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ABSTRACT: Aldehyde oxidases (AOXs) and xanthine dehydrogenases (XDHs) belong to the family of molybdo-flavoenzymes. Although AOXs are not identifiable in fungi, these enzymes are represented in certain protists and the majority of plants and vertebrates. The physiological functions and substrates of AOXs are unknown. Nevertheless, AOXs are major drug metabolizing enzymes, oxidizing a wide range of aromatic aldehydes and heterocyclic compounds of medical/toxicological importance. Using genome sequencing data, we predict the structures of AOX genes and pseudogenes, reconstructing their evolution. Fishes are the most primitive organisms with an AOX gene (AOXα), originating from the duplication of an ancestral XDH. Further evolution of fishes resulted in the duplication of AOXα into AOXβ and successive pseudogenization of AOXα. AOXβ is maintained in amphibians and it is the likely precursors of reptilian, avian, and mammalian AOX1. Amphibian AOXγ is a duplication of AOXβ and the likely ancestor of reptilian and avian AOX2, which, in turn, gave rise to mammalian AOX3L1. Subsequent gene duplications generated the two mammalian genes, AOX3 and AOX4. The evolution of mammalian AOX genes is dominated by pseudogenization and deletion events. Our analysis is relevant from a structural point of view, as it provides information on the residues characterizing the three domains of each mammalian AOX isoenzyme. We cloned the cDNAs encoding the AOX proteins of guinea pig and cynomolgus monkeys, two unique species as to the evolution of this enzyme family. We identify chimeric RNAs from the human AOX3 and AOX3L1 pseudogenes with potential to encode a novel microRNA.
Cellular and Molecular Life Sciences CMLS 12/2012; · 6.57 Impact Factor
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Maddalena Fratelli,
James Neil Fisher,
Gabriela Paroni,
Angela Maria Di Francesco,
Filomena Pierri,
Claudio Pisano,
Klaus Godl,
Sandra Marx,
Andreas Tebbe,
Claudia Valli,
Maurizio Gianni,
Matteo Stravalaci,
Marco Gobbi,
Mineko Terao,
Enrico Garattini
[show abstract]
[hide abstract]
ABSTRACT: ST1926 is an atypical retinoid and a promising anti-tumour agent with selective apoptotic activity on the leukaemic blast. The anti-tumour activity of the compound has been associated with its capacity to induce DNA double stranded breaks. Target profiling by affinity chromatography coupled to mass spectrometry led to the identification of histone H2A.Z as a protein capable of binding ST1926 specifically. The result was confirmed by studies involving Surface Plasmon Resonance (SPR). This indicates that H2A.Z is a primary target of ST1926 and links the perturbations of the histone pathway observed by microarray analysis to the DNA damage and apoptotic responses caused by the atypical retinoid. Comparison of the whole-genome gene-expression profiles of the ST1926-sensitive NB4 and the ST1926-resistant NB4.437r cell lines demonstrated differential expression of numerous genes. Network analysis of the data indicated enrichment of the cellular pathways controlling cAMP (cyclic adenosinemonophosphate)-dependent signal transduction, proteasome-dependent protein degradation and nuclear histones in NB4.437r cells. Pharmacological inhibition of cAMP-dependent protein kinase A with H89 partially reverted resistance of NB4.437r cells to ST1926. Conversely, inhibition of the proteasome with MG132 or bortezomib blocked the apoptotic response afforded by ST1926 in the NB4 cell line. This last effect was associated with a dramatic reduction in the DNA damage caused by the atypical retinoid. The results corroborate the idea that DNA damage is an important determinant of ST1926 apoptotic activity. More importantly, they demonstrate a proactive role of the proteasome in the DNA damaging and ensuing apoptotic response observed upon the challenge of acute myeloid leukaemia cells with ST1926.
European journal of cancer (Oxford, England: 1990) 12/2012; · 4.12 Impact Factor
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Denise Locatelli,
Mineko Terao, Maddalena Fratelli,
Adriana Zanetti,
Mami Kurosaki,
Monica Lupi,
Maria Monica Barzago,
Andrea Uggetti,
Silvia Capra,
Paolo D'Errico,
Giorgio S Battaglia,
Enrico Garattini
[show abstract]
[hide abstract]
ABSTRACT: Spinal muscular atrophy is a fatal genetic disease of motoneurons due to loss of full-length survival of motor neuron protein, the main product of the disease gene SMN1. Axonal SMN (a-SMN) is an alternatively spliced isoform of SMN1, generated by retention of intron 3. To study a-SMN function, we generated cellular clones for the expression of the protein in mouse motoneuron-like NSC34 cells. The model was instrumental in providing evidence that a-SMN decreases cell growth and plays an important role in the processes of axon growth and cellular motility. In our conditions, low levels of a-SMN expression were sufficient to trigger the observed biological effects, which were not modified by further increasing the amounts of the expressed protein. Differential transcriptome analysis led to the identification of novel a-SMN-regulated factors, i.e. the transcripts coding for the two chemokines, C-C motif ligands 2 and 7 (CCL2 and CCL7), as well as the neuronal and myotrophic factor, insulin-like growth factor-1 (IGF1). a-SMN-dependent induction of CCL2 and IGF1 mRNAs resulted in increased intracellular levels and secretion of the respective protein products. Induction of CCL2 contributes to the a-SMN effects, mediating part of the action on axon growth and random cell motility, as indicated by chemokine knockdown and re-addition studies. Our results shed new light on a-SMN function and the underlying molecular mechanisms. The data provide a rational framework to understand the role of a-SMN deficiency in the etiopathogenesis of spinal muscular atrophy.
Journal of Biological Chemistry 06/2012; 287(31):25782-94. · 4.77 Impact Factor
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Mineko Terao, Maddalena Fratelli,
Mami Kurosaki,
Adriana Zanetti,
Valeria Guarnaccia,
Gabriela Paroni,
Anna Tsykin,
Monica Lupi,
Maurizio Gianni,
Gregory J. Goodall,
Enrico Garattini
[show abstract]
[hide abstract]
ABSTRACT: Retinoids are promising agents for the treatment/prevention of breast carcinoma. We examined the role of microRNAs in mediating
the effects of all-trans-retinoic acid (ATRA), which suppresses the proliferation of estrogen receptor-positive (ERα+) breast carcinoma cells, such as MCF-7, but not estrogen receptor-negative cells, such as MDA-MB-231. We found that pro-oncogenic miR-21 is selectively induced by ATRA in ERα+ cells. Induction of miR-21 counteracts the anti-proliferative action of ATRA but has the potentially beneficial effect of
reducing cell motility. In ERα+ cells, retinoid-dependent induction of miR-21 is due to increased transcription of the MIR21 gene via ligand-dependent activation of the nuclear retinoid receptor, RARα. RARα is part of the transcription complex present
in the 5′-flanking region of the MIR21 gene. The receptor binds to two functional retinoic acid-responsive elements mapping upstream of the transcription initiation
site. Silencing of miR-21 enhances ATRA-dependent growth inhibition and senescence while reverting suppression of cell motility
afforded by the retinoid. Up-regulation of miR-21 results in retinoid-dependent inhibition of the established target, maspin.
Knockdown and overexpression of maspin in MCF-7 cells indicates that the protein is involved in ATRA-induced growth inhibition and contributes to the ATRA-dependent anti-motility
responses. Integration between whole genome analysis of genes differentially regulated by ATRA in MCF-7 and MDA-MB-231 cells, prediction of miR-21 regulated genes, and functional studies led to the identification of three novel direct miR-21
targets: the pro-inflammatory cytokine IL1B, the adhesion molecule ICAM-1 and PLAT, the tissue-type plasminogen activator.
Evidence for ICAM-1 involvement in retinoid-dependent inhibition of MCF-7 cell motility is provided.
Journal of Biological Chemistry 02/2011; 286(5):4027-4042. · 4.77 Impact Factor
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Mineko Terao, Maddalena Fratelli,
Mami Kurosaki,
Adriana Zanetti,
Valeria Guarnaccia,
Gabriela Paroni,
Anna Tsykin,
Monica Lupi,
Maurizio Gianni,
Gregory J Goodall,
Enrico Garattini
[show abstract]
[hide abstract]
ABSTRACT: Retinoids are promising agents for the treatment/prevention of breast carcinoma. We examined the role of microRNAs in mediating the effects of all-trans-retinoic acid (ATRA), which suppresses the proliferation of estrogen receptor-positive (ERα(+)) breast carcinoma cells, such as MCF-7, but not estrogen receptor-negative cells, such as MDA-MB-231. We found that pro-oncogenic miR-21 is selectively induced by ATRA in ERα(+) cells. Induction of miR-21 counteracts the anti-proliferative action of ATRA but has the potentially beneficial effect of reducing cell motility. In ERα(+) cells, retinoid-dependent induction of miR-21 is due to increased transcription of the MIR21 gene via ligand-dependent activation of the nuclear retinoid receptor, RARα. RARα is part of the transcription complex present in the 5'-flanking region of the MIR21 gene. The receptor binds to two functional retinoic acid-responsive elements mapping upstream of the transcription initiation site. Silencing of miR-21 enhances ATRA-dependent growth inhibition and senescence while reverting suppression of cell motility afforded by the retinoid. Up-regulation of miR-21 results in retinoid-dependent inhibition of the established target, maspin. Knockdown and overexpression of maspin in MCF-7 cells indicates that the protein is involved in ATRA-induced growth inhibition and contributes to the ATRA-dependent anti-motility responses. Integration between whole genome analysis of genes differentially regulated by ATRA in MCF-7 and MDA-MB-231 cells, prediction of miR-21 regulated genes, and functional studies led to the identification of three novel direct miR-21 targets: the pro-inflammatory cytokine IL1B, the adhesion molecule ICAM-1 and PLAT, the tissue-type plasminogen activator. Evidence for ICAM-1 involvement in retinoid-dependent inhibition of MCF-7 cell motility is provided.
Journal of Biological Chemistry 02/2011; 286(5):4027-42. · 4.77 Impact Factor
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Monica Binaschi,
Andrea Boldetti,
Maurizio Gianni,
Carlo Alberto Maggi,
Martina Gensini,
Mario Bigioni,
Massimo Parlani,
Alessandro Giolitti, Maddalena Fratelli,
Claudia Valli,
Mineko Terao,
Enrico Garattini
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ABSTRACT: Histone deacetylases are promising molecular targets for the development of antitumor agents. A novel series of histone deacetylase inhibitors of the hydroxamic acid type were synthesized for structure−activity studies. Thirteen tricyclic dibenzo-diazepine, -oxazepine, and -thiazepine analogues were studied and shown to induce variable degrees of histone H3/H4 and tubulin acetylation in a cellular model of myeloid leukemia sensitive to all-trans retinoic acid (ATRA). Multiparametric correlations between acetylation of the three substrates, tumor cell growth inhibition, and ATRA-dependent cytodifferentiation were performed, providing information on the chemical functionalities governing these activities. For two analogues, antitumor activity in the animal was demonstrated.Keywords (keywords): HDAC inhibitors; hydroxamic acid; retinoic acid; antiproliferative effect; differentiation
07/2010;
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ABSTRACT: Aldehyde oxidases (EC 1.2.3.1) are a small group of structurally conserved cytosolic proteins represented in both the animal and plant kingdoms. In vertebrates, aldehyde oxidases constitute the small sub-family of molybdo-flavoenzymes, along with the evolutionarily and structurally related protein, xanthine oxidoreductase. These enzymes require a molybdo-pterin cofactor (molybdenum cofactor, MoCo) and flavin adenine dinucleotide for their catalytic activity. Aldehyde oxidases have broad substrate specificity and catalyse the hydroxylation of N-heterocycles and the oxidation of aldehydes to the corresponding acid. In humans, a single aldehyde oxidase gene ( AOX1 ) and two pseudogenes clustering on a short stretch of chromosome 2q are known. In other mammals, a variable number of structurally conserved aldehyde oxidase genes has been described. Four genes ( Aox1 , Aox3 , Aox4 and Aox3l1 ), coding for an equivalent number of catalytically active enzymes, are present in the mouse and rat genomes. Although human AOX1 and its homologous proteins are best known as drug metabolising enzymes, the physiological substrate(s) and function(s) are as yet unknown. The present paper provides an update of the available information on the evolutionary history, tissue- and cell-specific distribution and function of mammalian aldehyde oxidases.
Human genomics 12/2009; 4(2):119-30.
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Mineko Terao,
Mami Kurosaki,
Maria Monica Barzago, Maddalena Fratelli,
Renzo Bagnati,
Antonio Bastone,
Chiara Giudice,
Eugenio Scanziani,
Alessandra Mancuso,
Cecilia Tiveron,
Enrico Garattini
[show abstract]
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ABSTRACT: The mouse aldehyde oxidase AOH2 (aldehyde oxidase homolog 2) is a molybdoflavoenzyme. Harderian glands are the richest source of AOH2, although the protein is detectable also in sebaceous glands, epidermis, and other keratinized epithelia. The levels of AOH2 in the Harderian gland and skin are controlled by genetic background, being maximal in CD1 and C57BL/6 and minimal in DBA/2, CBA, and 129/Sv strains. Testosterone is a negative regulator of AOH2 in Harderian glands. Purified AOH2 oxidizes retinaldehyde into retinoic acid, while it is devoid of pyridoxal-oxidizing activity. Aoh2(-/-) mice, the first aldehyde oxidase knockout animals ever generated, are viable and fertile. The data obtained for this knockout model indicate a significant role of AOH2 in the local synthesis and biodisposition of endogenous retinoids in the Harderian gland and skin. The Harderian gland's transcriptome of knockout mice demonstrates overall downregulation of direct retinoid-dependent genes as well as perturbations in pathways controlling lipid homeostasis and cellular secretion, particularly in sexually immature animals. The skin of knockout mice is characterized by thickening of the epidermis in basal conditions and after UV light exposure. This has correlates in the corresponding transcriptome, which shows enrichment and overall upregulation of genes involved in hypertrophic responses.
Molecular and cellular biology 12/2008; 29(2):357-77. · 6.06 Impact Factor
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Claudia Valli,
Gabriela Paroni,
Angela Maria Di Francesco,
Riccardo Riccardi,
Michele Tavecchio,
Eugenio Erba,
Andrea Boldetti,
Maurizio Gianni, Maddalena Fratelli,
Claudio Pisano,
Lucio Merlini,
Antonio Antoccia,
Chiara Cenciarelli,
Mineko Terao,
Enrico Garattini
[show abstract]
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ABSTRACT: Retinoid-related molecules (RRM) are novel agents with tumor-selective cytotoxic/antiproliferative activity, a different mechanism of action from classic retinoids and no cross-resistance with other chemotherapeutics. ST1926 and CD437 are prototypic RRMs, with the former currently undergoing phase I clinical trials. We show here that ST1926, CD437, and active congeners cause DNA damage. Cellular and subcellular COMET assays, H2AX phosphorylation (gamma-H2AX), and scoring of chromosome aberrations indicate that active RRMs produce DNA double-strand breaks (DSB) and chromosomal lesions in NB4, an acute myeloid leukemia (AML) cell line characterized by high sensitivity to RRMs. There is a direct quantitative correlation between the levels of DSBs and the cytotoxic/antiproliferative effects induced by RRMs. NB4.437r blasts, which are selectively resistant to RRMs, do not show any sign of DNA damage after treatment with ST1926, CD437, and analogues. DNA damage is the major mechanism underlying the antileukemic activity of RRMs in NB4 and other AML cell lines. In accordance with the S-phase specificity of the cytotoxic and antiproliferative responses of AML cells to RRMs, increases in DSBs are maximal during the S phase of the cell cycle. Induction of DSBs precedes inhibition of DNA replication and is associated with rapid activation of ataxia telangectasia mutated, ataxia telangectasia RAD3-related, and DNA-dependent protein kinases with subsequent stimulation of the p38 mitogen-activated protein kinase. Inhibition of ataxia telangectasia mutated and DNA-dependent protein kinases reduces phosphorylation of H2AX. Cells defective for homologous recombination are particularly sensitive to ST1926, indicating that this process is important for the protection of cells from the RRM-dependent DNA damage and cytotoxicity.
Molecular Cancer Therapeutics 10/2008; 7(9):2941-54. · 5.23 Impact Factor
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Edoardo Parrella,
Maurizio Giannì, Maddalena Fratelli,
Maria Monica Barzago,
Ivan Raska,
Luisa Diomede,
Mami Kurosaki,
Claudio Pisano,
Paolo Carminati,
Lucio Merlini,
Sabrina Dallavalle,
Michele Tavecchio,
Cecile Rochette-Egly,
Mineko Terao,
Enrico Garattini
[show abstract]
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ABSTRACT: The retinoid-related molecules (RRMs) ST1926 [(E)-3-(4'-hydroxy-3'-adamantylbiphenyl-4-yl)acrylic acid] and CD437 (6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid) are promising anticancer agents. We compared the retinoic acid receptor (RAR) trans-activating properties of the two RRMs and all-trans-retinoic acid (ATRA). ST1926 and CD437 are better RARgamma agonists than ATRA. We used three teratocarcinoma cell lines to evaluate the significance of RARgamma in the activity of RRMs: F9-wild type (WT); F9gamma-/-, lacking the RARgamma gene; F9gamma51, aF9gamma-/-derivative, complemented for the RARgamma deficit. Similar to ATRA, ST1926 and CD437 activate cytodifferentiation only in F9-WT cells. Unlike ATRA, ST1926 and CD437 arrest cells in the G2/M phase of the cell cycle and induce apoptosis in all F9 cell lines. Our data indicate that RARgamma and the classic retinoid pathway are not relevant for the antiproliferative and apoptotic activities of RRMs in vitro. Increases in cytosolic calcium are fundamental for apoptosis, in that intracellular calcium chelators abrogate the process. Comparison of the gene expression profiles associated with ST1926 and ATRA in F9-WT and F9gamma-/-indicates that the RRM activates a conspicuous nonretinoid response in addition to the classic and RAR-dependent pathway. The pattern of genes regulated by ST1926 selectively, in a RARgamma-independent manner, provides novel insights into the possible molecular determinants underlying the activity of RRMs in vitro. Furthermore, it suggests that RARgamma-dependent responses are relevant to the activity of RRMs in vivo. Indeed, the receptor hinders the antitumor activity in vivo, in that both syngeneic and immunosuppressed SCID mice bearing F9gamma-/- tumors have increased life spans after treatment with ST1926 and CD437 relative to their F9-WT counterparts.
Molecular Pharmacology 10/2006; 70(3):909-24. · 4.88 Impact Factor
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ABSTRACT: Aldehyde oxidases are molybdo-flavoenzymes structurally related to xanthine oxidoreductase. They catalyze the oxidation of aldehydes or N-heterocycles of physiological, pharmacological, and toxicological relevance. Rodents are characterized by four aldehyde oxidases as follows: AOX1 and aldehyde oxidase homologs 1-3 (AOH1, AOH2, and AOH3). Humans synthesize a single functional aldehyde oxidase, AOX1. Here we define the structure and the characteristics of the aldehyde oxidase genes and proteins in chicken and dog. The avian genome contains two aldehyde oxidase genes, AOX1 and AOH, mapping to chromosome 7. AOX1 and AOH are structurally very similar and code for proteins whose sequence was deduced from the corresponding cDNAs. AOX1 is the ortholog of the same gene in mammals, whereas AOH represents the likely ancestor of rodent AOH1, AOH2, and AOH3. The dog genome is endowed with two structurally conserved and active aldehyde oxidases clustering on chromosome 37. Cloning of the corresponding cDNAs and tissue distribution studies demonstrate that they are the orthologs of rodent AOH2 and AOH3. The vestiges of dog AOX1 and AOH1 are recognizable upstream of AOH2 and AOH3 on the same chromosome. Comparison of the complement and the structure of the aldehyde oxidase and xanthine oxidoreductase genes in vertebrates and other animal species indicates that they evolved through a series of duplication and inactivation events. Purification of the chicken AOX1 protein to homogeneity from kidney demonstrates that the enzyme possesses retinaldehyde oxidase activity. Unlike humans and most other mammals, dog and chicken are devoid of liver aldehyde oxidase activity.
Journal of Biological Chemistry 08/2006; 281(28):19748-61. · 4.77 Impact Factor
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Pietro Ghezzi,
Simona Casagrande,
Tania Massignan,
Manuela Basso,
Emanuele Bellacchio,
Luca Mollica,
Emiliano Biasini,
Rossella Tonelli,
Ivano Eberini,
Elisabetta Gianazza,
Wei Wei Dai, Maddalena Fratelli,
Mario Salmona,
Barbara Sherry,
Valentina Bonetto
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ABSTRACT: Using redox proteomics techniques to characterize the thiol status of proteins in human T lymphocytes, we identified cyclophilin A (CypA) as a specifically oxidized protein early after mitogen activation. CypA is an abundantly expressed cytosolic protein, target of the immunosuppressive drug cyclosporin A (CsA), for which a variety of functions has been described. In this study, we could identify CypA as a protein undergoing glutathionylation in vivo. Using MALDI-MS we identified Cys52 and Cys62 as targets of glutathionylation in T lymphocytes, and, using bioinformatic tools, we defined the reasons for the susceptibility of these residues to the modification. In addition, we found by circular dichroism spectroscopy that glutathionylation has an important impact on the secondary structure of CypA. Finally, we suggest that glutathionylation of CypA may have biological implications and that CypA may play a key role in redox regulation of immunity.
PROTEOMICS 03/2006; 6(3):817-25. · 4.51 Impact Factor
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ABSTRACT: Proteins can form reversible mixed disulfides with glutathione (GSH). It has been hypothesized that protein glutathionylation may represent a mechanism of redox regulation, in a fashion similar to that mediated by protein phosphorylation. We investigated whether GSH has a signaling role in the response of HL60 cells to hydrogen peroxide (H2O2), in addition to its obvious antioxidant role. We identified early changes in gene expression induced at different times by H2O2 treatment, under conditions that increase protein glutathionylation and minimal toxicity. We then investigated the effect of prior GSH depletion by buthionine sulfoximine and diethylmaleate on this response. The analysis revealed 2,016 genes regulated by H2O2. Of these, 215 genes showed GSH-dependent expression changes, classifiable into four clusters displaying down- or up-regulation by H2O2, either potentiated or inhibited by GSH depletion. The modulation of 20 selected genes was validated by real-time RT-PCR. The biological process categories overrepresented in the largest cluster (genes whose up-regulation was inhibited by GSH depletion) were NF-kappaB activation, transcription, and DNA methylation. This cluster also included several cytokine and chemokine ligands and receptors, the redox regulator thioredoxin interacting protein, and the histone deacetylase sirtuin. The cluster of genes whose up-regulation was potentiated by GSH depletion included two HSPs (HSP40 and HSP70) and the AP-1 transcription factor components Fos and FosB. This work demonstrates that GSH, in addition to its antioxidant and protective function against oxidative stress, has a specific signaling role in redox regulation.
Proceedings of the National Academy of Sciences 10/2005; 102(39):13998-4003. · 9.68 Impact Factor
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ABSTRACT: Although radical oxygen and nitrogen species are harmful molecules that destroy cell functions, many operate as mediators of important cell signaling pathways when not in excess. Oxidants can modify protein function through the covalent, reversible addition of glutathione to cysteine. This review addresses different proteomic methods of identifying glutathionylation targets and emphasizes ways of defining their pattern of modification in response to oxidative stimuli in cells. Finally, the literature on nonproteomic studies that investigate the functional changes induced by glutathionylation are reviewed and future studies are commented on.
Expert Review of Proteomics 11/2004; 1(3):365-76. · 3.68 Impact Factor
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Michael Brines,
Giovanni Grasso,
Fabio Fiordaliso,
Alessandra Sfacteria,
Pietro Ghezzi, Maddalena Fratelli,
Roberto Latini,
Qiao-Wen Xie,
John Smart,
Chiao-Ju Su-Rick,
Eileen Pobre,
Deborah Diaz,
Daniel Gomez,
Carla Hand,
Thomas Coleman,
Anthony Cerami
[show abstract]
[hide abstract]
ABSTRACT: The cytokine erythropoietin (Epo) is tissue-protective in preclinical models of ischemic, traumatic, toxic, and inflammatory injuries. We have recently characterized Epo derivatives that do not bind to the Epo receptor (EpoR) yet are tissue-protective. For example, carbamylated Epo (CEpo) does not stimulate erythropoiesis, yet it prevents tissue injury in a wide variety of in vivo and in vitro models. These observations suggest that another receptor is responsible for the tissue-protective actions of Epo. Notably, prior investigation suggests that EpoR physically interacts with the common beta receptor (betacR), the signal-transducing subunit shared by the granulocyte-macrophage colony stimulating factor, and the IL-3 and IL-5 receptors. However, because betacR knockout mice exhibit normal erythrocyte maturation, betacR is not required for erythropoiesis. We hypothesized that betacR in combination with the EpoR expressed by nonhematopoietic cells constitutes a tissue-protective receptor. In support of this hypothesis, membrane proteins prepared from rat brain, heart, liver, or kidney were greatly enriched in EpoR after passage over either Epo or CEpo columns but covalently bound in a complex with betacR. Further, antibodies against EpoR coimmunoprecipitated betacR from membranes prepared from neuronal-like P-19 cells that respond to Epo-induced tissue protection. Immunocytochemical studies of spinal cord neurons and cardiomyocytes protected by Epo demonstrated cellular colocalization of Epo betacR and EpoR. Finally, as predicted by the hypothesis, neither Epo nor CEpo was active in cardiomyocyte or spinal cord injury models performed in the betacR knockout mouse. These data support the concept that EpoR and betacR comprise a tissue-protective heteroreceptor.
Proceedings of the National Academy of Sciences 11/2004; 101(41):14907-12. · 9.68 Impact Factor
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Michael Brines,
Giovanni Grasso,
Fabio Fiordaliso,
Alessandra Sfacteria,
Pietro Ghezzi, Maddalena Fratelli,
Roberto Latini,
Qiao-wen Xie,
John Smart,
Chiao-ju Su-Rick,
Eileen Pobre,
Deborah Diaz,
Daniel Gomez,
Carla Hand,
Thomas Coleman,
Anthony Cerami
[show abstract]
[hide abstract]
ABSTRACT: The cytokine erythropoietin (Epo) is tissue-protective in preclinical models of ischemic, traumatic, toxic, and inflammatory
injuries. We have recently characterized Epo derivatives that do not bind to the Epo receptor (EpoR) yet are tissue-protective.
For example, carbamylated Epo (CEpo) does not stimulate erythropoiesis, yet it prevents tissue injury in a wide variety of
in vivo and in vitro models. These observations suggest that another receptor is responsible for the tissue-protective actions of Epo. Notably,
prior investigation suggests that EpoR physically interacts with the common β receptor (βcR), the signal-transducing subunit
shared by the granulocyte-macrophage colony stimulating factor, and the IL-3 and IL-5 receptors. However, because βcR knockout
mice exhibit normal erythrocyte maturation, βcR is not required for erythropoiesis. We hypothesized that βcR in combination
with the EpoR expressed by nonhematopoietic cells constitutes a tissue-protective receptor. In support of this hypothesis,
membrane proteins prepared from rat brain, heart, liver, or kidney were greatly enriched in EpoR after passage over either
Epo or CEpo columns but covalently bound in a complex with βcR. Further, antibodies against EpoR coimmunoprecipitated βcR
from membranes prepared from neuronal-like P-19 cells that respond to Epo-induced tissue protection. Immunocytochemical studies
of spinal cord neurons and cardiomyocytes protected by Epo demonstrated cellular colocalization of Epo βcR and EpoR. Finally,
as predicted by the hypothesis, neither Epo nor CEpo was active in cardiomyocyte or spinal cord injury models performed in
the βcR knockout mouse. These data support the concept that EpoR and βcR comprise a tissue-protective heteroreceptor.
Proceedings of the National Academy of Sciences 10/2004; 101(41):14907-14912. · 9.68 Impact Factor
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Marcel Leist,
Pietro Ghezzi,
Giovanni Grasso,
Roberto Bianchi,
Pia Villa, Maddalena Fratelli,
Costanza Savino,
Marina Bianchi,
Jacob Nielsen,
Jens Gerwien, [......],
Alessandra Sfacteria,
Serhat Erbayraktar,
Zubeyde Erbayraktar,
Necati Gokmen,
Osman Yilmaz,
Carla Cerami-Hand,
Qiao-Wen Xie,
Thomas Coleman,
Anthony Cerami,
Michael Brines
[show abstract]
[hide abstract]
ABSTRACT: Erythropoietin (EPO) is both hematopoietic and tissue protective, putatively through interaction with different receptors. We generated receptor subtype-selective ligands allowing the separation of EPO's bioactivities at the cellular level and in animals. Carbamylated EPO (CEPO) or certain EPO mutants did not bind to the classical EPO receptor (EPOR) and did not show any hematopoietic activity in human cell signaling assays or upon chronic dosing in different animal species. Nevertheless, CEPO and various nonhematopoietic mutants were cytoprotective in vitro and conferred neuroprotection against stroke, spinal cord compression, diabetic neuropathy, and experimental autoimmune encephalomyelitis at a potency and efficacy comparable to EPO.
Science 08/2004; 305(5681):239-42. · 31.20 Impact Factor
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ABSTRACT: Protein glutathionylation is a post-translational modification consisting of the formation of a mixed disulfide between protein cysteines and glutathione (GSH). To identify proteins undergoing glutathionylation in primary rat hepatocytes and in human HepG2 hepatoma cells, we radiolabeled the intracellular GSH pool with L-[(35)S] cysteine. Cells were then exposed to oxidative stress. Proteins were separated by two-dimensional gel electrophoresis under nonreducing conditions, and glutathionylated proteins were located by autoradiography and identified by mass spectrometry after tryptic digestion. Several proteins previously not known to undergo glutathionylation were thus recognized. Among the identified proteins some are the same or belong to the same functional class as those we have already identified in a previous paper on T cell blasts (actin, nucleophosmin, phosphogluconolactonase, myosin, profilin, cyclophilin A, stress 70 protein, ubiquitin in HepG2 cells and actin, peroxiredoxin 5, cytochrome C oxidase, heat shock cognate 70 in hepatocytes) while others are newly recognized (Ran specific GTPase activating protein, histidine triad nucleotide binding protein 2 in HepG2 cells and enoyl CoA hydratase in hepatocytes). The technique described proved equally applicable to a variety of cell types.
PROTEOMICS 08/2003; 3(7):1154-61. · 4.51 Impact Factor
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Serhat Erbayraktar,
Giovanni Grasso,
Alessandra Sfacteria,
Qiao-wen Xie,
Thomas Coleman,
Mads Kreilgaard,
Lars Torup,
Thomas Sager,
Zubeyde Erbayraktar,
Necati Gokmen, [......],
Marcel Leist,
Lone Helboe,
Jens Gerwein,
Søren Christensen,
Marie Aavang Geist,
Lars Østergaard Pedersen,
Carla Cerami-Hand,
Jean-Paul Wuerth,
Anthony Cerami,
Michael Brines
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ABSTRACT: Erythropoietin (EPO) is a tissue-protective cytokine preventing vascular spasm, apoptosis, and inflammatory responses. Although best known for its role in hematopoietic lineages, EPO also affects other tissues, including those of the nervous system. Enthusiasm for recombinant human erythropoietin (rhEPO) as a potential neuroprotective therapeutic must be tempered, however, by the knowledge it also enlarges circulating red cell mass and increases platelet aggregability. Here we examined whether erythropoietic and tissue-protective activities of rhEPO might be dissociated by a variation of the molecule. We demonstrate that asialoerythropoietin (asialoEPO), generated by total enzymatic desialylation of rhEPO, possesses a very short plasma half-life and is fully neuroprotective. In marked contrast with rhEPO, this molecule at doses and frequencies at which rhEPO exhibited erythropoiesis, did not increase the hematocrit of mice or rats. AsialoEPO appeared promptly within the cerebrospinal fluid after i.v. administration; intravenously administered radioiodine-labeled asialoEPO bound to neurons within the hippocampus and cortex in a pattern corresponding to the distribution of the EPO receptor. Most importantly, asialoEPO exhibits a broad spectrum of neuroprotective activities, as demonstrated in models of cerebral ischemia, spinal cord compression, and sciatic nerve crush. These data suggest that nonerythropoietic variants of rhEPO can cross the blood-brain barrier and provide neuroprotection.
Proceedings of the National Academy of Sciences 06/2003; 100(11):6741-6. · 9.68 Impact Factor
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ABSTRACT: Nonsteroidal antiinflammatory drugs (NSAIDs) are widely used in the treatment of chronic inflammatory states. In addition, they show promise for the prevention and therapy of colon cancers and of Alzheimer's disease, although their gastrointestinal toxicity is of concern for these indications. Nitric oxide-releasing NSAIDs are reported to be safer than their parent compounds. We report here that flurbiprofen nitroxybutyl ester inhibits nuclear factor-kappaB (NF-kappaB) activity and cell growth in L929 cells at a concentration of 100 microM, whereas flurbiprofen is inactive. Inhibition of cell growth is not due to the induction of apoptosis, but to a retardation of all phases of the cell cycle. NF-kappaB is implicated both in the control of immune and inflammatory response and in the control of cell proliferation and apoptosis. Therefore, its inhibition at low concentrations by an NSAID with low gastrointestinal toxicity could be important for all the above-mentioned therapeutic indications.
Antioxidants and Redox Signaling 05/2003; 5(2):229-35. · 8.46 Impact Factor
