Iolanda Spera

Università degli Studi di Bari Aldo Moro, Bari, Apulia, Italy

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Publications (7)29.59 Total impact

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    ABSTRACT: 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. Copyright © 2015. Published by Elsevier B.V.
    Full-text · Article · Apr 2015 · Biochimica et Biophysica Acta (BBA) - Bioenergetics
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    ABSTRACT: The role of glutamine synthetase (GS) during adipocyte differentiation is unclear. Here, we assess the impact of GS on the adipocytic response to a proinflammatory challenge at different differentiation stages. GS expression at the late stages of differentiation desensitized mature adipocytes to bacterial lipopolysaccharide (LPS) by increasing intracellular glutamine levels. Furthermore, LPS-activated mature adipocytes were unable to produce inflammatory mediators; LPS sensitivity was rescued following GS inhibition and the associated drop in intracellular glutamine levels. The ability of adipocytes to differentially respond to LPS during differentiation negatively correlates to GS expression and intracellular glutamine levels. Hence, modulation of intracellular glutamine levels by GS expression represents an endogenous mechanism through which mature adipocytes control the inflammatory response. Copyright © 2014. Published by Elsevier B.V.
    Full-text · Article · Nov 2014 · FEBS Letters
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    ABSTRACT: Purpose: Oxidative stress plays a key role in cardiac diseases, although the sources of reactive oxygen species (ROS) have not been defined conclusively. Recent studies demonstrated that the mitochondrial enzymes monoamine oxidases (MAO) are a major source of ROS in reperfusion injury and decompensated hypertrophy. The present study characterized the molecular mechanisms responsible for the increased activity of MAO. Based upon available information, the activity of these enzymes depends mostly on substrate availability. Therefore, we aimed at identifying the major substrates of MAO in hearts undergoing oxidative stress. Mass spectrometry was used to identify and quantitate potential substrates by comparing their contents in the absence and the presence of MAO inhibition. Methods and Results: Firstly, we applied a metabolomic profiling method to investigate changes in amine contents in isolated mouse hearts, by means of a LC-MS/MS approach in the precursor ion scanning mode. Maximal oxidative stress was induced by perfusing isolated mouse hearts with 1 mM hydrogen peroxide for 15 min. Addition of 0.5 mM pargyline to the perfusion buffer 10 min before hydrogen peroxide resulted in a significant increased content of the typical MAO substrates serotonin and epinephrine, along with histamine and its product N1-methyl histamine. N1-methyl histamine was found to be the more aboundant metabolite and its content displayed a 180% increase in pargyline-treated hearts, as compared to the untreated ones. The accumulation of MAO substrates upon pargyline treatment correlated with a reduced MAO-dependent production of hydrogen peroxide. In fact we observed a decreased extent of (i) oxidation of myofibrillar proteins, as detected by disulfide bond formation in tropomyosin (Western blot under non reducing conditions), and (ii) ROS levels in tissue, as detected by dihydroethidine (DHE) staining. Surprisingly, these findings imply that the profound injury induced by H2O2 administration is not due to a direct action. Indeed, H2O2 perfusion appears to trigger an amplification pathway whereby the increase in MAO activity due to a larger substrate availability is the end-effector of the initial oxidative stress. Conclusions: This study provides the first information on endogenous substrates of MAO becoming available under conditions of oxidative stress that is then amplified by the increased MAO activity. The identification of histamine and N1-methyl histamine, that are involved in neurotransmission and immune response, suggests a significant trafficking of MAO substrates between myocytes and non-myocyte cells in the heart.
    Preview · Article · Jul 2014 · Cardiovascular Research
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    ABSTRACT: Pathological changes occur in areas of CNS tissue remote from inflammatory lesions in multiple sclerosis (MS) and its animal model experimental allergic encephalomyelitis (EAE). To determine if oxidative stress is a significant contributor to this non-inflammatory pathology, cortex tissues from mice with clinical signs of EAE were examined for evidence of inflammation and oxidative stress. Histology and gene expression analysis showed little evidence of immune/inflammatory cell invasion but reductions in natural antioxidant levels and increased protein oxidation that paralleled disease severity. Two-dimensional oxyblots and mass-spectrometry-based protein fingerprinting identified glutamine synthetase (GS) as a particular target of oxidation. Oxidation of GS was associated with reductions in enzyme activity and increased glutamate/glutamine levels. The possibility that this may cause neurodegeneration through glutamate excitotoxicity is supported by evidence of increasing cortical Ca(2+) levels in cortex extracts from animals with greater disease severity. These findings indicate that oxidative stress occurs in brain areas that are not actively undergoing inflammation in EAE and that this can lead to a neurodegenerative process due to the susceptibility of GS to oxidative inactivation.
    Full-text · Article · Jun 2011 · Neuroscience
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    ABSTRACT: In Down's syndrome there is evidence that increased gene expression coding for specific cystathionine beta-synthase translates directly into biochemical aberrations, which result in a biochemical and metabolic imbalance of the methyl status. This event is destined to impact mitochondrial function since methylation is a necessary event in mitochondria and relies on the availability and uptake of the methyl donor S-adenosylmethionine. Indeed mitochondrial dysfunctions have been widely described in Down's syndrome, but they have never been correlated to a possible mitochondrial methyl unbalance. In the present study we find that the mitochondrial levels of S-adenosylmethionine are reduced in Down's syndrome compared to control cells demonstrating the effect of the methyl unbalance on mitochondria. The possible role of methylation in mitochondria is discussed and some preliminary results on a possible methylation target are presented.
    Full-text · Article · Mar 2011 · Molecular Genetics and Metabolism
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    ABSTRACT: Mitochondrial carriers are a family of transport proteins that shuttle metabolites, nucleotides, and coenzymes across the mitochondrial membrane. The function of only a few of the 35 Saccharomyces cerevisiae mitochondrial carriers still remains to be uncovered. In this study, we have functionally defined and characterized the S. cerevisiae mitochondrial carrier Yhm2p. The YHM2 gene was overexpressed in S. cerevisiae, and its product was purified and reconstituted into liposomes. Its transport properties, kinetic parameters, and targeting to mitochondria show that Yhm2p is a mitochondrial transporter for citrate and oxoglutarate. Reconstituted Yhm2p also transported oxaloacetate, succinate, and fumarate to a lesser extent, but virtually not malate and isocitrate. Yhm2p catalyzed only a counter-exchange transport that was saturable and inhibited by sulfhydryl-blocking reagents but not by 1,2,3-benzenetricarboxylate (a powerful inhibitor of the citrate/malate carrier). The physiological role of Yhm2p is to increase the NADPH reducing power in the cytosol (required for biosynthetic and antioxidant reactions) and probably to act as a key component of the citrate-oxoglutarate NADPH redox shuttle between mitochondria and cytosol. This protein function is based on observations documenting a decrease in the NADPH/NADP+ and GSH/GSSG ratios in the cytosol of ΔYHM2 cells as well as an increase in the NADPH/NADP+ ratio in their mitochondria compared with wild-type cells. Our proposal is also supported by the growth defect displayed by the ΔYHM2 strain and more so by the ΔYHM2ΔZWF1 strain upon H2O2 exposure, implying that Yhm2p has an antioxidant function.
    Full-text · Article · Jun 2010 · Journal of Biological Chemistry
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    ABSTRACT: Mitochondrial carriers are a family of transport proteins that shuttle metabolites, nucleotides, and coenzymes across the mitochondrial membrane. The function of only a few of the 35 Saccharomyces cerevisiae mitochondrial carriers still remains to be uncovered. In this study, we have functionally defined and characterized the S. cerevisiae mitochondrial carrier Yhm2p. The YHM2 gene was overexpressed in S. cerevisiae, and its product was purified and reconstituted into liposomes. Its transport properties, kinetic parameters, and targeting to mitochondria show that Yhm2p is a mitochondrial transporter for citrate and oxoglutarate. Reconstituted Yhm2p also transported oxaloacetate, succinate, and fumarate to a lesser extent, but virtually not malate and isocitrate. Yhm2p catalyzed only a counter-exchange transport that was saturable and inhibited by sulfhydryl-blocking reagents but not by 1,2,3-benzenetricarboxylate (a powerful inhibitor of the citrate/malate carrier). The physiological role of Yhm2p is to increase the NADPH reducing power in the cytosol (required for biosynthetic and antioxidant reactions) and probably to act as a key component of the citrate-oxoglutarate NADPH redox shuttle between mitochondria and cytosol. This protein function is based on observations documenting a decrease in the NADPH/NADP+ and GSH/GSSG ratios in the cytosol of ΔYHM2 cells as well as an increase in the NADPH/NADP+ ratio in their mitochondria compared with wild-type cells. Our proposal is also supported by the growth defect displayed by the ΔYHM2 strain and more so by the ΔYHM2ΔZWF1 strain upon H2O2 exposure, implying that Yhm2p has an antioxidant function.
    Full-text · Article · Apr 2010 · Journal of Biological Chemistry