, it has to be stressed that endothelial nitric oxide synthase (eNOS) and especially inducible NOS (iNOS) have been described to associate with mitochondria, thus producing NO close to the mitochondrial outer

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membrane which can rapidly diffuse in the intermembrane space promptly reacting with C136 of

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CACT, which is located at the bottom of the substrate binding cavity of the protein, but faces the aqueous environment which is in communication with the intermembrane space (Fig. 8 and refs. [12, 35]). The intermembrane environment is favourable to the reaction of NO with the Cys residue

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since the ratio GSH/GSSG is lower than in the matrix and cytosol [36, 37]. In addition, during oxidative stress as in ischemia/reperfusion, the ratio GSH/GSSG decreases in mitochondria [38].

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This correlates well with more favourable nitrosylation conditions for CACT in a pathological context. It has also been described that NO can reach local higher levels than the average

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concentration in tissues [29] and that association of iNOS with the mitochondrial outer membrane increases under pathological conditions, such as sepsis and inflammation [39], correlating well with the proposed role of CACT S-nitrosylation. Moreover increase of NO is reported in diabetics rats [40]. Indeed, reducing intramitochondrial acetyl-CoA accumulation may have a beneficial effect in type 2 diabetes [41]. Very interestingly, mitochondrial Carnitine Acetyl transferase (CrAT), which shares some substrates (acetyl-carnitine and carnitine) with CACT, shows relationships with NO signalling. On the one hand, silencing this enzyme resulted in decreased NO signalling [42]; on the other hand, nitration of CrAT decreases its activity [43]. Nitrosylated peptides of CPT1 and CPT2, which are also involved in carnitine system, have been found in proteomic analysis [2], indicating that these enzymes might be target of nitrosylation. However, no data on activity modulation have been reported, so far. It has to be stressed that CPT2 is associated to CACT [16], thus nitrosylation 14

ACCEPTED MANUSCRIPT might be a coordinated mechanisms of modulation of the transporter/enzyme complex. Another enzyme complex of fatty acid metabolism, the fatty acid synthase, is activated by nitrosylation. This

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correlated well with the inhibition of CACT. Thus, the two opposite metabolic pathways are

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regulated in a concerted manner [44]. The lower inhibition observed in intact mitochondria with

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respect to the reconstituted protein may be due to the presence of redox buffering systems present in the intermembrane space which partially remove the released NO before its reaction with the CACT C136. Under pathological conditions, as for example ischemia/reperfusion, the mitochondrial redox

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potential switch towards more oxidized conditions, thus favouring nitrosylation (see above and ref.

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38). Our present findings agree well with a stepwise process previously described, leading from the physiological inhibition of Complex IV at low NO concentrations, to the pathophysiological inhibition of Complex I at higher NO concentrations [26]. Inhibition of CACT would have a

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synergistic effect with Complex I, thus being useful in the cytoprotection exerted by inhibition of the enzyme [33, 45]. In addition, the unidirectional function of CACT, that plays the role of

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providing mitochondria with cytosolically synthesized carnitine [22, 46], is also strongly inhibited by NO. Inhibition may have the effect of lowering the intramitochondrial carnitine concentration

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and this, in turn, may contribute in reducing acyl-carnitine flux into mitochondria whose transport rate depends on the intramitochondrial concentration of free carnitine [47]. In the light of the results here presented, the proposed use of GSNO for correcting VLCAD defects [28] has to be reconsidered. The inhibition of CACT causing impairment of β-oxidation may also contribute to a metabolic switch towards glycolytic metabolism [48, 49] that has an important role under ischemic conditions [50]. This last consideration is important for further applications of the reported results. The mechanism of inhibition of CACT by NO releasing compounds may have importance in driving design of novel drugs which my facilitate the above cited metabolic switch by inhibiting the CACT. Such perspectives, among other reasons, support the great interest recently emerged, in searching for inhibitors of the CACT [51]. In conclusion, NO represents the physiological inhibitor acting via nitrosylation of C136, which was previously identified as a crucial residue of CACT by 15

ACCEPTED MANUSCRIPT studies performed with chemical compounds such as NEM. Funding

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This work was supported by funds from: Programma Operativo Nazionale [01_00937] - "Modelli sperimentali biotecnologici integrati per lo sviluppo e la selezione di molecole di interesse per la

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salute dell’uomo", MIUR (Italian Ministery of Instruction, University and Research).

Legend to Figures

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Fig. 1. Effect of GSNO on the carnitine antiport mediated by CACT. (A) Native rat CACT and (B) recombinant WT rat CACT reconstituted in proteoliposomes. Transport was measured adding

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0.1 mM [3H]carnitine at time zero, and terminated at the indicated times as described in section 2.3.

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0.2 mM GSNO (●) or external buffer (■) were added to the proteoliposomes 2 min before the start.

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2 mM DTE was added at 10 min after the start of the transport assay to the proteliposomes treated with GSNO (○) or to the control (□). Data are means ± SD from three independent experiments. The inset describes the time-dependence of inhibition of transport, measured as described in section

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2.3. Inhibition was assayed after incubation with 0.2 mM GSNO of proteoliposome aliquots at the indicated times as residual transport activity, with respect to control sample, i.e., without GSNO incubation. Fig. 2. Effect of GSNO on the carnitine efflux from proteoliposomes. Efflux of 15 mM [3H]carnitine from pre-labeled proteoliposomes was measured as described in section 2.5 in the presence of external 10 mM Pipes pH 7.0 (○), 10 mM Pipes pH 7.0 plus 0.2 mM GSNO (●), 0.5 mM carnitine (□) or 0.5 mM carnitine plus 0.2 mM GSNO (■) added 2 min before starting the transport reaction. The values are means ± SD from three independent experiments. Fig. 3. Dependence of inhibition on GSNO concentration. A) Dose-response curves for the 16

ACCEPTED MANUSCRIPT inhibition of the reconstituted CACT. The carnitine/carnitine antiport rate was measured as described in section 2.5, adding 0.1 mM [3H]carnitine to proteoliposomes containing 15 mM

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internal carnitine, reconstituted with rat recombinant WT (○) or native (●) CACT. GSNO, at the

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indicated concentrations, was added 2 min before the transport assay. Percent residual activities

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with respect to the control are reported. Transport activity of the controls were 0.36 ± 0.016 mmol/g protein/min or 0.64 ± 0.086 µmol/g protein/min for the recombinant or the native rat liver protein, respectively. The values are means ± S.D. from three independent experiments. B) Kinetic analysis

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of the effect of GSNO on the reconstituted CACT. The carnitine/carnitine antiport rate was

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measured, as described in section 2.3, adding [3H]carnitine at the indicated concentrations to proteoliposomes reconstituted with WT CACT and containing 15 mM internal carnitine, in the absence (○) or in the presence of 0.2 mM GSNO added together with the labeled substrate (●).

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Experimental data are plotted according to Lineweaver-Burk as reciprocal transport rate vs reciprocal carnitine concentrations. Reported values are means ± S.D. from three independent

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experiments.

Fig. 4. Interference between GSNO and NEM on the binding to CACT. Proteoliposomes,

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reconstituted with native CACT, were incubated for 2 min with each of the reagents: 0.2 mM GSNO (NO), 0.2 mM NEM and 2 mM DTE subsequently added as indicated in the figure. Carnitine/carnitine antiport was then measured in all the samples by adding 0.1 mM [3H]carnitine as described in section 2.3. Percent residual activity was calculated for each experiment with respect to its control sample (referred as 100%). The average specific activity of the control samples of the three experiments analyzed was 4.7 ± 0.69 µmol/10 min/g protein. The values are means ± S.D. from three independent experiments and are all significantly different from the control (100%), as estimated by the Student’s t test (> p, 0.05), calculated on the basis of comparison among control values (100 %) and the independent assays of inhibited samples. Fig. 5. Inhibition of WT and Cys mutants of CACT. Proteoliposomes, reconstituted with 17

ACCEPTED MANUSCRIPT recombinant WT CACT or CACT mutants. Transport was measured by adding 0.1 mM [3H]carnitine to proteoliposomes and stopped after 30 min. 0.2 mM GSNO was added to the

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proteoliposomes reconstituted with the various mutants and incubated for 2 min. Percent residual

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activity with respect to the control (absence of GSNO) is reported. In the legend, the Cys residues

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substituted in the mutant proteins are indicated. The values are means ± S.D. from three independent experiments. Significantly different from inhibited WT CACT, as estimated by the Student’s t test (*p, 0.05; **p, 0.01). Relative transport activities for each mutant with respect to the

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WT (3.7 ± 0.29 mmol/g protein/30 min) were: C136S 100%; C155S 58%; C136S/C155S, 63%;

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C23S/C58S/C89S/C283S, 44%; C23V/C58V/C89S/C155V/C283S 86%; C23V/C58V/C89S/C136V/C283S 94%; C23V/C58V/C89S/C136V/C155V/C283S (C-less) 20%.

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Fig. 6. Sequence alignment of the mitochondrial carnitine carrier subfamily. The amino acid

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sequence of the CAC proteins from the different species were aligned using the Clustal W software. The sequences of the third transmembrane α-helix (H3) and the first part of the connecting

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hydrophilic segment, containing C136 of the rat protein, are reported. The amino acids corresponding to Cys 136 of rat are highlighted in gray; conserved Cys are in bold.

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Fig. 7. Nitrosylation of the CACT in intact mitochondria. (A): upper panel, western blot analysis of CACT immunoprecipitated with a very specific anti-CACT antibody [16] and immunostained with an antibody that recognizes nitrosylated Cys residues (anti-NO-Cys); lower panel, same nitrocellulose membrane stained by ponceau red. Intact mitochondria were treated with 0.2 mM GSNO and/or 2 mM DTE as indicated in the figure by (+). Similar data were obtained in three experiments. (B) Pools of mitochondria treated or not (control) with GSNO were used for transport assay, in 20 min, as described in section 2.3. The values are means ± S.D. from three independent experiments. Fig. 8. Position of C136 in the structural model of CACT. Ribbon diagram of the CACT structural model from (A) lateral or (B) top view, that is, from intermembrane space; (C) space 18

ACCEPTED MANUSCRIPT filled diagram of the top view. The homology model was constructed as previously described [15]. The protein is constituted by six α-helices crossing the mitochondrial inner-membrane (H1-H6, in

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grey), connected by more hydrophilic segments. The structure arbours a central water filled cavity

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where the substrate binds. C136, highlighted in red, is located in the bottom of the cavity.

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Highlights  The mitochondrial Carnitine/Acylcarnitine Carrier was strongly inhibited by GSNO.  The native and the recombinant carriers exhibited similar responses to GSNO.  The inhibition was caused by reversible S-nitrosylation of C136 of the transporter.  S-nitrosylation of the carrier was revealed after GSNO treatment of intact mitochondria.

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