Publications by authors named "Ciro L Pierri"

12 Publications

  • Page 1 of 1

Glufosinate constrains synchronous and metachronous metastasis by promoting anti-tumor macrophages.

EMBO Mol Med 2020 10 4;12(10):e11210. Epub 2020 Sep 4.

Laboratory of Tumor Inflammation and Angiogenesis, Center for Cancer Biology (CCB), VIB, Leuven, Belgium.

Glutamine synthetase (GS) generates glutamine from glutamate and controls the release of inflammatory mediators. In macrophages, GS activity, driven by IL10, associates to the acquisition of M2-like functions. Conditional deletion of GS in macrophages inhibits metastasis by boosting the formation of anti-tumor, M1-like, tumor-associated macrophages (TAMs). From this basis, we evaluated the pharmacological potential of GS inhibitors in targeting metastasis, identifying glufosinate as a specific human GS inhibitor. Glufosinate was tested in both cultured macrophages and on mice bearing metastatic lung, skin and breast cancer. We found that glufosinate rewires macrophages toward an M1-like phenotype both at the primary tumor and metastatic site, countering immunosuppression and promoting vessel sprouting. This was also accompanied to a reduction in cancer cell intravasation and extravasation, leading to synchronous and metachronous metastasis growth inhibition, but no effects on primary tumor growth. Glufosinate treatment was well-tolerated, without liver and brain toxicity, nor hematopoietic defects. These results identify GS as a druggable enzyme to rewire macrophage functions and highlight the potential of targeting metabolic checkpoints in macrophages to treat cancer metastasis.
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http://dx.doi.org/10.15252/emmm.201911210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7539200PMC
October 2020

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

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

A Variant of GJD2, Encoding for Connexin 36, Alters the Function of Insulin Producing β-Cells.

PLoS One 2016 9;11(3):e0150880. Epub 2016 Mar 9.

Department of Cell Physiology and Metabolism, University of Geneva Faculty of Medicine, Geneva, Switzerland.

Signalling through gap junctions contributes to control insulin secretion and, thus, blood glucose levels. Gap junctions of the insulin-producing β-cells are made of connexin 36 (Cx36), which is encoded by the GJD2 gene. Cx36-null mice feature alterations mimicking those observed in type 2 diabetes (T2D). GJD2 is also expressed in neurons, which share a number of common features with pancreatic β-cells. Given that a synonymous exonic single nucleotide polymorphism of human Cx36 (SNP rs3743123) associates with altered function of central neurons in a subset of epileptic patients, we investigated whether this SNP also caused alterations of β-cell function. Transfection of rs3743123 cDNA in connexin-lacking HeLa cells resulted in altered formation of gap junction plaques and cell coupling, as compared to those induced by wild type (WT) GJD2 cDNA. Transgenic mice expressing the very same cDNAs under an insulin promoter revealed that SNP rs3743123 expression consistently lead to a post-natal reduction of islet Cx36 levels and β-cell survival, resulting in hyperglycemia in selected lines. These changes were not observed in sex- and age-matched controls expressing WT hCx36. The variant GJD2 only marginally associated to heterogeneous populations of diabetic patients. The data document that a silent polymorphism of GJD2 is associated with altered β-cell function, presumably contributing to T2D pathogenesis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0150880PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4784816PMC
August 2016

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

Pathogenic potential of SLC25A15 mutations assessed by transport assays and complementation of Saccharomyces cerevisiae ORT1 null mutant.

Mol Genet Metab 2015 May 17;115(1):27-32. Epub 2015 Mar 17.

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

HHH syndrome is an autosomal recessive urea cycle disorder caused by alterations in the SLC25A15 gene encoding the mitochondrial ornithine carrier 1, which catalyzes the transport of cytosolic ornithine into the mitochondria in exchange for intramitochondrial citrulline. In this study the functional effects of several SLC25A15 missense mutations p.G27R, p.M37R, p.N74A, p.F188L, p.F188Y, p.S200K, p.R275Q and p.R275K have been tested by transport assays in reconstituted liposomes and complementation of Saccharomyces cerevisiae ORT1 null mutant in arginine-less synthetic complete medium. The HHH syndrome-causing mutations p.G27R, p.M37R, p.F188L and p.R275Q had impaired transport and did not complement ORT1∆ cells (except p.M37R slightly after 5 days in solid medium). The experimentally produced mutations p.N74A, p.S200K and p.R275K exhibited normal or considerable transport activity and complemented ORT1∆ cells after 3 days (p.N74A, p.S200K) or 5 days (p.R275K) incubation. Furthermore, the experimentally produced p.F188Y mutation displayed a substantial transport activity but did not complement the ORT1∆ cells in both liquid and solid media. In view of the disagreement in the results obtained between the two methods, it is recommended that the method of complementing the S. cerevisiae ORT1 knockout strain is used complimentary with the measurement of the catalytic activity, in order to distinguish HHH syndrome-causing mutations from isomorphisms.
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http://dx.doi.org/10.1016/j.ymgme.2015.03.003DOI Listing
May 2015

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

Evolution, structure and function of mitochondrial carriers: a review with new insights.

Plant J 2011 Apr;66(1):161-81

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

The mitochondrial carriers (MC) constitute a large family (MCF) of inner membrane transporters displaying different substrate specificities, patterns of gene expression and even non-mitochondrial organelle localization. In Arabidopsis thaliana 58 genes encode these six trans-membrane domain proteins. The number in other sequenced plant genomes varies from 37 to 125, thus being larger than that of Saccharomyces cerevisiae and comparable with that of Homo sapiens. In addition to displaying highly similar secondary structures, the proteins of the MCF can be subdivided into subfamilies on the basis of substrate specificity and the presence of specific symmetry-related amino acid triplets. We assessed the predictive power of these triplets by comparing predictions with experimentally determined data for Arabidopsis MCs, and applied these predictions to the not yet functionally characterized mitochondrial carriers of the grass, Brachypodium distachyon, and the alga, Ostreococcus lucimarinus. We additionally studied evolutionary aspects of the plant MCF by comparing sequence data of the Arabidopsis MCF with those of Saccharomyces cerevisiae and Homo sapiens, then with those of Brachypodium distachyon and Ostreococcus lucimarinus, employing intra- and inter-genome comparisons. Finally, we discussed the importance of the approaches of global gene expression analysis and in vivo characterizations in order to address the relevance of these vital carrier proteins.
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http://dx.doi.org/10.1111/j.1365-313X.2011.04516.xDOI Listing
April 2011

Novel hydroxycinnamoyl-coenzyme A quinate transferase genes from artichoke are involved in the synthesis of chlorogenic acid.

Plant Physiol 2010 Jul 29;153(3):1224-38. Epub 2010 Apr 29.

Institute of Plant Genetics, National Research Council, 70126 Bari, Italy.

Artichoke (Cynara cardunculus subsp. scolymus) extracts have high antioxidant capacity, due primarily to flavonoids and phenolic acids, particularly chlorogenic acid (5-caffeoylquinic acid [CGA]), dicaffeoylquinic acids, and caffeic acid, which are abundant in flower bracts and bioavailable to humans in the diet. The synthesis of CGA can occur following different routes in plant species, and hydroxycinnamoyl-coenzyme A transferases are important enzymes in these pathways. Here, we report on the isolation and characterization of two novel genes both encoding hydroxycinnamoyl-coenzyme A quinate transferases (HQT) from artichoke. The recombinant proteins (HQT1 and HQT2) were assayed after expression in Escherichia coli, and both showed higher affinity for quinate over shikimate. Their preferences for acyl donors, caffeoyl-coenzyme A or p-coumaroyl-coenzyme A, were examined. Modeling and docking analyses were used to propose possible pockets and residues involved in determining substrate specificities in the HQT enzyme family. Quantitative real-time polymerase chain reaction analysis of gene expression indicated that HQT1 might be more directly associated with CGA content. Transient and stable expression of HQT1 in Nicotiana resulted in a higher production of CGA and cynarin (1,3-dicaffeoylquinic acid). These findings suggest that several isoforms of HQT contribute to the synthesis of CGA in artichoke according to physiological needs and possibly following various metabolic routes.
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http://dx.doi.org/10.1104/pp.109.150144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2899911PMC
July 2010

AGC1 deficiency associated with global cerebral hypomyelination.

N Engl J Med 2009 Jul;361(5):489-95

Center for Inherited Metabolic Diseases, Karolinska University Hospital, Stockholm, Sweden.

The mitochondrial aspartate-glutamate carrier isoform 1 (AGC1), specific to neurons and muscle, supplies aspartate to the cytosol and, as a component of the malate-aspartate shuttle, enables mitochondrial oxidation of cytosolic NADH, thought to be important in providing energy for neurons in the central nervous system. We describe AGC1 deficiency, a novel syndrome characterized by arrested psychomotor development, hypotonia, and seizures in a child with a homozygous missense mutation in the solute carrier family 25, member 12, gene SLC25A12, which encodes the AGC1 protein. Functional analysis of the mutant AGC1 protein showed abolished activity. The child had global hypomyelination in the cerebral hemispheres, suggesting that impaired efflux of aspartate from neuronal mitochondria prevents normal myelin formation.
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http://dx.doi.org/10.1056/NEJMoa0900591DOI Listing
July 2009

Identification of novel mutations in the SLC25A15 gene in hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome: a clinical, molecular, and functional study.

Hum Mutat 2009 May;30(5):741-8

Molecular Medicine and Metabolism, Istituto di Ricovero e Cura a Carattere Scientifico Children's Hospital Bambino Gesù, Rome, Italy.

Hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome is an autosomal recessive disorder of the urea cycle. With the exception of the French-Canadian founder effect, no common mutation has been detected in other populations. In this study, we collected 16 additional HHH cases and expanded the spectrum of SLC25A15/ORC1 mutations. Eleven novel mutations were identified including six new missense and one microrearrangement. We also measured the transport properties of the recombinant purified proteins in reconstituted liposomes for four new and two previously reported missense mutations and proved that the transport activities of these mutant forms of ORC1 were reduced as compared with the wild-type protein; residual activity ranged between 4% and 19%. Furthermore, we designed three-dimensional (3D)-modeling of mutant ORC1 proteins. While modeling the changes in silico allowed us to obtain new information on the pathomechanisms underlying HHH syndrome, we found no clear-cut genotype-phenotype correlations. Although patient metabolic alterations responded well to low-protein therapy, predictions concerning the long-term evolution of HHH syndrome remain uncertain. The preference for a hepatic rather than a neurological presentation at onset also continues, largely, to elude us. Neither modifications in oxidative metabolism-related energy, such as those expected in different mtDNA haplogroups, nor sequence variants in SLC25A2/ORC2 seem to be crucial. Other factors, including protein stability and function, and ORC1-ORC2 structural interactions should be further investigated.
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http://dx.doi.org/10.1002/humu.20930DOI Listing
May 2009

alpha-Isopropylmalate, a leucine biosynthesis intermediate in yeast, is transported by the mitochondrial oxalacetate carrier.

J Biol Chem 2008 Oct 5;283(42):28445-53. Epub 2008 Aug 5.

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

In Saccharomyces cerevisiae, alpha-isopropylmalate (alpha-IPM), which is produced in mitochondria, must be exported to the cytosol where it is required for leucine biosynthesis. Recombinant and reconstituted mitochondrial oxalacetate carrier (Oac1p) efficiently transported alpha-IPM in addition to its known substrates oxalacetate, sulfate, and malonate and in contrast to other di- and tricarboxylate transporters as well as the previously proposed alpha-IPM transporter. Transport was saturable with a half-saturation constant of 75 +/- 4 microm for alpha-IPM and 0.31 +/- 0.04 mm for beta-IPM and was inhibited by the substrates of Oac1p. Though not transported, alpha-ketoisocaproate, the immediate precursor of leucine in the biosynthetic pathway, inhibited Oac1p activity competitively. In contrast, leucine, alpha-ketoisovalerate, valine, and isoleucine neither inhibited nor were transported by Oac1p. Consistent with the function of Oac1p as an alpha-IPM transporter, cells lacking the gene for this carrier required leucine for optimal growth on fermentable carbon sources. Single deletions of other mitochondrial carrier genes or of LEU4, which is the only other enzyme that can provide the cytosol with alpha-IPM (in addition to Oac1p) exhibited no growth defect, whereas the double mutant DeltaOAC1DeltaLEU4 did not grow at all on fermentable substrates in the absence of leucine. The lack of growth of DeltaOAC1DeltaLEU4 cells was partially restored by adding the leucine biosynthetic cytosolic intermediates alpha-ketoisocaproate and alpha-IPM to these cells as well as by complementing them with one of the two unknown human mitochondrial carriers SLC25A34 and SLC25A35. Oac1p is important for leucine biosynthesis on fermentable carbon sources catalyzing the export of alpha-IPM, probably in exchange for oxalacetate.
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http://dx.doi.org/10.1074/jbc.M804637200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2661403PMC
October 2008