Publications by authors named "Angelo Vozza"

21 Publications

  • Page 1 of 1

KRAS-regulated glutamine metabolism requires UCP2-mediated aspartate transport to support pancreatic cancer growth.

Nat Metab 2020 12 23;2(12):1373-1381. Epub 2020 Nov 23.

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

The oncogenic KRAS mutation has a critical role in the initiation of human pancreatic ductal adenocarcinoma (PDAC) since it rewires glutamine metabolism to increase reduced nicotinamide adenine dinucleotide phosphate (NADPH) production, balancing cellular redox homeostasis with macromolecular synthesis. Mitochondrial glutamine-derived aspartate must be transported into the cytosol to generate metabolic precursors for NADPH production. The mitochondrial transporter responsible for this aspartate efflux has remained elusive. Here, we show that mitochondrial uncoupling protein 2 (UCP2) catalyses this transport and promotes tumour growth. UCP2-silenced KRAS cell lines display decreased glutaminolysis, lower NADPH/NADP and glutathione/glutathione disulfide ratios and higher reactive oxygen species levels compared to wild-type counterparts. UCP2 silencing reduces glutaminolysis also in KRAS PDAC cells but does not affect their redox homeostasis or proliferation rates. In vitro and in vivo, UCP2 silencing strongly suppresses KRAS PDAC cell growth. Collectively, these results demonstrate that UCP2 plays a vital role in PDAC, since its aspartate transport activity connects the mitochondrial and cytosolic reactions necessary for KRAS rewired glutamine metabolism, and thus it should be considered a key metabolic target for the treatment of this refractory tumour.
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http://dx.doi.org/10.1038/s42255-020-00315-1DOI Listing
December 2020

Cloning, Purification, and Characterization of the Catalytic C-Terminal Domain of the Human 3-Hydroxy-3-methyl glutaryl-CoA Reductase: An Effective, Fast, and Easy Method for Testing Hypocholesterolemic Compounds.

Mol Biotechnol 2020 Feb;62(2):119-131

Department of Pharmacy, Health, and Nutritional Sciences, University of Calabria, 87036, Arcavacata di Rende, Cosenza, Italy.

3-hydroxy-3-methyl glutaryl-CoA reductase, also known as HMGR, plays a crucial role in regulating cholesterol biosynthesis and represents the main pharmacological target of statins. In mammals, this enzyme localizes to the endoplasmic reticulum membrane. HMGR includes different regions, an integral N-terminal domain connected by a linker-region to a cytosolic C-terminal domain, the latter being responsible for enzymatic activity. The aim of this work was to design a simple strategy for cloning, expression, and purification of the catalytic C-terminal domain of the human HMGR (cf-HMGR), in order to spectrophotometrically test its enzymatic activity. The recombinant cf-HMGR protein was heterologously expressed in Escherichia coli, purified by Ni-agarose affinity chromatography and reconstituted in its active form. MALDI mass spectrometry was adopted to monitor purification procedure as a technique orthogonal to the classical Western blot analysis. Protein identity was validated by MS and MS/MS analysis, confirming about 82% of the recombinant sequence. The specific activity of the purified and dialyzed cf-HMGR preparation was enriched about 85-fold with respect to the supernatant obtained from cell lysate. The effective, cheap, and easy method here described could be useful for screening statin-like molecules, so simplifying the search for new drugs with hypocholesterolemic effects.
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http://dx.doi.org/10.1007/s12033-019-00230-1DOI Listing
February 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

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

ISCA1 mutation in a patient with infantile-onset leukodystrophy causes defects in mitochondrial [4Fe-4S] proteins.

Hum Mol Genet 2018 10;27(20):3650

Laboratory of Molecular Medicine, Unit of Muscular and Neurodegenerative Disorders, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy.

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http://dx.doi.org/10.1093/hmg/ddy273DOI Listing
October 2018

ISCA1 mutation in a patient with infantile-onset leukodystrophy causes defects in mitochondrial [4Fe-4S] proteins.

Hum Mol Genet 2018 08;27(15):2739-2754

Laboratory of Molecular Medicine, Unit of Muscular and Neurodegenerative Disorders, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy.

Multiple mitochondrial dysfunction syndromes (MMDS) comprise a group of severe autosomal recessive diseases characterized by impaired respiration and lipoic acid metabolism, resulting in infantile-onset mitochondrial encephalopathy, non-ketotic hyperglycinemia, myopathy, lactic acidosis and early death. Four different MMDS have been analyzed in detail according to the genes involved in the disease, MMDS1 (NFU1), MMDS2 (BOLA3), MMDS3 (IBA57) and MMDS4 (ISCA2). MMDS5 has recently been described in a clinical case report of patients carrying a mutation in ISCA1, but with no further functional analysis. ISCA1 encodes a mitochondrial protein essential for the assembly of [4Fe-4S] clusters in key metabolic and respiratory enzymes. Here, we describe a patient with a severe early onset leukodystrophy, multiple defects of respiratory complexes and a severe impairment of lipoic acid synthesis. A homozygous missense mutation in ISCA1 (c.29T>G; p.V10G) identified by targeted MitoExome sequencing resulted in dramatic reduction of ISCA1 protein level. The mutation located in the uncleaved presequence severely affected both mitochondrial import and stability of ISCA1. Down-regulation of ISCA1 in HeLa cells by RNAi impaired the biogenesis of mitochondrial [4Fe-4S] proteins, yet could be complemented by expression of wild-type ISCA1. In contrast, the ISCA1 p.V10G mutant protein only partially complemented the defects, closely resembling the biochemical phenotypes observed for ISCA1 patient fibroblasts. Collectively, our comprehensive clinical and biochemical investigations show that the ISCA1 p.V10G mutation functionally impaired mitochondrial [4Fe-4S] protein assembly and hence was causative for the observed clinical defects.
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http://dx.doi.org/10.1093/hmg/ddy183DOI Listing
August 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

Biochemical characterization of a new mitochondrial transporter of dephosphocoenzyme A in Drosophila melanogaster.

Biochim Biophys Acta Bioenerg 2017 Feb 9;1858(2):137-146. Epub 2016 Nov 9.

Department of Biosciences, Biotechnologies and Biopharmaceutics, Laboratory of Biochemistry and Molecular Biology, University of Bari, via Orabona 4, 70125 Bari, Italy. Electronic address:

CoA is an essential cofactor that holds a central role in cell metabolism. Although its biosynthetic pathway is conserved across the three domains of life, the subcellular localization of the eukaryotic biosynthetic enzymes and the mechanism behind the cytosolic and mitochondrial CoA pools compartmentalization are still under debate. In humans, the transport of CoA across the inner mitochondrial membrane has been ascribed to two related genes, SLC25A16 and SLC25A42 whereas in D. melanogaster genome only one gene is present, CG4241, phylogenetically closer to SLC25A42. CG4241 encodes two alternatively spliced isoforms, dPCoAC-A and dPCoAC-B. Both isoforms were expressed in Escherichia coli, but only dPCoAC-A was successfully reconstituted into liposomes, where transported dPCoA and, to a lesser extent, ADP and dADP but not CoA, which was a powerful competitive inhibitor. The expression of both isoforms in a Saccharomyces cerevisiae strain lacking the endogenous putative mitochondrial CoA carrier restored the growth on respiratory carbon sources and the mitochondrial levels of CoA. The results reported here and the proposed subcellular localization of some of the enzymes of the fruit fly CoA biosynthetic pathway, suggest that dPCoA may be synthesized and phosphorylated to CoA in the matrix, but it can also be transported by dPCoAC to the cytosol, where it may be phosphorylated to CoA by the monofunctional dPCoA kinase. Thus, dPCoAC may connect the cytosolic and mitochondrial reactions of the CoA biosynthetic pathway without allowing the two CoA pools to get in contact.
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http://dx.doi.org/10.1016/j.bbabio.2016.11.006DOI Listing
February 2017

Novel mutations in IBA57 are associated with leukodystrophy and variable clinical phenotypes.

J Neurol 2017 Jan 26;264(1):102-111. Epub 2016 Oct 26.

Unit of Muscular and Neurodegenerative Disorders, Laboratory of Molecular Medicine, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy.

Defects of the Fe/S cluster biosynthesis represent a subgroup of diseases affecting the mitochondrial energy metabolism. In the last years, mutations in four genes (NFU1, BOLA3, ISCA2 and IBA57) have been related to a new group of multiple mitochondrial dysfunction syndromes characterized by lactic acidosis, hyperglycinemia, multiple defects of the respiratory chain complexes, and impairment of four lipoic acid-dependent enzymes: α-ketoglutarate dehydrogenase complex, pyruvic dehydrogenase, branched-chain α-keto acid dehydrogenase complex and the H protein of the glycine cleavage system. Few patients have been reported with mutations in IBA57 and with variable clinical phenotype. Herein, we describe four unrelated patients carrying novel mutations in IBA57. All patients presented with combined or isolated defect of complex I and II. Clinical features varied widely, ranging from fatal infantile onset of the disease to acute and severe psychomotor regression after the first year of life. Brain MRI was characterized by cavitating leukodystrophy. The identified mutations were never reported previously and all had a dramatic effect on IBA57 stability. Our study contributes to expand the array of the genotypic variation of IBA57 and delineates the leukodystrophic pattern of IBA57 deficient patients.
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http://dx.doi.org/10.1007/s00415-016-8312-zDOI Listing
January 2017

New insights about the structural rearrangements required for substrate translocation in the bovine mitochondrial oxoglutarate carrier.

Biochim Biophys Acta 2016 11 30;1864(11):1473-80. Epub 2016 Jul 30.

Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036 Arcavacata di Rende, Cosenza, Italy; Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Via Orabona 4, 70125 Bari, Italy. Electronic address:

The oxoglutarate carrier (OGC) belongs to the mitochondrial carrier family and plays a key role in important metabolic pathways. Here, site-directed mutagenesis was used to conservatively replace lysine 122 by arginine, in order to investigate new structural rearrangements required for substrate translocation. K122R mutant was kinetically characterized, exhibiting a significant Vmax reduction with respect to the wild-type (WT) OGC, whereas Km value was unaffected, implying that this substitution does not interfere with 2-oxoglutarate binding site. Moreover, K122R mutant was more inhibited by several sulfhydryl reagents with respect to the WT OGC, suggesting that the reactivity of some cysteine residues towards these Cys-specific reagents is increased in this mutant. Different sulfhydryl reagents were employed in transport assays to test the effect of the cysteine modifications on single-cysteine OGC mutants named C184, C221, C224 (constructed in the WT background) and K122R/C184, K122R/C221, K122R/C224 (constructed in the K122R background). Cysteines 221 and 224 were more deeply influenced by some sulfhydryl reagents in the K122R background. Furthermore, the presence of 2-oxoglutarate significantly enhanced the degree of inhibition of K122R/C221, K122R/C224 and C224 activity by the sulfhydryl reagent 2-Aminoethyl methanethiosulfonate hydrobromide (MTSEA), suggesting that cysteines 221 and 224, together with K122, take part to structural rearrangements required for the transition from the c- to the m-state during substrate translocation. Our results are interpreted in the light of the homology model of BtOGC, built by using as a template the X-ray structure of the bovine ADP/ATP carrier isoform 1 (AAC1).
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http://dx.doi.org/10.1016/j.bbapap.2016.07.009DOI Listing
November 2016

Functional characterization and organ distribution of three mitochondrial ATP-Mg/Pi carriers in Arabidopsis thaliana.

Biochim Biophys Acta 2015 Oct 2;1847(10):1220-30. Epub 2015 Jul 2.

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

The Arabidopsis thaliana genome contains 58 membrane proteins belonging to the mitochondrial carrier family. Three members of this family, here named AtAPC1, AtAPC2, and AtAPC3, exhibit high structural similarities to the human mitochondrial ATP-Mg(2+)/phosphate carriers. Under normal physiological conditions the AtAPC1 gene was expressed at least five times more than the other two AtAPC genes in flower, leaf, stem, root and seedlings. However, in stress conditions the expression levels of AtAPC1 and AtAPC3 change. Direct transport assays with recombinant and reconstituted AtAPC1, AtAPC2 and AtAPC3 showed that they transport phosphate, AMP, ADP, ATP, adenosine 5'-phosphosulfate and, to a lesser extent, other nucleotides. AtAPC2 and AtAPC3 also had the ability to transport sulfate and thiosulfate. All three AtAPCs catalyzed a counter-exchange transport that was saturable and inhibited by pyridoxal-5'-phosphate. The transport activities of AtAPCs were also inhibited by the addition of EDTA or EGTA and stimulated by the addition of Ca(2+). Given that phosphate and sulfate can be recycled via their own specific carriers, these findings indicate that AtAPCs can catalyze net transfer of adenine nucleotides across the inner mitochondrial membrane in exchange for phosphate (or sulfate), and that this transport is regulated both at the transcriptional level and by Ca(2+).
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http://dx.doi.org/10.1016/j.bbabio.2015.06.015DOI Listing
October 2015

Riboflavin responsive mitochondrial myopathy is a new phenotype of dihydrolipoamide dehydrogenase deficiency. The chaperon-like effect of vitamin B2.

Mitochondrion 2014 Sep 22;18:49-57. Epub 2014 Sep 22.

Division of Metabolism, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy. Electronic address:

Dihydrolipoamide dehydrogenase (DLD, E3) is a flavoprotein common to pyruvate, α-ketoglutarate and branched-chain α-keto acid dehydrogenases. We found two novel DLD mutations (p.I40Lfs*4; p.G461E) in a 19 year-old patient with lactic acidosis and a complex amino- and organic aciduria consistent with DLD deficiency, manifesting progressive exertional fatigue. Muscle biopsy showed mitochondrial proliferation and lack of DLD cross-reacting material. Riboflavin supplementation determined the complete resolution of exercise intolerance with the partial restoration of the DLD protein and disappearance of mitochondrial proliferation in the muscle. Morphological and functional studies support the riboflavin chaperon-like role in stabilizing DLD protein with rescue of its expression in the muscle.
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http://dx.doi.org/10.1016/j.mito.2014.09.006DOI Listing
September 2014

UCP2 transports C4 metabolites out of mitochondria, regulating glucose and glutamine oxidation.

Proc Natl Acad Sci U S A 2014 Jan 6;111(3):960-5. Epub 2014 Jan 6.

Department of Biosciences, Biotechnologies, and Biopharmaceutics and Center of Excellence in Comparative Genomics, University of Bari, 70125 Bari, Italy.

Uncoupling protein 2 (UCP2) is involved in various physiological and pathological processes such as insulin secretion, stem cell differentiation, cancer, and aging. However, its biochemical and physiological function is still under debate. Here we show that UCP2 is a metabolite transporter that regulates substrate oxidation in mitochondria. To shed light on its biochemical role, we first studied the effects of its silencing on the mitochondrial oxidation of glucose and glutamine. Compared with wild-type, UCP2-silenced human hepatocellular carcinoma (HepG2) cells, grown in the presence of glucose, showed a higher inner mitochondrial membrane potential and ATP:ADP ratio associated with a lower lactate release. Opposite results were obtained in the presence of glutamine instead of glucose. UCP2 reconstituted in lipid vesicles catalyzed the exchange of malate, oxaloacetate, and aspartate for phosphate plus a proton from opposite sides of the membrane. The higher levels of citric acid cycle intermediates found in the mitochondria of siUCP2-HepG2 cells compared with those found in wild-type cells in addition to the transport data indicate that, by exporting C4 compounds out of mitochondria, UCP2 limits the oxidation of acetyl-CoA-producing substrates such as glucose and enhances glutaminolysis, preventing the mitochondrial accumulation of C4 metabolites derived from glutamine. Our work reveals a unique regulatory mechanism in cell bioenergetics and provokes a substantial reconsideration of the physiological and pathological functions ascribed to UCP2 based on its purported uncoupling properties.
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http://dx.doi.org/10.1073/pnas.1317400111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3903233PMC
January 2014

Overexpression in E. coli and purification of the L. pneumophila Lpp2981 protein.

Mol Biotechnol 2014 Feb;56(2):157-65

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

The Lpp2981 gene from Legionella pneumophila, the causative agent of Legionnaire's disease, was cloned into the pMWT7 plasmid. The construct was used to express this gene in Escherichia coli. Five different bacterial strains were tested to overexpress the gene but without success. Sequence analysis revealed a cluster of four rare codons near the 5'-end of the gene. These codons were replaced with those commonly used in E. coli. The mutated Lpp2981 gene was successfully expressed in all the E. coli strains tested. The expressed protein (with an apparent molecular mass of 30 kDa) was collected in the insoluble fraction of the cell lysate, purified as inclusion bodies and functionally reconstituted into liposomes. The highest level of overexpression was obtained in E. coli C0214 after 6 h of induction with isopropyl-β-D-thiogalactopyranoside at 37 °C, yielding 74 mg of purified protein per liter of culture. We conclude that the clustering of rare codons at the 5'-end of the open-reading frame is a critical factor for the heterologous expression of Lpp2981 in E. coli.
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http://dx.doi.org/10.1007/s12033-013-9691-3DOI Listing
February 2014

A new Caucasian case of neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD): a clinical, molecular, and functional study.

Mol Genet Metab 2011 Dec 25;104(4):501-6. Epub 2011 Aug 25.

Department of Pharmaco-Biology, Laboratory of Biochemistry and Molecular Biology, University of Bari, Bari, Italy.

Citrin is the liver-specific isoform of the mitochondrial aspartate/glutamate carrier (AGC2). AGC2 deficiency is an autosomal recessive disorder with two age related phenotypes: neonatal intrahepatic cholestasis (NICCD, OMIM#605814) and adult-onset type II citrullinemia (CTLN2, OMIM#603471). NICCD arises within the first few weeks of life resulting in prolonged cholestasis and metabolic abnormalities including aminoacidemia and galactosuria. Usually symptoms disappear within the first year of life, thus making a diagnosis difficult after this time. In this study we report a new Caucasian case of NICCD, a seven week old Romanian boy with prolonged jaundice. Sequencing of the AGC2 gene showed a novel homozygous missense double-nucleotide (doublet) mutation, which produces the change of the glycine at position 437 into glutamate. Functional studies, carried out on the recombinant mutant protein, for the first time demonstrated, that NICCD is caused by a reduced transport activity of AGC2. The presence of AGC2 deficiency in other ethnic groups besides Asian population suggests further consideration for NICCD diagnosis of any neonate with an unexplained cholestasis; a prompt diagnosis is crucial to resolve the metabolic decompensation with an appropriate dietary treatment.
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http://dx.doi.org/10.1016/j.ymgme.2011.08.022DOI Listing
December 2011

The biochemical properties of the mitochondrial thiamine pyrophosphate carrier from Drosophila melanogaster.

FEBS J 2010 Mar 27;277(5):1172-81. Epub 2010 Jan 27.

Department of Pharmaco-Biology, University of Calabria, Arcavacata di Rende, Cosenza, Italy.

The mitochondrial carriers are a family of transport proteins that shuttle metabolites, nucleotides and cofactors across the inner mitochondrial membrane. The genome of Drosophila melanogaster encodes at least 46 members of this family. Only five of these have been characterized, whereas the transport functions of the remainder cannot be assessed with certainty. In the present study, we report the functional identification of two D. melanogaster genes distantly related to the human and yeast thiamine pyrophosphate carrier (TPC) genes as well as the corresponding expression pattern throughout development. Furthermore, the functional characterization of the D. melanogaster mitochondrial thiamine pyrophosphate carrier protein (DmTpc1p) is described. DmTpc1p was over-expressed in bacteria, the purified protein was reconstituted into liposomes, and its transport properties and kinetic parameters were characterized. Reconstituted DmTpc1p transports thiamine pyrophosphate and, to a lesser extent, pyrophosphate, ADP, ATP and other nucleotides. The expression of DmTpc1p in Saccharomyces cerevisiaeTPC1 null mutant abolishes the growth defect on fermentable carbon sources. The main role of DmTpc1p is to import thiamine pyrophosphate into mitochondria by exchange with intramitochondrial ATP and/or ADP.
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http://dx.doi.org/10.1111/j.1742-4658.2009.07550.xDOI Listing
March 2010

Transcription of the mitochondrial citrate carrier gene: identification of a silencer and its binding protein ZNF224.

Biochem Biophys Res Commun 2009 Aug 6;386(1):186-91. Epub 2009 Jun 6.

Department of Pharmaco-Biology, University of Bari, Bari, Italy.

In the last few years, we have been functionally characterizing the promoter of the human mitochondrial citrate carrier (CIC). In this study we show that CIC silencer activity extends over 26 bp (-595/-569), which specifically bind a protein present in HepG2 cell nuclear extracts. This transcription factor was purified by DNA affinity and identified as ZNF224. Overexpression of ZNF224 decreases LUC transgene activity in cells transfected with a construct containing the CIC silencer region, whereas ZNF224 silencing activates reporter transcription in cells transfected with the same construct. Moreover, overexpression and silencing of ZNF224 diminishes and enhances, respectively, CIC transcript and protein levels. Finally, ZNF224 is abundantly expressed in fetal tissues contrary to CIC. It is suggested that CIC transcriptional repression by ZNF224 explains, at least in part, the low expression of CIC in fetal tissues in which fatty acid synthesis is low.
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http://dx.doi.org/10.1016/j.bbrc.2009.06.003DOI Listing
August 2009

Cytopathic effects of the cytomegalovirus-encoded apoptosis inhibitory protein vMIA.

J Cell Biol 2006 Sep 18;174(7):985-96. Epub 2006 Sep 18.

Centre National de la Recherche Scientifique, FRE2939, Institut Gustave Roussy, F-94805 Villejuif, France.

Replication of human cytomegalovirus (CMV) requires the expression of the viral mitochondria-localized inhibitor of apoptosis (vMIA). vMIA inhibits apoptosis by recruiting Bax to mitochondria, resulting in its neutralization. We show that vMIA decreases cell size, reduces actin polymerization, and induces cell rounding. As compared with vMIA-expressing CMV, vMIA-deficient CMV, which replicates in fibroblasts expressing the adenoviral apoptosis suppressor E1B19K, induces less cytopathic effects. These vMIA effects can be separated from its cell death-inhibitory function because vMIA modulates cellular morphology in Bax-deficient cells. Expression of vMIA coincided with a reduction in the cellular adenosine triphosphate (ATP) level. vMIA selectively inhibited one component of the ATP synthasome, namely, the mitochondrial phosphate carrier. Exposure of cells to inhibitors of oxidative phosphorylation produced similar effects, such as an ATP level reduced by 30%, smaller cell size, and deficient actin polymerization. Similarly, knockdown of the phosphate carrier reduced cell size. Our data suggest that the cytopathic effect of CMV can be explained by vMIA effects on mitochondrial bioenergetics.
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http://dx.doi.org/10.1083/jcb.200604069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2064390PMC
September 2006

Identification of mitochondrial carriers in Saccharomyces cerevisiae by transport assay of reconstituted recombinant proteins.

Biochim Biophys Acta 2006 Sep-Oct;1757(9-10):1249-62. Epub 2006 May 23.

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

The inner membranes of mitochondria contain a family of carrier proteins that are responsible for the transport in and out of the mitochondrial matrix of substrates, products, co-factors and biosynthetic precursors that are essential for the function and activities of the organelle. This family of proteins is characterized by containing three tandem homologous sequence repeats of approximately 100 amino acids, each folded into two transmembrane alpha-helices linked by an extensive polar loop. Each repeat contains a characteristic conserved sequence. These features have been used to determine the extent of the family in genome sequences. The genome of Saccharomyces cerevisiae contains 34 members of the family. The identity of five of them was known before the determination of the genome sequence, but the functions of the remaining family members were not. This review describes how the functions of 15 of these previously unknown transport proteins have been determined by a strategy that consists of expressing the genes in Escherichia coli or Saccharomyces cerevisiae, reconstituting the gene products into liposomes and establishing their functions by transport assay. Genetic and biochemical evidence as well as phylogenetic considerations have guided the choice of substrates that were tested in the transport assays. The physiological roles of these carriers have been verified by genetic experiments. Various pieces of evidence point to the functions of six additional members of the family, but these proposals await confirmation by transport assay. The sequences of many of the newly identified yeast carriers have been used to characterize orthologs in other species, and in man five diseases are presently known to be caused by defects in specific mitochondrial carrier genes. The roles of eight yeast mitochondrial carriers remain to be established.
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http://dx.doi.org/10.1016/j.bbabio.2006.05.023DOI Listing
December 2006

Identification of the mitochondrial GTP/GDP transporter in Saccharomyces cerevisiae.

J Biol Chem 2004 May 3;279(20):20850-7. Epub 2004 Mar 3.

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

The genome of Saccharomyces cerevisiae contains 35 members of a family of transport proteins that, with a single exception, are found in the inner membranes of mitochondria. The transport functions of the 16 biochemically identified mitochondrial carriers are concerned with shuttling substrates, biosynthetic intermediates, and cofactors across the inner membrane. Here the identification and functional characterization of the mitochondrial GTP/GDP carrier (Ggc1p) is described. The ggc1 gene was overexpressed in bacteria. The purified protein was reconstituted into liposomes, and its transport properties and kinetic parameters were characterized. It transported GTP and GDP and, to a lesser extent, the corresponding deoxynucleotides and the structurally related ITP and IDP by a counter-exchange mechanism. Transport was saturable with an apparent K(m) of 1 microm for GTP and 5 microm for GDP. It was strongly inhibited by pyridoxal 5'-phosphate, bathophenanthroline, tannic acid, and bromcresol purple but little affected by the inhibitors of the ADP/ATP carrier carboxyatractyloside and bongkrekate. Furthermore, in contrast to the ADP/ATP carrier, the Ggc1p-mediated GTP/GDP heteroexchange is H(+)-compensated and thus electroneutral. Cells lacking the ggc1 gene had reduced levels of GTP and increased levels of GDP in their mitochondria. Furthermore, the knock-out of ggc1 results in lack of growth on nonfermentable carbon sources and complete loss of mitochondrial DNA. The physiological role of Ggc1p in S. cerevisiae is probably to transport GTP into mitochondria, where it is required for important processes such as nucleic acid and protein synthesis, in exchange for intramitochondrially generated GDP.
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http://dx.doi.org/10.1074/jbc.M313610200DOI Listing
May 2004