Giovanni Musci | Università del Molise (original) (raw)
Papers by Giovanni Musci
Pharmaceutics
SARS-CoV-2 causes COVID-19, a predominantly pulmonary disease characterized by a burst of pro-inf... more SARS-CoV-2 causes COVID-19, a predominantly pulmonary disease characterized by a burst of pro-inflammatory cytokines and an increase in free iron. The viral glycoprotein Spike mediates fusion to the host cell membrane, but its role as a virulence factor is largely unknown. Recently, the antiviral activity of lactoferrin against SARS-CoV-2 was demonstrated in vitro and shown to occur via binding to cell surface receptors, and its putative interaction with Spike was suggested by in silico analyses. We investigated the anti-SARS-CoV-2 activity of bovine and human lactoferrins in epithelial and macrophagic cells using a Spike-decorated pseudovirus. Lactoferrin inhibited pseudoviral fusion and counteracted the deleterious effects of Spike on iron and inflammatory homeostasis by restoring basal levels of iron-handling proteins and of proinflammatory cytokines IL-1β and IL-6. Using pull-down assays, we experimentally proved for the first time that lactoferrin binds to Spike, immediately su...
International Journal of Molecular Sciences, 2021
The ferroxidase ceruloplasmin (CP) plays a crucial role in iron homeostasis in vertebrates togeth... more The ferroxidase ceruloplasmin (CP) plays a crucial role in iron homeostasis in vertebrates together with the iron exporter ferroportin. Mutations in the CP gene give rise to aceruloplasminemia, a rare neurodegenerative disease for which no cure is available. Many aspects of the (patho)physiology of CP are still unclear and would benefit from the availability of recombinant protein for structural and functional studies. Furthermore, recombinant CP could be evaluated for enzyme replacement therapy for the treatment of aceruloplasminemia. We report the production and preliminary characterization of high-quality recombinant human CP in glycoengineered Pichia pastoris SuperMan5. A modified yeast strain lacking the endogenous ferroxidase has been generated and employed as host for heterologous expression of the secreted isoform of human CP. Highly pure biologically active protein has been obtained by an improved two-step purification procedure. Glycan analysis indicates that predominant g...
Bio-Algorithms and Med-Systems, 2017
Ferroportin (Fpn) is a membrane protein representing the major cellular iron exporter, essential ... more Ferroportin (Fpn) is a membrane protein representing the major cellular iron exporter, essential for metal translocation from cells into plasma. Despite its pivotal role in human iron homeostasis, many questions on Fpn structure and biology remain unanswered. In this work, we present two novel and more reliable structural models of human Fpn (hFpn; inward-facing and outward-facing conformations) obtained using as templates the recently solved crystal structures of a bacterial homologue of hFpn,
The Journal of Neuroscience, 1997
Hydra feeding response is a very primitive olfactory-like behavior present in a multicellular org... more Hydra feeding response is a very primitive olfactory-like behavior present in a multicellular organism. We investigated the role of nitric oxide (NO) in the induction and control of hydra feeding response. Under basal conditions, hydra specimens produce detectable amounts of nitrite (NO 2 Ϫ), the breakdown product of NO. When hydra were incubated with reduced glutathione (GSH), the typical activator of feeding response, an increase of basal NO production was observed. This effect was inhibited by glutamic or ␣-aminoadipic acids, two GSH antagonists, which block GSH-induced feeding response, and by the NO synthase (NOS) inhibitor L-NAME. Moreover, we found that hydra possess a calcium-dependent (but calmodulin-independent) NOS isoform. By using exogenous NO donors and NOS inhibitors, we demonstrated that NO stimulus can participate both in triggering tentacular movements and in recruiting neighbor tentacles during hydra feeding response. By using dbt 2-cGMP, an analog to cGMP, we observed that the NO effect was independent of cGMP pathway. Our results strongly implicate NO involvement in hydra very primitive feeding behavior, thus confirming its preservation throughout evolution.
Biometals : an international journal on the role of metal ions in biology, biochemistry, and medicine, Jan 16, 2018
In the last 20 years, several new genes and proteins involved in iron metabolism in eukaryotes, p... more In the last 20 years, several new genes and proteins involved in iron metabolism in eukaryotes, particularly related to pathological states both in animal models and in humans have been identified, and we are now starting to unveil at the molecular level the mechanisms of iron absorption, the regulation of iron transport and the homeostatic balancing processes. In this review, we will briefly outline the general scheme of iron metabolism in humans and then focus our attention on the cellular iron export system formed by the permease ferroportin and the ferroxidase ceruloplasmin. We will finally summarize data on the role of the iron binding protein lactoferrin on the regulation of the ferroportin/ceruloplasmin couple and of other proteins involved in iron homeostasis in inflamed human macrophages.
Frontiers in immunology, 2017
Human lactoferrin (hLf), an 80-kDa multifunctional iron-binding cationic glycoprotein, is constit... more Human lactoferrin (hLf), an 80-kDa multifunctional iron-binding cationic glycoprotein, is constitutively secreted by exocrine glands and by neutrophils during inflammation. hLf is recognized as a key element in the host immune defense system. The in vitro and in vivo experiments are carried out with bovine Lf (bLf), which shares high sequence homology and identical functions with hLf, including anti-inflammatory activity. Here, in "pure" M1 human macrophages, obtained by stimulation with a mixture of 10 pg/ml LPS and 20 ng/ml IFN-γ, as well as in a more heterogeneous macrophage population, challenged with high-dose of LPS (1 µg/ml), the effect of bLf on the expression of the main proteins involved in iron and inflammatory homeostasis, namely ferroportin (Fpn), membrane-bound ceruloplasmin (Cp), cytosolic ferritin (Ftn), transferrin receptor 1, and cytokines has been investigated. The increase of IL-6 and IL-1β cytokines, following the inflammatory treatments, is associated...
![Research paper thumbnail of Pichia pastoris Fep1 is a [2Fe-2S] protein with a Zn finger that displays an unusual oxygen-dependent role in cluster binding](https://attachments.academia-assets.com/96110552/thumbnails/1.jpg)
](Scientific reports, Aug 22, 2016
Fep1, the iron-responsive GATA factor from the methylotrophic yeast Pichia pastoris, has been cha... more Fep1, the iron-responsive GATA factor from the methylotrophic yeast Pichia pastoris, has been characterised both in vivo and in vitro. This protein has two Cys2-Cys2 type zinc fingers and a set of four conserved cysteines arranged in a Cys-X5-Cys-X8-Cys-X2-Cys motif located between the two zinc fingers. Electronic absorption and resonance Raman spectroscopic analyses in anaerobic and aerobic conditions indicate that Fep1 binds iron in the form of a [2Fe-2S] cluster. Site-directed mutagenesis shows that replacement of the four cysteines with serine inactivates this transcriptional repressor. Unexpectedly, the inactive mutant is still able to bind a [2Fe-2S] cluster, employing two cysteine residues belonging to the first zinc finger. These two cysteine residues can act as alternative cluster ligands selectively in aerobically purified Fep1 wild type, suggesting that oxygen could play a role in Fep1 function by causing differential localization of the [Fe-S] cluster.
Cellular and molecular biology (Noisy-le-Grand, France), Jan 17, 2007
We have firstly investigated the toxicological activity by hemolytic assay of crude extract obtai... more We have firstly investigated the toxicological activity by hemolytic assay of crude extract obtained by sonication of holotrichous isorhiza isolated nematocysts of the Scyphozoan Pelagia noctiluca, collected in the Strait of Messina. The hemolytic activity was both time- and dose-dependent on fish, rabbit, chicken and human red blood cells. At lowest doses rabbit and chicken erythrocytes were the most sensitive, whereas those of eel were the most resistant to the crude extract. Different storage conditions, such as -20 degrees C, -80 degrees C for up to 6 months and lyophilization, did not affect the stability of crude venom. Moreover, neither treatment at 4 degrees C, 20 degrees C and 37 degrees C for different time periods ranging between 30 min and 24 h, nor harsh thermal treatment at 80 degrees C and 100 degrees C affected the hemolytic power. The crude venom resulted even stable towards proteolysis and alkaline pH values.
The FASEB Journal, 2002
The amyloid peptides Aβ1-42 and Aβ25-35 strongly inhibited the activity of constitutive neuronal ... more The amyloid peptides Aβ1-42 and Aβ25-35 strongly inhibited the activity of constitutive neuronal and endothelial nitric oxide synthases (i.e., NOS-I and NOS-III, respectively) in cellfree assays. The molecular mechanism of NOS inhibition by Aβ fragments was studied in detail with Aβ25-35. The inhibitory ability was mostly NADPH-dependent and specific for the soluble form of Aβ25-35. Optical, fluorescence, and NMR spectroscopy showed that the soluble, but not aggregated, Aβ25-35 interacted with NADPH, thus suggesting that a direct recruitment of NADPH may result in diminished availability of the redox cofactor for NOS functioning. To assess the physiological relevance of our findings, rat neuronal-like PC12 and glioma C6 cell lines were used as cellular models. After Aβ25-35 internalization into cells was verified, the activity of constitutive NOS was measured using the DAF-2DA detection system and found to be severely impaired upon Aβ25-35 uptake. Consistent with previous results on the molecular cross-talk between NOS isoforms, repression of constitutive NOS by Aβ25-35 resulted in enhanced expression of inducible NOS (NOS-II) mRNA in C6 cells. Our results represent the first evidence that amyloid fragments impair constitutive NOS activity in cell-free and cellular systems, providing a possible molecular mechanism for the onset and/or maintenance of Alzheimer's disease.
The EMBO Journal, 2006
Ferritin is a cytosolic molecule comprised of subunits that self-assemble into a nanocage capable... more Ferritin is a cytosolic molecule comprised of subunits that self-assemble into a nanocage capable of containing up to 4500 iron atoms. Iron stored within ferritin can be mobilized for use within cells or exported from cells. Expression of ferroportin (Fpn) results in export of cytosolic iron and ferritin degradation. Fpn-mediated iron loss from ferritin occurs in the cytosol and precedes ferritin degradation by the proteasome. Depletion of ferritin iron induces the monoubiquitination of ferritin subunits. Ubiquitination is not required for iron release but is required for disassembly of ferritin nanocages, which is followed by degradation of ferritin by the proteasome. Specific mammalian machinery is not required to extract iron from ferritin. Iron can be removed from ferritin when ferritin is expressed in Saccharomyces cerevisiae, which does not have endogenous ferritin. Expressed ferritin is monoubiquitinated and degraded by the proteasome. Exposure of ubiquitination defective mammalian cells to the iron chelator desferrioxamine leads to degradation of ferritin in the lysosome, which can be prevented by inhibitors of autophagy. Thus, ferritin degradation can occur through two different mechanisms.
Proceedings of the National Academy of Sciences, 2005
Mutations in the iron exporter ferroportin (Fpn) (IREG1, SLC40A1 , and MTP1) result in hemochroma... more Mutations in the iron exporter ferroportin (Fpn) (IREG1, SLC40A1 , and MTP1) result in hemochromatosis type IV, a disorder with a dominant genetic pattern of inheritance and heterogeneous clinical presentation. Most patients develop iron loading of Kupffer cells with relatively low saturation of plasma transferrin, but others present with high transferrin saturation and iron-loaded hepatocytes. We show that known human mutations introduced into mouse Fpn-GFP generate proteins that either are defective in cell surface localization or have a decreased ability to be internalized and degraded in response to hepcidin. Studies using coimmunoprecipitation of epitope-tagged Fpn and size-exclusion chromatography demonstrated that Fpn is multimeric. Both WT and mutant Fpn participate in the multimer, and mutant Fpn can affect the localization of WT Fpn, its stability, and its response to hepcidin. The behavior of mutant Fpn in cell culture and the ability of mutant Fpn to act as a dominant ne...
Neuroscience Letters, 2010
Previously, we demonstrated that IL-1β was able to increase iron efflux from glial cells through ... more Previously, we demonstrated that IL-1β was able to increase iron efflux from glial cells through a coordinate induction of both ferroportin-1 (Fpn) and ceruloplasmin (Cp) synthesis. In this study, we have investigated the signaling pathways that are involved in the transcriptional activation of the Cp and Fpn. Our data show that the expression of Cp and Fpn in response to IL-1β requires the activation of MAP kinase pathways as a consequence of an IL-1β receptor stimulation. Moreover, we have observed that IL-1β regulates the expression of Cp and Fpn genes through (i) p38 MAPK-mediated activation of C/EBP transcription factor, (ii) ERK1/2-, JNK1- and partially p38 MAPK-dependent activation of AP-1, and through (iii) activation of NF-κB partially mediated by p38 MAPK.
Neuroscience Letters, 2010
Astrocytes accumulate iron under chronic oxidative stress conditions in ageing and neurological d... more Astrocytes accumulate iron under chronic oxidative stress conditions in ageing and neurological disorders. The soybean isoflavone genistein possesses antioxidant properties and selective estrogen-like activities. Here, a possible role of genistein in modulation of iron transport was explored in glial cells. Genistein significantly increased iron export through estrogen receptor--dependent p38 MAPK activation. Evidence is presented that this effect is associated to a p38 MAPK-triggered up-regulation of the iron export system made by ceruloplasmin and ferroportin-1, a pathway requiring activation of the transcription factor C/EBP.
Neuroscience Letters, 2004
A number of pathologies, including neurodegeneration and inflammation, have been associated with ... more A number of pathologies, including neurodegeneration and inflammation, have been associated with iron dysmetabolism in the brain. Hence, systems involved in iron homeostasis at the cellular level have aroused considerable interest in recent years. The iron exporter ferroportin-1 (FP) and the multicopper oxidase ceruloplasmin (CP) are essential for iron efflux from cells. By using RT-PCR, we demonstrate that FP and CP gene expression is up-regulated by treatment with the pro-inflammatory cytokine IL-1b in rat C6 cells, taken as a glial cellular model. Following stimulation with IL-1b, a higher expression level of CP and FP was also confirmed by Western blotting. Moreover, IL-1b has been found to increase iron efflux from C6 cells, suggesting that both proteins may play a crucial role in iron homeostasis in pathological brain conditions, such as inflammatory and/or neurodegenerative diseases.
Neuroscience Letters, 2011
Previously, we demonstrated that IL-1β was able to increase iron efflux from glial cells through ... more Previously, we demonstrated that IL-1β was able to increase iron efflux from glial cells through a coordinate induction of both ferroportin-1 (Fpn) and ceruloplasmin (Cp) synthesis. In this study, we have investigated the signaling pathways that are involved in the transcriptional activation of the Cp and Fpn. Our data show that the expression of Cp and Fpn in response to
Molecular Biology of the Cell, 2007
Ferroportin (Fpn) is the only known iron exporter in vertebrates. Hepcidin, a peptide secreted by... more Ferroportin (Fpn) is the only known iron exporter in vertebrates. Hepcidin, a peptide secreted by the liver in response to iron or inflammation, binds to Fpn, inducing its internalization and degradation. We show that after binding of hepcidin, Fpn is tyrosine phosphorylated at the plasma membrane. Mutants of human Fpn that do not get internalized or that are internalized slowly show either absent or impaired phosphorylation. We identify adjacent tyrosines as the phosphorylation sites and show that mutation of both tyrosines prevents hepcidin-mediated Fpn internalization. Once internalized, Fpn is dephosphorylated and subsequently ubiquitinated. An inability to ubiquitinate Fpn does not prevent hepcidin-induced internalization, but it inhibits the degradation of Fpn. Ubiquitinated Fpn is trafficked through the multivesicular body pathway en route to degradation in the late endosome/lysosome. Depletion of proteins involved in multivesicular body trafficking (Endosome Sorting Complex ...
Mechanisms of Ageing and Development, 2006
We present here a brief description of the relationships among metals, nitric oxide metabolism, a... more We present here a brief description of the relationships among metals, nitric oxide metabolism, and ageing. In particular, we will discuss the interactions occurring between redox (copper, iron) and non-redox (zinc) metals and nitric oxide, the metal- and nitric oxide-catalyzed formation of thiol adducts (nitrosothiols, mixed disulphides) and the possible involvement of these species in the ageing process.
Journal of Biological Chemistry, 2005
Saccharomyces cerevisiae transcriptionally regulates the expression of the plasma membrane high a... more Saccharomyces cerevisiae transcriptionally regulates the expression of the plasma membrane high affinity iron transport system in response to iron need. This transport system is comprised of the products of the FET3 and FTR1 genes. We show that Fet3p and Ftr1p are post-translationally regulated by iron. Incubation of cells in high iron leads to the internalization and degradation of both Fet3p and Ftr1p. Yeast strains defective in endocytosis (Δend4) show a reduced iron-induced loss of Fet3p-Ftr1p. In cells with a deletion in the vacuolar protease PEP4, high iron medium leads to the accumulation of Fet3p and Ftr1p in the vacuole. Iron-induced degradation of Fet3p-Ftr1p is significantly reduced in strains containing a deletion of a gene, VTA1, which is involved in multivesicular body (MVB) sorting in yeast. Sorting through the MVB can involve ubiquitination. We demonstrate that Ftr1p is ubiquitinated, whereas Fet3p is not ubiquitinated. Iron-induced internalization and degradation of...
Pharmaceutics
SARS-CoV-2 causes COVID-19, a predominantly pulmonary disease characterized by a burst of pro-inf... more SARS-CoV-2 causes COVID-19, a predominantly pulmonary disease characterized by a burst of pro-inflammatory cytokines and an increase in free iron. The viral glycoprotein Spike mediates fusion to the host cell membrane, but its role as a virulence factor is largely unknown. Recently, the antiviral activity of lactoferrin against SARS-CoV-2 was demonstrated in vitro and shown to occur via binding to cell surface receptors, and its putative interaction with Spike was suggested by in silico analyses. We investigated the anti-SARS-CoV-2 activity of bovine and human lactoferrins in epithelial and macrophagic cells using a Spike-decorated pseudovirus. Lactoferrin inhibited pseudoviral fusion and counteracted the deleterious effects of Spike on iron and inflammatory homeostasis by restoring basal levels of iron-handling proteins and of proinflammatory cytokines IL-1β and IL-6. Using pull-down assays, we experimentally proved for the first time that lactoferrin binds to Spike, immediately su...
International Journal of Molecular Sciences, 2021
The ferroxidase ceruloplasmin (CP) plays a crucial role in iron homeostasis in vertebrates togeth... more The ferroxidase ceruloplasmin (CP) plays a crucial role in iron homeostasis in vertebrates together with the iron exporter ferroportin. Mutations in the CP gene give rise to aceruloplasminemia, a rare neurodegenerative disease for which no cure is available. Many aspects of the (patho)physiology of CP are still unclear and would benefit from the availability of recombinant protein for structural and functional studies. Furthermore, recombinant CP could be evaluated for enzyme replacement therapy for the treatment of aceruloplasminemia. We report the production and preliminary characterization of high-quality recombinant human CP in glycoengineered Pichia pastoris SuperMan5. A modified yeast strain lacking the endogenous ferroxidase has been generated and employed as host for heterologous expression of the secreted isoform of human CP. Highly pure biologically active protein has been obtained by an improved two-step purification procedure. Glycan analysis indicates that predominant g...
Bio-Algorithms and Med-Systems, 2017
Ferroportin (Fpn) is a membrane protein representing the major cellular iron exporter, essential ... more Ferroportin (Fpn) is a membrane protein representing the major cellular iron exporter, essential for metal translocation from cells into plasma. Despite its pivotal role in human iron homeostasis, many questions on Fpn structure and biology remain unanswered. In this work, we present two novel and more reliable structural models of human Fpn (hFpn; inward-facing and outward-facing conformations) obtained using as templates the recently solved crystal structures of a bacterial homologue of hFpn,
The Journal of Neuroscience, 1997
Hydra feeding response is a very primitive olfactory-like behavior present in a multicellular org... more Hydra feeding response is a very primitive olfactory-like behavior present in a multicellular organism. We investigated the role of nitric oxide (NO) in the induction and control of hydra feeding response. Under basal conditions, hydra specimens produce detectable amounts of nitrite (NO 2 Ϫ), the breakdown product of NO. When hydra were incubated with reduced glutathione (GSH), the typical activator of feeding response, an increase of basal NO production was observed. This effect was inhibited by glutamic or ␣-aminoadipic acids, two GSH antagonists, which block GSH-induced feeding response, and by the NO synthase (NOS) inhibitor L-NAME. Moreover, we found that hydra possess a calcium-dependent (but calmodulin-independent) NOS isoform. By using exogenous NO donors and NOS inhibitors, we demonstrated that NO stimulus can participate both in triggering tentacular movements and in recruiting neighbor tentacles during hydra feeding response. By using dbt 2-cGMP, an analog to cGMP, we observed that the NO effect was independent of cGMP pathway. Our results strongly implicate NO involvement in hydra very primitive feeding behavior, thus confirming its preservation throughout evolution.
Biometals : an international journal on the role of metal ions in biology, biochemistry, and medicine, Jan 16, 2018
In the last 20 years, several new genes and proteins involved in iron metabolism in eukaryotes, p... more In the last 20 years, several new genes and proteins involved in iron metabolism in eukaryotes, particularly related to pathological states both in animal models and in humans have been identified, and we are now starting to unveil at the molecular level the mechanisms of iron absorption, the regulation of iron transport and the homeostatic balancing processes. In this review, we will briefly outline the general scheme of iron metabolism in humans and then focus our attention on the cellular iron export system formed by the permease ferroportin and the ferroxidase ceruloplasmin. We will finally summarize data on the role of the iron binding protein lactoferrin on the regulation of the ferroportin/ceruloplasmin couple and of other proteins involved in iron homeostasis in inflamed human macrophages.
Frontiers in immunology, 2017
Human lactoferrin (hLf), an 80-kDa multifunctional iron-binding cationic glycoprotein, is constit... more Human lactoferrin (hLf), an 80-kDa multifunctional iron-binding cationic glycoprotein, is constitutively secreted by exocrine glands and by neutrophils during inflammation. hLf is recognized as a key element in the host immune defense system. The in vitro and in vivo experiments are carried out with bovine Lf (bLf), which shares high sequence homology and identical functions with hLf, including anti-inflammatory activity. Here, in "pure" M1 human macrophages, obtained by stimulation with a mixture of 10 pg/ml LPS and 20 ng/ml IFN-γ, as well as in a more heterogeneous macrophage population, challenged with high-dose of LPS (1 µg/ml), the effect of bLf on the expression of the main proteins involved in iron and inflammatory homeostasis, namely ferroportin (Fpn), membrane-bound ceruloplasmin (Cp), cytosolic ferritin (Ftn), transferrin receptor 1, and cytokines has been investigated. The increase of IL-6 and IL-1β cytokines, following the inflammatory treatments, is associated...
Scientific reports, Aug 22, 2016
Fep1, the iron-responsive GATA factor from the methylotrophic yeast Pichia pastoris, has been cha... more Fep1, the iron-responsive GATA factor from the methylotrophic yeast Pichia pastoris, has been characterised both in vivo and in vitro. This protein has two Cys2-Cys2 type zinc fingers and a set of four conserved cysteines arranged in a Cys-X5-Cys-X8-Cys-X2-Cys motif located between the two zinc fingers. Electronic absorption and resonance Raman spectroscopic analyses in anaerobic and aerobic conditions indicate that Fep1 binds iron in the form of a [2Fe-2S] cluster. Site-directed mutagenesis shows that replacement of the four cysteines with serine inactivates this transcriptional repressor. Unexpectedly, the inactive mutant is still able to bind a [2Fe-2S] cluster, employing two cysteine residues belonging to the first zinc finger. These two cysteine residues can act as alternative cluster ligands selectively in aerobically purified Fep1 wild type, suggesting that oxygen could play a role in Fep1 function by causing differential localization of the [Fe-S] cluster.
Cellular and molecular biology (Noisy-le-Grand, France), Jan 17, 2007
We have firstly investigated the toxicological activity by hemolytic assay of crude extract obtai... more We have firstly investigated the toxicological activity by hemolytic assay of crude extract obtained by sonication of holotrichous isorhiza isolated nematocysts of the Scyphozoan Pelagia noctiluca, collected in the Strait of Messina. The hemolytic activity was both time- and dose-dependent on fish, rabbit, chicken and human red blood cells. At lowest doses rabbit and chicken erythrocytes were the most sensitive, whereas those of eel were the most resistant to the crude extract. Different storage conditions, such as -20 degrees C, -80 degrees C for up to 6 months and lyophilization, did not affect the stability of crude venom. Moreover, neither treatment at 4 degrees C, 20 degrees C and 37 degrees C for different time periods ranging between 30 min and 24 h, nor harsh thermal treatment at 80 degrees C and 100 degrees C affected the hemolytic power. The crude venom resulted even stable towards proteolysis and alkaline pH values.
The FASEB Journal, 2002
The amyloid peptides Aβ1-42 and Aβ25-35 strongly inhibited the activity of constitutive neuronal ... more The amyloid peptides Aβ1-42 and Aβ25-35 strongly inhibited the activity of constitutive neuronal and endothelial nitric oxide synthases (i.e., NOS-I and NOS-III, respectively) in cellfree assays. The molecular mechanism of NOS inhibition by Aβ fragments was studied in detail with Aβ25-35. The inhibitory ability was mostly NADPH-dependent and specific for the soluble form of Aβ25-35. Optical, fluorescence, and NMR spectroscopy showed that the soluble, but not aggregated, Aβ25-35 interacted with NADPH, thus suggesting that a direct recruitment of NADPH may result in diminished availability of the redox cofactor for NOS functioning. To assess the physiological relevance of our findings, rat neuronal-like PC12 and glioma C6 cell lines were used as cellular models. After Aβ25-35 internalization into cells was verified, the activity of constitutive NOS was measured using the DAF-2DA detection system and found to be severely impaired upon Aβ25-35 uptake. Consistent with previous results on the molecular cross-talk between NOS isoforms, repression of constitutive NOS by Aβ25-35 resulted in enhanced expression of inducible NOS (NOS-II) mRNA in C6 cells. Our results represent the first evidence that amyloid fragments impair constitutive NOS activity in cell-free and cellular systems, providing a possible molecular mechanism for the onset and/or maintenance of Alzheimer's disease.
The EMBO Journal, 2006
Ferritin is a cytosolic molecule comprised of subunits that self-assemble into a nanocage capable... more Ferritin is a cytosolic molecule comprised of subunits that self-assemble into a nanocage capable of containing up to 4500 iron atoms. Iron stored within ferritin can be mobilized for use within cells or exported from cells. Expression of ferroportin (Fpn) results in export of cytosolic iron and ferritin degradation. Fpn-mediated iron loss from ferritin occurs in the cytosol and precedes ferritin degradation by the proteasome. Depletion of ferritin iron induces the monoubiquitination of ferritin subunits. Ubiquitination is not required for iron release but is required for disassembly of ferritin nanocages, which is followed by degradation of ferritin by the proteasome. Specific mammalian machinery is not required to extract iron from ferritin. Iron can be removed from ferritin when ferritin is expressed in Saccharomyces cerevisiae, which does not have endogenous ferritin. Expressed ferritin is monoubiquitinated and degraded by the proteasome. Exposure of ubiquitination defective mammalian cells to the iron chelator desferrioxamine leads to degradation of ferritin in the lysosome, which can be prevented by inhibitors of autophagy. Thus, ferritin degradation can occur through two different mechanisms.
Proceedings of the National Academy of Sciences, 2005
Mutations in the iron exporter ferroportin (Fpn) (IREG1, SLC40A1 , and MTP1) result in hemochroma... more Mutations in the iron exporter ferroportin (Fpn) (IREG1, SLC40A1 , and MTP1) result in hemochromatosis type IV, a disorder with a dominant genetic pattern of inheritance and heterogeneous clinical presentation. Most patients develop iron loading of Kupffer cells with relatively low saturation of plasma transferrin, but others present with high transferrin saturation and iron-loaded hepatocytes. We show that known human mutations introduced into mouse Fpn-GFP generate proteins that either are defective in cell surface localization or have a decreased ability to be internalized and degraded in response to hepcidin. Studies using coimmunoprecipitation of epitope-tagged Fpn and size-exclusion chromatography demonstrated that Fpn is multimeric. Both WT and mutant Fpn participate in the multimer, and mutant Fpn can affect the localization of WT Fpn, its stability, and its response to hepcidin. The behavior of mutant Fpn in cell culture and the ability of mutant Fpn to act as a dominant ne...
Neuroscience Letters, 2010
Previously, we demonstrated that IL-1β was able to increase iron efflux from glial cells through ... more Previously, we demonstrated that IL-1β was able to increase iron efflux from glial cells through a coordinate induction of both ferroportin-1 (Fpn) and ceruloplasmin (Cp) synthesis. In this study, we have investigated the signaling pathways that are involved in the transcriptional activation of the Cp and Fpn. Our data show that the expression of Cp and Fpn in response to IL-1β requires the activation of MAP kinase pathways as a consequence of an IL-1β receptor stimulation. Moreover, we have observed that IL-1β regulates the expression of Cp and Fpn genes through (i) p38 MAPK-mediated activation of C/EBP transcription factor, (ii) ERK1/2-, JNK1- and partially p38 MAPK-dependent activation of AP-1, and through (iii) activation of NF-κB partially mediated by p38 MAPK.
Neuroscience Letters, 2010
Astrocytes accumulate iron under chronic oxidative stress conditions in ageing and neurological d... more Astrocytes accumulate iron under chronic oxidative stress conditions in ageing and neurological disorders. The soybean isoflavone genistein possesses antioxidant properties and selective estrogen-like activities. Here, a possible role of genistein in modulation of iron transport was explored in glial cells. Genistein significantly increased iron export through estrogen receptor--dependent p38 MAPK activation. Evidence is presented that this effect is associated to a p38 MAPK-triggered up-regulation of the iron export system made by ceruloplasmin and ferroportin-1, a pathway requiring activation of the transcription factor C/EBP.
Neuroscience Letters, 2004
A number of pathologies, including neurodegeneration and inflammation, have been associated with ... more A number of pathologies, including neurodegeneration and inflammation, have been associated with iron dysmetabolism in the brain. Hence, systems involved in iron homeostasis at the cellular level have aroused considerable interest in recent years. The iron exporter ferroportin-1 (FP) and the multicopper oxidase ceruloplasmin (CP) are essential for iron efflux from cells. By using RT-PCR, we demonstrate that FP and CP gene expression is up-regulated by treatment with the pro-inflammatory cytokine IL-1b in rat C6 cells, taken as a glial cellular model. Following stimulation with IL-1b, a higher expression level of CP and FP was also confirmed by Western blotting. Moreover, IL-1b has been found to increase iron efflux from C6 cells, suggesting that both proteins may play a crucial role in iron homeostasis in pathological brain conditions, such as inflammatory and/or neurodegenerative diseases.
Neuroscience Letters, 2011
Previously, we demonstrated that IL-1β was able to increase iron efflux from glial cells through ... more Previously, we demonstrated that IL-1β was able to increase iron efflux from glial cells through a coordinate induction of both ferroportin-1 (Fpn) and ceruloplasmin (Cp) synthesis. In this study, we have investigated the signaling pathways that are involved in the transcriptional activation of the Cp and Fpn. Our data show that the expression of Cp and Fpn in response to
Molecular Biology of the Cell, 2007
Ferroportin (Fpn) is the only known iron exporter in vertebrates. Hepcidin, a peptide secreted by... more Ferroportin (Fpn) is the only known iron exporter in vertebrates. Hepcidin, a peptide secreted by the liver in response to iron or inflammation, binds to Fpn, inducing its internalization and degradation. We show that after binding of hepcidin, Fpn is tyrosine phosphorylated at the plasma membrane. Mutants of human Fpn that do not get internalized or that are internalized slowly show either absent or impaired phosphorylation. We identify adjacent tyrosines as the phosphorylation sites and show that mutation of both tyrosines prevents hepcidin-mediated Fpn internalization. Once internalized, Fpn is dephosphorylated and subsequently ubiquitinated. An inability to ubiquitinate Fpn does not prevent hepcidin-induced internalization, but it inhibits the degradation of Fpn. Ubiquitinated Fpn is trafficked through the multivesicular body pathway en route to degradation in the late endosome/lysosome. Depletion of proteins involved in multivesicular body trafficking (Endosome Sorting Complex ...
Mechanisms of Ageing and Development, 2006
We present here a brief description of the relationships among metals, nitric oxide metabolism, a... more We present here a brief description of the relationships among metals, nitric oxide metabolism, and ageing. In particular, we will discuss the interactions occurring between redox (copper, iron) and non-redox (zinc) metals and nitric oxide, the metal- and nitric oxide-catalyzed formation of thiol adducts (nitrosothiols, mixed disulphides) and the possible involvement of these species in the ageing process.
Journal of Biological Chemistry, 2005
Saccharomyces cerevisiae transcriptionally regulates the expression of the plasma membrane high a... more Saccharomyces cerevisiae transcriptionally regulates the expression of the plasma membrane high affinity iron transport system in response to iron need. This transport system is comprised of the products of the FET3 and FTR1 genes. We show that Fet3p and Ftr1p are post-translationally regulated by iron. Incubation of cells in high iron leads to the internalization and degradation of both Fet3p and Ftr1p. Yeast strains defective in endocytosis (Δend4) show a reduced iron-induced loss of Fet3p-Ftr1p. In cells with a deletion in the vacuolar protease PEP4, high iron medium leads to the accumulation of Fet3p and Ftr1p in the vacuole. Iron-induced degradation of Fet3p-Ftr1p is significantly reduced in strains containing a deletion of a gene, VTA1, which is involved in multivesicular body (MVB) sorting in yeast. Sorting through the MVB can involve ubiquitination. We demonstrate that Ftr1p is ubiquitinated, whereas Fet3p is not ubiquitinated. Iron-induced internalization and degradation of...