Acylcarnitine Transporter, a Novel Target of

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The mitochondrial carnitine/acylcarnitine transporter, a novel target of mercury toxicity Annamaria Tonazzi, Nicola Giangregorio, Lara Console, Mariafrancesca Scalise, Daniele La Russa, Caterina Notaristefano, Elvira Brunelli, Donatella Barca, and Cesare Indiveri Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.5b00050 • Publication Date (Web): 07 Apr 2015 Downloaded from http://pubs.acs.org on April 11, 2015

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Chemical Research in Toxicology

The mitochondrial carnitine/acylcarnitine transporter, a novel target of mercury toxicity Annamaria Tonazzi†‡ǁ, Nicola Giangregorio†‡ǁ, Lara Console§, Mariafrancesca Scalise§, Daniele La Russa§, Caterina Notaristefano‡, Elvira Brunelli§, Donatella Barca§, Cesare Indiveri†§* †

CNR Institute of Biomembranes and Bioenergetics, via Amendola 165/A, 70126 Bari, Italy Department of Bioscience, Biotechnology and Biopharmaceutics, University of 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 ǁ These authors contributed equally to this work. ‡

Keywords: mercury; methylmercury; mitochondria; glutathione; zebrafish; carnitine

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TOC


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Abstract

The effect of Hg2+ and CH3Hg+ on the mitochondrial carnitine/acylcarnitine transporter (CACT) has been studied on the recombinant protein and on the CACT extracted from HeLa cells or Zebrafish and reconstituted in proteoliposomes. Transport was abolished upon treatment of the recombinant CACT in proteoliposomes by Hg2+ or CH3Hg+. Inhibition was reversed by the SH reducing agent 1,4-Dithioerythritol, GSH and N-acetylcysteine. IC50 for Hg2+and CH3Hg+ of 90 nM and 137 nM, respectively, were measured by dose-response analyses. Inhibition was abolished in the C-less CACT mutant. Strong reduction of inhibition by both reagents was observed in the C136A and some reduction in the C155A mutants. Inhibition similar to the WT was observed in the C23V/C58V/C89S /C155V/C283S mutant, containing only C136. Optimal inhibition by Hg2+was found in the four replacement mutant C23V/C58V/C89S/C283S containing both C136 and C155 indicating cross-reaction of Hg2+with the two Cys residues. Inhibition kinetic analysis showed mixed inhibition by Hg2+ or competitive inhibition by CH3Hg+. HeLa cells or Zebrafish were treated with the more potent inhibitor. 10 µM HgCl2 caused clear impairment of viability of HeLa cells. Transport assay in proteoliposomes with CACT extracted from treated cells showed that the transporter was inactivated and DTE rescued the activity. Nearly identical results were observed with Zebrafish upon extraction of the CACT from liver of the treated animals that, indeed, showed accumulation of the mercurial compound.

Introduction

Mercury compounds are present in the environment as products of industry, cigarette smoke and contaminated sea foods .1, 2 The most common forms of these toxicants are Hg2+ as inorganic compound and CH3Hg+ (methyl mercury) as the main organic compound.3 It is well known that these compounds interact with proteins forming covalent bonds with SH groups of Cys residues .4-6 The great attention in identifying new cellular targets of mercury, is due to its ability to induce oxidative damage, inflammation and neurodegeneration.1, 2, 7 Interestingly, some data about mercury 3 ACS Paragon Plus Environment


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toxicity on mitochondrial metabolism are available. Initial studies evidenced that CH3Hg+ affected phosphorylation, transport and oxidative processes.8 More recent studies described alterations of the mitochondrial energetics induced by mercury. 9, 10 The most acknowledged mitochondrial target for mercury are thioredoxin and thioredoxin reductase both in vitro and in vivo causing cell growth impairment. The IC50 of HgCl2 and CH3Hg+ for these mitochondrial targets are 22 µM and 2 µM, respectively.11 The action of mercury on mitochondrial metabolism was also associated to occurrence of autism, even though this issue is still controversial due to lack of an unequivocal link with the pathology.12 The mechanism of impairment of mitochondrial function by mercury is thus, still underneath. Therefore, it was interesting to investigate other mitochondrial targets of organic and inorganic mercury. Owing to the reaction mechanism of mercury compounds, protein targets should possess reactive SH groups of Cys. Among many mitochondrial proteins containing Cys residues the mitochondrial carnitine/acylcarnitine transporter (CACT) exhibits extremely reactive Cys residues which have been well characterized for their role in transport function and regulation, 13

according to pioneering work on inhibition by mercurials of carnitine transport in intact

mitochondria . 14 This protein belongs to the mitochondrial carrier family and is an essential component of the fatty acid catabolism. It has been well studied with respect to its function and structure/function relationships. The main reaction catalyzed by CACT is an antiport of acylcarnitines with carnitine that, physiologically, provides acyl groups to mitochondria for βoxidation, recycling carnitine, an essential cofactor for this metabolic process. It is well known that impairment of CACT causes alteration of the energetic metabolism as definitively demonstrated by the occurrence of the secondary carnitine deficiency, an inherited lethal syndrome, which is caused by defects of CACT .13 The structure/function relationships of the CACT have been well defined by site directed mutagenesis using both the rat and human recombinant proteins. The main advances concerning this issue are the identification of the amino acid residues involved in the substrate binding and transport pathway and the Cys residues which are responsible of the targeting by selective chemical reagents such as NEM and MTS.13 These data led to define the topology of the 4 ACS Paragon Plus Environment

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protein structure, i.e., its orientation in membrane and their mutual relationships. Moreover, the structural homology model of the transporters was also obtained using as template the ADP/ATP carrier structure.15 The model was validated by site directed mutagenesis of selective exposed amino acid residues. Interestingly, involvement of CACT in pharmacology has been recently highlighted: the interaction and inactivation by mildronate, β-lactams and omeprazole have been described accounting for side effects of these compounds.16-18 Moreover, interaction of some mercury compounds, such as mersalyl and HgCl2 were previously described for CACT extracted from liver mitochondria.19 In those studies it was suggested that those compounds could interact with Cys residues of the transporter. A comprehensive study has been carried out in the present work, describing the molecular determinants of the potent inhibition of CACT by HgCl2 and CH3Hg+. Furthermore, correlation of the molecular action with in vivo effects is provided using different experimental approaches.

Materials and Methods

Materials Sephadexes G-50, G-75 and G-200 were purchased from Pharmacia, l-[methyl-3H]carnitine from Amersham, egg-yolk phospholipids (l-α-phosphatidylcholine from fresh turkey egg yolk),65% nitric acid from Merck, Suprapur, Pipes, Triton X-100, cardiolipin, L-carnitine, HgCl2, CH3Hg+(as CH3HgCl)and N-dodecanoylsarcosine (sarkosyl) from Sigma, St. Louis, MO. All other reagents were of analytical grade.

Methods

Site-directed mutagenesis, overexpression and isolation of the CACT proteins



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The CACT cDNA, cloned in the pMW7 expression vector, was used as a template to introduce mutation in CACT protein. Mutations were performed with complementary mutagenic primers using the overlap extension method 20, 21 and the High Fidelity PCR System. The PCR products were purified, digested with NdeI and HindIII and ligated into the pMW7 expression vector. The CACT cDNA was analyzed by DNA sequencing and, except for the desired base changes, corresponded to the CACT coding sequence. To obtain the overexpression of CACT recombinant protein, E. coli C0214 cells were transformed with CACT- pMW7. Isolation of inclusion bodies, solubilization and purification of WT and mutant proteins were performed as previously described.21

Animal maintenance Thirty adult zebrafish (Danio rerio) measuring 0.35±0.5 cm and weighing 0.43±0.06 g were obtained from a fish farm (COF SAS, Bologna, Italy), and acclimated to laboratory conditions for one week before the experiment. During the acclimation period, animals were kept in 60 L aquaria filled with tap water. Water parameters were: temperature 28±0.5°C, pH 7.2, conductivity 347 µS/cm, hardness 34°F, alkalinity 230 mg/L CaCO3; ion concentrations were (mg/L): Cl- 5, SO42- 24, Ca2+ 50, Mg2+ 14, Na+ 4, K+ 1, NO3- 1, NO2-

Acylcarnitine Transporter, a Novel Target of

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