Publications by authors named "Vito Porcelli"

24 Publications

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Phospholipidomics of peripheral blood mononuclear cells (PBMCs): the tricky case of children with autism spectrum disorder (ASD) and their healthy siblings.

Anal Bioanal Chem 2020 Oct 1;412(25):6859-6874. Epub 2020 Aug 1.

Department of Chemistry, University of Bari Aldo Moro, via Orabona 4, 70126, Bari, Italy.

Autism spectrum disorder (ASD) is a broad and heterogeneous group of neurological developmental disorders characterized by impaired social interaction and communication, restricted and repetitive behavioural patterns, and altered sensory processing. Currently, no reliable ASD molecular biomarkers are available. Since immune dysregulation has been supposed to be related with ASD onset and dyslipidaemia has been recognized as an early symptom of biological perturbation, lipid extracts from peripheral blood mononuclear cells (PBMCs), consisting primarily of lymphocytes (T cells, B cells, and NK cells) and monocytes, of 38 children with ASD and their non-autistic siblings were investigated by hydrophilic interaction liquid chromatography (HILIC) coupled with electrospray ionization and Fourier-transform mass spectrometry (ESI-FTMS). Performances of two freeware software for data extraction and processing were compared with acquired reliable data regardless of the used informatics. A reduction of variables from 1460 by the untargeted XCMS to 324 by the semi-untargeted Alex software was attained. All-ion fragmentation (AIF) MS scans along with Alex software were successfully applied to obtain information related to fatty acyl chain composition of six glycerophospholipid classes occurring in PBMC. Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were explored to verify the occurrence of significant differences in the lipid pool composition of ASD children compared with 36 healthy siblings. After rigorous statistical validation, we conclude that phospholipids extracted from PBMC of children affected by ASD do not exhibit diagnostic biomarkers. Yet interindividual variability comes forth from this study as the dominant effect in keeping with the existing phenotypic and etiological heterogeneity among ASD individuals. Graphical abstract.
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http://dx.doi.org/10.1007/s00216-020-02817-zDOI Listing
October 2020

Biochemical and functional characterization of a mitochondrial citrate carrier in Arabidopsis thaliana.

Biochem J 2020 05;477(9):1759-1777

Departamento de Biologia Vegetal, Universidade Federal de Viçosa, 36570-900 Viçosa, Minas Gerais, Brazil.

A homolog of the mitochondrial succinate/fumarate carrier from yeast (Sfc1p) has been found in the Arabidopsis genome, named AtSFC1. The AtSFC1 gene was expressed in Escherichia coli, and the gene product was purified and reconstituted in liposomes. Its transport properties and kinetic parameters demonstrated that AtSFC1 transports citrate, isocitrate and aconitate and, to a lesser extent, succinate and fumarate. This carrier catalyzes a fast counter-exchange transport as well as a low uniport of substrates, exhibits a higher transport affinity for tricarboxylates than dicarboxylates, and is inhibited by pyridoxal 5'-phosphate and other inhibitors of mitochondrial carriers to various degrees. Gene expression analysis indicated that the AtSFC1 transcript is mainly present in heterotrophic tissues, and fusion with a green-fluorescent protein localized AtSFC1 to the mitochondria. Furthermore, 35S-AtSFC1 antisense lines were generated and characterized at metabolic and physiological levels in different organs and at various developmental stages. Lower expression of AtSFC1 reduced seed germination and impaired radicle growth, a phenotype that was related to reduced respiration rate. These findings demonstrate that AtSFC1 might be involved in storage oil mobilization at the early stages of seedling growth and in nitrogen assimilation in root tissue by catalyzing citrate/isocitrate or citrate/succinate exchanges.
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http://dx.doi.org/10.1042/BCJ20190785DOI Listing
May 2020

CUGC for hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome.

Eur J Hum Genet 2020 07 2;28(7):982-987. Epub 2020 Apr 2.

Division of Metabolism and Research Unit of Metabolic Biochemistry, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy.

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http://dx.doi.org/10.1038/s41431-020-0616-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7316770PMC
July 2020

A Mutation in the Mitochondrial Aspartate/Glutamate Carrier Leads to a More Oxidizing Intramitochondrial Environment and an Inflammatory Myopathy in Dutch Shepherd Dogs.

J Neuromuscul Dis 2019 ;6(4):485-501

Department of Pathology, School of Medicine, University of California San Diego, La Jolla, CA, USA.

Background: Inflammatory myopathies are characterized by infiltration of inflammatory cells into muscle. Typically, immune-mediated disorders such as polymyositis, dermatomyositis and inclusion body myositis are diagnosed.

Objective: A small family of dogs with early onset muscle weakness and inflammatory muscle biopsies were investigated for an underlying genetic cause.

Methods: Following the histopathological diagnosis of inflammatory myopathy, mutational analysis including whole genome sequencing, functional transport studies of the mutated and wild-type proteins, and metabolomic analysis were performed.

Results: Whole genome resequencing identified a pathological variant in the SLC25A12 gene, resulting in a leucine to proline substitution at amino acid 349 in the mitochondrial aspartate-glutamate transporter known as the neuron and muscle specific aspartate glutamate carrier 1 (AGC1). Functionally reconstituting recombinant wild-type and mutant AGC1 into liposomes demonstrated a dramatic decrease in AGC1 transport activity and inability to transfer reducing equivalents from the cytosol into mitochondria. Targeted, broad-spectrum metabolomic analysis from affected and control muscles demonstrated a proinflammatory milieu and strong support for oxidative stress.

Conclusions: This study provides the first description of a metabolic mechanism in which ablated mitochondrial glutamate transport markedly reduced the import of reducing equivalents into mitochondria and produced a highly oxidizing and proinflammatory muscle environment and an inflammatory myopathy.
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http://dx.doi.org/10.3233/JND-190421DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6918910PMC
April 2020

Mitochondrial Carriers for Aspartate, Glutamate and Other Amino Acids: A Review.

Int J Mol Sci 2019 Sep 10;20(18). Epub 2019 Sep 10.

Department of Biosciences, Biotechnologies and Biopharmaceutics, Laboratory of Biochemistry and Molecular Biology, University of Bari Aldo Moro, Via E. Orabona 4, 70125 Bari, Italy.

Members of the mitochondrial carrier (MC) protein family transport various molecules across the mitochondrial inner membrane to interlink steps of metabolic pathways and biochemical processes that take place in different compartments; i.e., are localized partly inside and outside the mitochondrial matrix. MC substrates consist of metabolites, inorganic anions (such as phosphate and sulfate), nucleotides, cofactors and amino acids. These compounds have been identified by in vitro transport assays based on the uptake of radioactively labeled substrates into liposomes reconstituted with recombinant purified MCs. By using this approach, 18 human, plant and yeast MCs for amino acids have been characterized and shown to transport aspartate, glutamate, ornithine, arginine, lysine, histidine, citrulline and glycine with varying substrate specificities, kinetics, influences of the pH gradient, and capacities for the antiport and uniport mode of transport. Aside from providing amino acids for mitochondrial translation, the transport reactions catalyzed by these MCs are crucial in energy, nitrogen, nucleotide and amino acid metabolism. In this review we dissect the transport properties, phylogeny, regulation and expression levels in different tissues of MCs for amino acids, and summarize the main structural aspects known until now about MCs. The effects of their disease-causing mutations and manipulation of their expression levels in cells are also considered as clues for understanding their physiological functions.
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http://dx.doi.org/10.3390/ijms20184456DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6769469PMC
September 2019

CRAT missense variants cause abnormal carnitine acetyltransferase function in an early-onset case of Leigh syndrome.

Hum Mutat 2020 01 23;41(1):110-114. Epub 2019 Sep 23.

Department of Biosciences, Biotechnology and Biopharmaceutics, University of Bari, Bari, Italy.

Leigh syndrome, or subacute necrotizing encephalomyelopathy, is one of the most severe pediatric disorders of the mitochondrial energy metabolism. By performing whole-exome sequencing in a girl affected by Leigh syndrome and her parents, we identified two heterozygous missense variants (p.Tyr110Cys and p.Val569Met) in the carnitine acetyltransferase (CRAT) gene, encoding an enzyme involved in the control of mitochondrial short-chain acyl-CoA concentrations. Biochemical assays revealed carnitine acetyltransferase deficiency in the proband-derived fibroblasts. Functional analyses of recombinant-purified CRAT proteins demonstrated that both missense variants, located in the acyl-group binding site of the enzyme, severely impair its catalytic function toward acetyl-CoA, and the p.Val569Met variant also toward propionyl-CoA and octanoyl-CoA. Although a single recessive variant in CRAT has been recently associated with neurodegeneration with brain iron accumulation (NBIA), this study reports the first kinetic analysis of naturally occurring CRAT variants and demonstrates the genetic basis of carnitine acetyltransferase deficiency in a case of mitochondrial encephalopathy.
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http://dx.doi.org/10.1002/humu.23901DOI Listing
January 2020

The human uncoupling proteins 5 and 6 (UCP5/SLC25A14 and UCP6/SLC25A30) transport sulfur oxyanions, phosphate and dicarboxylates.

Biochim Biophys Acta Bioenerg 2019 09 26;1860(9):724-733. Epub 2019 Jul 26.

Department of Biosciences, Biotechnologies and Biopharmaceutics, Laboratory of Biochemistry and Molecular Biology, University of Bari Aldo Moro, Via E. Orabona 4, 70125 Bari, Italy; Center of Excellence in Comparative Genomics, University of Bari, via Orabona 4, 70125 Bari, Italy; CNR Institute of Biomembranes, Bioenergetics and Molecular Biotechnologies (IBIOM), 70126 Bari, Italy.

The human genome encodes 53 members of the solute carrier family 25 (SLC25), also called the mitochondrial carrier family. In this work, two members of this family, UCP5 (BMCP1, brain mitochondrial carrier protein 1 encoded by SLC25A14) and UCP6 (KMCP1, kidney mitochondrial carrier protein 1 encoded by SLC25A30) have been thoroughly characterized biochemically. They were overexpressed in bacteria, purified and reconstituted in phospholipid vesicles. Their transport properties and kinetic parameters demonstrate that UCP5 and UCP6 transport inorganic anions (sulfate, sulfite, thiosulfate and phosphate) and, to a lesser extent, a variety of dicarboxylates (e.g. malonate, malate and citramalate) and, even more so, aspartate and (only UCP5) glutamate and tricarboxylates. Both carriers catalyzed a fast counter-exchange transport and a very low uniport of substrates. Transport was saturable and inhibited by mercurials and other mitochondrial carrier inhibitors at various degrees. The transport affinities of UCP5 and UCP6 were higher for sulfate and thiosulfate than for any other substrate, whereas the specific activity of UCP5 was much higher than that of UCP6. It is proposed that a main physiological role of UCP5 and UCP6 is to catalyze the export of sulfite and thiosulfate (the HS degradation products) from the mitochondria, thereby modulating the level of the important signal molecule HS.
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http://dx.doi.org/10.1016/j.bbabio.2019.07.010DOI Listing
September 2019

Molecular identification and functional characterization of a novel glutamate transporter in yeast and plant mitochondria.

Biochim Biophys Acta Bioenerg 2018 11 8;1859(11):1249-1258. Epub 2018 Aug 8.

Laboratory of Biochemistry and Molecular Biology, Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari Aldo Moro, Bari, Italy; CNR Institute of Biomembranes, Bioenergetics and Molecular Biotechnologies (IBIOM), Bari, Italy. Electronic address:

The genome of Saccharomyces cerevisiae encodes 35 members of the mitochondrial carrier family (MCF) and 58 MCF members are coded by the genome of Arabidopsis thaliana, most of which have been functionally characterized. Here two members of this family, Ymc2p from S. cerevisiae and BOU from Arabidopsis, have been thoroughly characterized. These proteins were overproduced in bacteria and reconstituted into liposomes. Their transport properties and kinetic parameters demonstrate that Ymc2p and BOU transport glutamate, and to a much lesser extent L-homocysteinesulfinate, but not other amino acids and many other tested metabolites. Transport catalyzed by both carriers was saturable, inhibited by mercuric chloride and dependent on the proton gradient across the proteoliposomal membrane. The growth phenotype of S. cerevisiae cells lacking the genes ymc2 and agc1, which encodes the only other S. cerevisiae carrier capable to transport glutamate besides aspartate, was fully complemented by expressing Ymc2p, Agc1p or BOU. Mitochondrial extracts derived from ymc2Δagc1Δ cells, reconstituted into liposomes, exhibited no glutamate transport at variance with wild-type, ymc2Δ and agc1Δ cells, showing that S. cerevisiae cells grown in the presence of acetate do not contain additional mitochondrial transporters for glutamate besides Ymc2p and Agc1p. Furthermore, mitochondria isolated from wild-type, ymc2Δ and agc1Δ strains, but not from the double mutant ymc2Δagc1Δ strain, swell in isosmotic ammonium glutamate showing that glutamate is transported by Ymc2p and Agc1p together with a H. It is proposed that the function of Ymc2p and BOU is to transport glutamate across the mitochondrial inner membrane and thereby play a role in intermediary metabolism, C1 metabolism and mitochondrial protein synthesis.
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http://dx.doi.org/10.1016/j.bbabio.2018.08.001DOI Listing
November 2018

Structure/function relationships of the human mitochondrial ornithine/citrulline carrier by Cys site-directed mutagenesis. Relevance to mercury toxicity.

Int J Biol Macromol 2018 Dec 16;120(Pt A):93-99. Epub 2018 Aug 16.

CNR Institute of Biomembranes, Bioenergetics and Molecular Biotechnology, via Amendola 165/A, 70126 Bari, Italy; Department DiBEST (Biologia, Ecologia, Scienze della Terra) Unit of Biochemistry and Molecular Biotechnology, University of Calabria, Via Bucci 4C, 87036 Arcavacata di Rende, Italy. Electronic address:

The effect of SH reagents on the human mitochondrial ornithine/citrulline carrier (hORC) was studied. Site-directed Cys mutants were employed to gain information on structure/function relationships. The substitutions of each Cys by Ala did not alter the hORC activity measured as [H]ornithine/ornithine antiport in proteoliposomes. N‑ethylmaleimide inhibited the transport of WT with IC50 of 149 μM. C51A, C50A and C132A showed a much higher IC50. MTSEA and MTSET also inhibited the WT with IC50 of 0.40 μM and 1.60 μM, respectively. C51A and C132A showed much higher IC50 values for both reagents. The triple mutant C50/51/132A showed an IC50 for the three reagents that was higher than that of the single mutants. The data strongly suggests that C132, C50 and C51 are involved in inhibition of hORC. Inhibition of WT and mutants by CuPhenanthroline, an S-S forming reagent, suggested that C132 may form disulfides with C50 or C51, impairing the transporter function. The structure/function relationships information deriving from the inhibition studies, were corroborated by the homology structural model of the transporter. The effect of HgCl and methyl mercury was also tested on hORC in the light of their capacity to bind thiol residues. Both reagents potently inhibit the transporter.
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http://dx.doi.org/10.1016/j.ijbiomac.2018.08.069DOI Listing
December 2018

Monoamine oxidase-dependent histamine catabolism accounts for post-ischemic cardiac redox imbalance and injury.

Biochim Biophys Acta Mol Basis Dis 2018 09 25;1864(9 Pt B):3050-3059. Epub 2018 Jun 25.

Department of Biomedical Sciences, University of Padova, Italy; Venetian Institute of Molecular Medicine (VIMM), Padova, Italy. Electronic address:

Monoamine oxidase (MAO), a mitochondrial enzyme that oxidizes biogenic amines generating hydrogen peroxide, is a major source of oxidative stress in cardiac injury. However, the molecular mechanisms underlying its overactivation in pathological conditions are still poorly characterized. Here, we investigated whether the enhanced MAO-dependent hydrogen peroxide production can be due to increased substrate availability using a metabolomic profiling method. We identified N-methylhistamine -the main catabolite of histamine- as an important substrate fueling MAO in Langendorff mouse hearts, directly perfused with a buffer containing hydrogen peroxide or subjected to ischemia/reperfusion protocol. Indeed, when these hearts were pretreated with the MAO inhibitor pargyline we observed N-methylhistamine accumulation along with reduced oxidative stress. Next, we showed that synaptic terminals are the major source of N-methylhistamine. Indeed, in vivo sympathectomy caused a decrease of N-methylhistamine levels, which was associated with a marked protection in post-ischemic reperfused hearts. As far as the mechanism is concerned, we demonstrate that exogenous histamine is transported into isolated cardiomyocytes and triggers a rise in the levels of reactive oxygen species (ROS). Once again, pargyline pretreatment induced intracellular accumulation of N-methylhistamine along with decrease in ROS levels. These findings uncover a receptor-independent mechanism for histamine in cardiomyocytes. In summary, our study reveals a novel and important pathophysiological causative link between MAO activation and histamine availability during pathophysiological conditions such as oxidative stress/cardiac injury.
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http://dx.doi.org/10.1016/j.bbadis.2018.06.018DOI Listing
September 2018

Effect of diazoxide on Friedreich ataxia models.

Hum Mol Genet 2018 03;27(6):992-1001

Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, 70125 Bari, Italy.

Friedreich ataxia (FRDA) is an inherited recessive disorder caused by a deficiency in the mitochondrial protein frataxin. There is currently no effective treatment for FRDA available, especially for neurological deficits. In this study, we tested diazoxide, a drug commonly used as vasodilator in the treatment of acute hypertension, on cellular and animal models of FRDA. We first showed that diazoxide increases frataxin protein levels in FRDA lymphoblastoid cell lines, via the mammalian target of rapamycin (mTOR) pathway. We then explored the potential therapeutic effect of diazoxide in frataxin-deficient transgenic YG8sR mice and we found that prolonged oral administration of 3 mpk/d diazoxide was found to be safe, but produced variable effects concerning efficacy. YG8sR mice showed improved beam walk coordination abilities and footprint stride patterns, but a generally reduced locomotor activity. Moreover, they showed significantly increased frataxin expression, improved aconitase activity, and decreased protein oxidation in cerebellum and brain mitochondrial tissue extracts. Further studies are needed before this drug should be considered for FRDA clinical trials.
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http://dx.doi.org/10.1093/hmg/ddy016DOI Listing
March 2018

An overview of combined D-2- and L-2-hydroxyglutaric aciduria: functional analysis of CIC variants.

J Inherit Metab Dis 2018 03 13;41(2):169-180. Epub 2017 Dec 13.

Metabolic Laboratory, Department of Clinical Chemistry, Amsterdam Neuroscience, VU Medical Center Metabolic Unit PK 1X009, Postbus 7057, 1007 MB, Amsterdam, The Netherlands.

Combined D-2- and L-2-hydroxyglutaric aciduria (D/L-2-HGA) is a devastating neurometabolic disorder, usually lethal in the first years of life. Autosomal recessive mutations in the SLC25A1 gene, which encodes the mitochondrial citrate carrier (CIC), were previously detected in patients affected with combined D/L-2-HGA. We showed that transfection of deficient fibroblasts with wild-type SLC25A1 restored citrate efflux and decreased intracellular 2-hydroxyglutarate levels, confirming that deficient CIC is the cause of D/L-2-HGA. We developed and implemented a functional assay and applied it to all 17 missense variants detected in a total of 26 CIC-deficient patients, including eight novel cases, showing reduced activities of varying degrees. In addition, we analyzed the importance of residues affected by these missense variants using our existing scoring system. This allowed not only a clinical and biochemical overview of the D/L-2-HGA patients but also phenotype-genotype correlation studies.
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http://dx.doi.org/10.1007/s10545-017-0106-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5830478PMC
March 2018

SLC25A10 biallelic mutations in intractable epileptic encephalopathy with complex I deficiency.

Hum Mol Genet 2018 02;27(3):499-504

Department of Biosciences, Biotechnology and Biopharmaceutics, University of Bari, 70125 Bari, Italy.

Mitochondrial diseases are a plethora of inherited neuromuscular disorders sharing defects in mitochondrial respiration, but largely different from one another for genetic basis and pathogenic mechanism. Whole exome sequencing was performed in a familiar trio (trio-WES) with a child affected by severe epileptic encephalopathy associated with respiratory complex I deficiency and mitochondrial DNA depletion in skeletal muscle. By trio-WES we identified biallelic mutations in SLC25A10, a nuclear gene encoding a member of the mitochondrial carrier family. Genetic and functional analyses conducted on patient fibroblasts showed that SLC25A10 mutations are associated with reduction in RNA quantity and aberrant RNA splicing, and to absence of SLC25A10 protein and its transporting function. The yeast SLC25A10 ortholog knockout strain showed defects in mitochondrial respiration and mitochondrial DNA content, similarly to what observed in the patient skeletal muscle, and growth susceptibility to oxidative stress. Albeit patient fibroblasts were depleted in the main antioxidant molecules NADPH and glutathione, transport assays demonstrated that SLC25A10 is unable to transport glutathione. Here, we report the first recessive mutations of SLC25A10 associated to an inherited severe mitochondrial neurodegenerative disorder. We propose that SLC25A10 loss-of-function causes pathological disarrangements in respiratory-demanding conditions and oxidative stress vulnerability.
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http://dx.doi.org/10.1093/hmg/ddx419DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5886107PMC
February 2018

Toward the Standardization of Mitochondrial Proteomics: The Italian Mitochondrial Human Proteome Project Initiative.

J Proteome Res 2017 12 13;16(12):4319-4329. Epub 2017 Sep 13.

Department of Science and High Technology, Università degli Studi dell'Insubria , Busto Arsizio I-21052, Italy.

The Mitochondrial Human Proteome Project aims at understanding the function of the mitochondrial proteome and its crosstalk with the proteome of other organelles. Being able to choose a suitable and validated enrichment protocol of functional mitochondria, based on the specific needs of the downstream proteomics analysis, would greatly help the researchers in the field. Mitochondrial fractions from ten model cell lines were prepared using three enrichment protocols and analyzed on seven different LC-MS/MS platforms. All data were processed using neXtProt as reference database. The data are available for the Human Proteome Project purposes through the ProteomeXchange Consortium with the identifier PXD007053. The processed data sets were analyzed using a suite of R routines to perform a statistical analysis and to retrieve subcellular and submitochondrial localizations. Although the overall number of identified total and mitochondrial proteins was not significantly dependent on the enrichment protocol, specific line to line differences were observed. Moreover, the protein lists were mapped to a network representing the functional mitochondrial proteome, encompassing mitochondrial proteins and their first interactors. More than 80% of the identified proteins resulted in nodes of this network but with a different ability in coisolating mitochondria-associated structures for each enrichment protocol/cell line pair.
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http://dx.doi.org/10.1021/acs.jproteome.7b00350DOI Listing
December 2017

Down-regulation of the mitochondrial aspartate-glutamate carrier isoform 1 AGC1 inhibits proliferation and N-acetylaspartate synthesis in Neuro2A cells.

Biochim Biophys Acta Mol Basis Dis 2017 06 21;1863(6):1422-1435. Epub 2017 Feb 21.

Institute of Biomembranes and Bioenergetics, Consiglio Nazionale delle Ricerche, Bari 70126, Italy. Electronic address:

The mitochondrial aspartate-glutamate carrier isoform 1 (AGC1) catalyzes a Ca-stimulated export of aspartate to the cytosol in exchange for glutamate, and is a key component of the malate-aspartate shuttle which transfers NADH reducing equivalents from the cytosol to mitochondria. By sustaining the complete glucose oxidation, AGC1 is thought to be important in providing energy for cells, in particular in the CNS and muscle where this protein is mainly expressed. Defects in the AGC1 gene cause AGC1 deficiency, an infantile encephalopathy with delayed myelination and reduced brain N-acetylaspartate (NAA) levels, the precursor of myelin synthesis in the CNS. Here, we show that undifferentiated Neuro2A cells with down-regulated AGC1 display a significant proliferation deficit associated with reduced mitochondrial respiration, and are unable to synthesize NAA properly. In the presence of high glutamine oxidation, cells with reduced AGC1 restore cell proliferation, although oxidative stress increases and NAA synthesis deficit persists. Our data suggest that the cellular energetic deficit due to AGC1 impairment is associated with inappropriate aspartate levels to support neuronal proliferation when glutamine is not used as metabolic substrate, and we propose that delayed myelination in AGC1 deficiency patients could be attributable, at least in part, to neuronal loss combined with lack of NAA synthesis occurring during the nervous system development.
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http://dx.doi.org/10.1016/j.bbadis.2017.02.022DOI Listing
June 2017

Asymmetric dimethylarginine is transported by the mitochondrial carrier SLC25A2.

Amino Acids 2016 Feb 24;48(2):427-36. Epub 2015 Sep 24.

Laboratory of Biochemistry and Molecular Biology, Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Via Orabona 4, 70125, Bari, Italy.

Asymmetric dimethyl L-arginine (ADMA) is generated within cells and in mitochondria when proteins with dimethylated arginine residues are degraded. The aim of this study was to identify the carrier protein(s) that transport ADMA across the inner mitochondrial membrane. It was found that the recombinant, purified mitochondrial solute carrier SLC25A2 when reconstituted into liposomes efficiently transports ADMA in addition to its known substrates arginine, lysine, and ornithine and in contrast to the other known mitochondrial amino acid transporters SLC25A12, SLC25A13, SLC25A15, SLC25A18, SLC25A22, and SLC25A29. The widely expressed SLC25A2 transported ADMA across the liposomal membrane in both directions by both unidirectional transport and exchange against arginine or lysine. The SLC25A2-mediated ADMA transport followed first-order kinetics, was nearly as fast as the transport of the best SLC25A2 substrates known so far, and was highly specific as symmetric dimethylarginine (SDMA) was not transported at all. Furthermore, ADMA inhibited SLC25A2 activity with an inhibition constant of 0.38 ± 0.04 mM, whereas SDMA inhibited it poorly. We propose that a major function of SLC25A2 is to export ADMA from mitochondria missing the mitochondrial ADMA-metabolizing enzyme AGXT2. There is evidence that ADMA can also be imported into mitochondria, e.g., in kidney proximal tubulus cells, to be metabolized by AGXT2. SLC25A2 may also mediate this transport function.
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http://dx.doi.org/10.1007/s00726-015-2096-9DOI Listing
February 2016

Mitochondrial transporters for ornithine and related amino acids: a review.

Amino Acids 2015 Sep 23;47(9):1763-77. Epub 2015 May 23.

Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Via E. Orabona 4, 70125, Bari, Italy.

Among the members of the mitochondrial carrier family, there are transporters that catalyze the translocation of ornithine and related substrates, such as arginine, homoarginine, lysine, histidine, and citrulline, across the inner mitochondrial membrane. The mitochondrial carriers ORC1, ORC2, and SLC25A29 from Homo sapiens, BAC1 and BAC2 from Arabidopsis thaliana, and Ort1p from Saccharomyces cerevisiae have been biochemically characterized by transport assays in liposomes. All of them transport ornithine and amino acids with side chains terminating at least with one amine. There are, however, marked differences in their substrate specificities including their affinity for ornithine (KM values in the mM to μM range). These differences are most likely reflected by minor differences in the substrate binding sites of these carriers. The physiological role of the above-mentioned mitochondrial carriers is to link several metabolic pathways that take place partly in the cytosol and partly in the mitochondrial matrix and to provide basic amino acids for mitochondrial translation. In the liver, human ORC1 catalyzes the citrulline/ornithine exchange across the mitochondrial inner membrane, which is required for the urea cycle. Human ORC1, ORC2, and SLC25A29 are likely to be involved in the biosynthesis and transport of arginine, which can be used as a precursor for the synthesis of NO, agmatine, polyamines, creatine, glutamine, glutamate, and proline, as well as in the degradation of basic amino acids. BAC1 and BAC2 are implicated in some processes similar to those of their human counterparts and in nitrogen and amino acid metabolism linked to stress conditions and the development of plants. Ort1p is involved in the biosynthesis of arginine and polyamines in yeast.
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http://dx.doi.org/10.1007/s00726-015-1990-5DOI Listing
September 2015

Acetylation of human mitochondrial citrate carrier modulates mitochondrial citrate/malate exchange activity to sustain NADPH production during macrophage activation.

Biochim Biophys Acta 2015 Aug 24;1847(8):729-38. Epub 2015 Apr 24.

Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari "Aldo Moro", Bari, Italy; Center of Excellence in Comparative Genomics, University of Bari "Aldo Moro", Bari, Italy. Electronic address:

The mitochondrial citrate-malate exchanger (CIC), a known target of acetylation, is up-regulated in activated immune cells and plays a key role in the production of inflammatory mediators. However, the role of acetylation in CIC activity is elusive. We show that CIC is acetylated in activated primary human macrophages and U937 cells and the level of acetylation is higher in glucose-deprived compared to normal glucose medium. Acetylation enhances CIC transport activity, leading to a higher citrate efflux from mitochondria in exchange with malate. Cytosolic citrate levels do not increase upon activation of cells grown in deprived compared to normal glucose media, indicating that citrate, transported from mitochondria at higher rates from acetylated CIC, is consumed at higher rates. Malate levels in the cytosol are lower in activated cells grown in glucose-deprived compared to normal glucose medium, indicating that this TCA intermediate is rapidly recycled back into the cytosol where it is used by the malic enzyme. Additionally, in activated cells CIC inhibition increases the NADP+/NADPH ratio in glucose-deprived cells; this ratio is unchanged in glucose-rich grown cells due to the activity of the pentose phosphate pathway. Consistently, the NADPH-producing isocitrate dehydrogenase level is higher in activated glucose-deprived as compared to glucose rich cells. These results demonstrate that, in the absence of glucose, activated macrophages increase CIC acetylation to enhance citrate efflux from mitochondria not only to produce inflammatory mediators but also to meet the NADPH demand through the actions of isocitrate dehydrogenase and malic enzyme.
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http://dx.doi.org/10.1016/j.bbabio.2015.04.009DOI Listing
August 2015

The human gene SLC25A29, of solute carrier family 25, encodes a mitochondrial transporter of basic amino acids.

J Biol Chem 2014 May 20;289(19):13374-84. Epub 2014 Mar 20.

From the Department of Biosciences, Biotechnologies and Biopharmaceutics, Laboratory of Biochemistry and Molecular Biology and.

The human genome encodes 53 members of the solute carrier family 25 (SLC25), also called the mitochondrial carrier family, many of which have been shown to transport carboxylates, amino acids, nucleotides, and cofactors across the inner mitochondrial membrane, thereby connecting cytosolic and matrix functions. In this work, a member of this family, SLC25A29, previously reported to be a mitochondrial carnitine/acylcarnitine- or ornithine-like carrier, has been thoroughly characterized biochemically. The SLC25A29 gene was overexpressed in Escherichia coli, and the gene product was purified and reconstituted in phospholipid vesicles. Its transport properties and kinetic parameters demonstrate that SLC25A29 transports arginine, lysine, homoarginine, methylarginine and, to a much lesser extent, ornithine and histidine. Carnitine and acylcarnitines were not transported by SLC25A29. This carrier catalyzed substantial uniport besides a counter-exchange transport, exhibited a high transport affinity for arginine and lysine, and was saturable and inhibited by mercurial compounds and other inhibitors of mitochondrial carriers to various degrees. The main physiological role of SLC25A29 is to import basic amino acids into mitochondria for mitochondrial protein synthesis and amino acid degradation.
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http://dx.doi.org/10.1074/jbc.M114.547448DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4036346PMC
May 2014

Mutations in the Mitochondrial Citrate Carrier SLC25A1 are Associated with Impaired Neuromuscular Transmission.

J Neuromuscul Dis 2014;1(1):75-90

Institute of Genetic Medicine, MRC Centre for Neuromuscular Diseases, Newcastle University, Newcastle upon Tyne, UK.

Background And Objective: Congenital myasthenic syndromes are rare inherited disorders characterized by fatigable weakness caused by malfunction of the neuromuscular junction. We performed whole exome sequencing to unravel the genetic aetiology in an English sib pair with clinical features suggestive of congenital myasthenia.

Methods: We used homozygosity mapping and whole exome sequencing to identify the candidate gene variants. Mutant protein expression and function were assessed and a knockdown zebrafish model was generated to assess neuromuscular junction development.

Results: We identified a novel homozygous missense mutation in the gene, encoding the mitochondrial citrate carrier. Mutant SLC25A1 showed abnormal carrier function. has recently been linked to a severe, often lethal clinical phenotype. Our patients had a milder phenotype presenting primarily as a neuromuscular (NMJ) junction defect. Of note, a previously reported patient with different compound heterozygous missense mutations of has since been shown to suffer from a neuromuscular transmission defect. Using knockdown of SLC25A1 expression in zebrafish, we were able to mirror the human disease in terms of variable brain, eye and cardiac involvement. Importantly, we show clear abnormalities in the neuromuscular junction, regardless of the severity of the phenotype.

Conclusions: Based on the axonal outgrowth defects seen in SLC25A1 knockdown zebrafish, we hypothesize that the neuromuscular junction impairment may be related to pre-synaptic nerve terminal abnormalities. Our findings highlight the complex machinery required to ensure efficient neuromuscular function, beyond the proteomes exclusive to the neuromuscular synapse.
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http://dx.doi.org/10.3233/JND-140021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4746751PMC
January 2014

Agenesis of corpus callosum and optic nerve hypoplasia due to mutations in SLC25A1 encoding the mitochondrial citrate transporter.

J Med Genet 2013 Apr 7;50(4):240-5. Epub 2013 Feb 7.

Monique and Jacques Roboh Department of Genetic Research, Hadassah, Hebrew University Medical Center, Jerusalem, Israel.

Background: Agenesis of corpus callosum has been associated with several defects of the mitochondrial respiratory chain and the citric acid cycle. We now report the results of the biochemical and molecular studies of a patient with severe neurodevelopmental disease manifesting by agenesis of corpus callosum and optic nerve hypoplasia.

Methods And Results: A mitochondrial disease was suspected in this patient based on the prominent excretion of 2-hydroxyglutaric acid and Krebs cycle intermediates in urine and the finding of increased reactive oxygen species content and decreased mitochondrial membrane potential in her fibroblasts. Whole exome sequencing disclosed compound heterozygosity for two pathogenic variants in the SLC25A1 gene, encoding the mitochondrial citrate transporter. These variants, G130D and R282H, segregated in the family and were extremely rare in controls. The mutated residues were highly conserved throughout evolution and in silico modeling investigations indicated that the mutations would have a deleterious effect on protein function, affecting either substrate binding to the transporter or its translocation mechanism. These predictions were validated by the observation that a yeast strain harbouring the mutations at equivalent positions in the orthologous protein exhibited a growth defect under stress conditions and by the loss of activity of citrate transport by the mutated proteins reconstituted into liposomes.

Conclusions: We report for the first time a patient with a mitochondrial citrate carrier deficiency. Our data support a role for citric acid cycle defects in agenesis of corpus callosum as already reported in patients with aconitase or fumarate hydratase deficiency.
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http://dx.doi.org/10.1136/jmedgenet-2012-101485DOI Listing
April 2013

Rapamycin reduces oxidative stress in frataxin-deficient yeast cells.

Mitochondrion 2012 Jan 18;12(1):156-61. Epub 2011 Jul 18.

Laboratory of Biochemistry and Molecular Biology, Department of Pharmaco-Biology, University of Bari, Via E. Orabona 4, 70125 Bari, Italy.

Friedreich ataxia (FRDA) is a common form of ataxia caused by decreased expression of the mitochondrial protein frataxin. Oxidative damage of mitochondria is thought to play a key role in the pathogenesis of the disease. Therefore, a possible therapeutic strategy should be directed to an antioxidant protection against mitochondrial damage. Indeed, treatment of FRDA patients with the antioxidant idebenone has been shown to improve neurological functions. The yeast frataxin knock-out model of the disease shows mitochondrial iron accumulation, iron-sulfur cluster defects and high sensitivity to oxidative stress. By flow cytometry analysis we studied reactive oxygen species (ROS) production of yeast frataxin mutant cells treated with two antioxidants, N-acetyl-L-cysteine and a mitochondrially-targeted analog of vitamin E, confirming that mitochondria are the main site of ROS production in this model. Furthermore we found a significant reduction of ROS production and a decrease in the mitochondrial mass in mutant cells treated with rapamycin, an inhibitor of TOR kinases, most likely due to autophagy of damaged mitochondria.
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http://dx.doi.org/10.1016/j.mito.2011.07.001DOI Listing
January 2012

The mitochondrial aspartate/glutamate carrier AGC1 and calcium homeostasis: physiological links and abnormalities in autism.

Mol Neurobiol 2011 Aug 21;44(1):83-92. Epub 2011 Jun 21.

Laboratory of Molecular Psychiatry & Neurogenetics, University Campus Bio-Medico, Via Alvaro del Portillo 21, 00128 Rome, Italy.

Autism spectrum disorder (ASD) is a severe, complex neurodevelopmental disorder characterized by impairments in reciprocal social interaction and communication, and restricted and stereotyped patterns of interests and behaviors. Recent evidence has unveiled an important role for calcium (Ca(2+)) signaling in the pathogenesis of ASD. Post-mortem studies of autistic brains have pointed toward abnormalities in mitochondrial function as possible downstream consequences of altered Ca(2+) signaling, abnormal synapse formation, and dysreactive immunity. SLC25A12, an ASD susceptibility gene, encodes the Ca(2+)-regulated mitochondrial aspartate-glutamate carrier, isoform 1 (AGC1). AGC1 is an important component of the malate/aspartate shuttle, a crucial system supporting oxidative phosphorylation and adenosine triphosphate (ATP) production. Here, we review the physiological roles of AGC1, its links to calcium homeostasis, and its involvement in autism pathogenesis.
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http://dx.doi.org/10.1007/s12035-011-8192-2DOI Listing
August 2011

Computational approaches for protein function prediction: a combined strategy from multiple sequence alignment to molecular docking-based virtual screening.

Biochim Biophys Acta 2010 Sep 28;1804(9):1695-712. Epub 2010 Apr 28.

Department of Pharmaco-Biology, Laboratory of Biochemistry and Molecular Biology, University of Bari, Va E. Orabona, 4 - 70125 Bari, Italy.

The functional characterization of proteins represents a daily challenge for biochemical, medical and computational sciences. Although finally proved on the bench, the function of a protein can be successfully predicted by computational approaches that drive the further experimental assays. Current methods for comparative modeling allow the construction of accurate 3D models for proteins of unknown structure, provided that a crystal structure of a homologous protein is available. Binding regions can be proposed by using binding site predictors, data inferred from homologous crystal structures, and data provided from a careful interpretation of the multiple sequence alignment of the investigated protein and its homologs. Once the location of a binding site has been proposed, chemical ligands that have a high likelihood of binding can be identified by using ligand docking and structure-based virtual screening of chemical libraries. Most docking algorithms allow building a list sorted by energy of the lowest energy docking configuration for each ligand of the library. In this review the state-of-the-art of computational approaches in 3D protein comparative modeling and in the study of protein-ligand interactions is provided. Furthermore a possible combined/concerted multistep strategy for protein function prediction, based on multiple sequence alignment, comparative modeling, binding region prediction, and structure-based virtual screening of chemical libraries, is described by using suitable examples. As practical examples, Abl-kinase molecular modeling studies, HPV-E6 protein multiple sequence alignment analysis, and some other model docking-based characterization reports are briefly described to highlight the importance of computational approaches in protein function prediction.
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http://dx.doi.org/10.1016/j.bbapap.2010.04.008DOI Listing
September 2010