Previous studies by us and other groups characterized protein expression variation following long... more Previous studies by us and other groups characterized protein expression variation following long-term moderate training, whereas the effects of single bursts of exercise are less known. Making use of a proteomic approach, we investigated the effects of acute swimming exercise (ASE) on protein expression and carbonylation patterns in two hind limb muscles: the Extensor Digitorum Longus (EDL) and the Soleus, mostly composed of fast-twitch and slow-twitch fibres, respectively. Carbonylation is one of the most common oxidative modifications of proteins and a marker of oxidative stress. In fact, several studies suggest that physical activity and the consequent increase in oxygen consumption can lead to increase in reactive oxygen and nitrogen species (RONS) production, hence the interest in examining the impact of RONS on skeletal muscle proteins following ASE. Results indicate that protein expression is unaffected by ASE in both muscle types. Unexpectedly, the protein carbonylation level was reduced following ASE. In particular, the analysis found 31 and 5 spots, in Soleus and EDL muscles respectively, whose carbonylation is reduced after ASE. Lipid peroxidation levels in Soleus were markedly reduced as well. Most of the decarbonylated proteins are involved either in the regulation of muscle contractions or in the regulation of energy metabolism. A number of hypotheses may be advanced to account for such results, which will be addressed in future studies.
Severe eosinophilic asthma is characterized by chronic airway inflammation, oxidative stress, and... more Severe eosinophilic asthma is characterized by chronic airway inflammation, oxidative stress, and elevated proinflammatory cytokines, especially IL-5. Mepolizumab and benralizumab are both humanized IgG antibodies directed against IL-5 signaling, directly acting on eosinophils count. Together with the complexity of severe asthma classification and patient selection for the targeted treatment, there is also the urgency to clarify the follow-up of therapy to identify biomarkers, in addition to eosinophils, for the optimal duration of treatment, persistence of effectiveness, and safety. To this purpose, here we performed a follow-up study using differential proteomic analysis on serum samples after 1 and 6 months of both therapies and sera from healthy patients. Statistical analysis by PCA and heatmap analyses were performed, and identified proteins were used for enrichment analysis by MetaCore software. The analysis highlighted 82 differences among all considered conditions. In partic...
In the longtime challenge of identifying specific, easily detectable and reliable biomarkers of I... more In the longtime challenge of identifying specific, easily detectable and reliable biomarkers of IPF, BALF proteomics is providing interesting new insights into its pathogenesis. To the best of our knowledge, the present study is the first shotgun proteomic investigation of EVs isolated from BALF of IPF patients. Our main aim was to characterize the proteome of the vesicular component of BALF and to explore its individual impact on the pathogenesis of IPF. To this purpose, ultracentrifugation was chosen as the EVs isolation technique, and their purification was assessed by TEM, 2DE and LC-MS/MS. Our 2DE data and scatter plots showed considerable differences between the proteome of EVs and that of whole BALF and of its fluid component. Analysis of protein content and protein functions evidenced that EV proteins are predominantly involved in cytoskeleton remodeling, adenosine signaling, adrenergic signaling, C-peptide signaling and lipid metabolism. Our findings may suggest a wider sys...
Crude homogenates of the nematode Caenorhabditis elegans exhibit maximal proteolytic activity und... more Crude homogenates of the nematode Caenorhabditis elegans exhibit maximal proteolytic activity under acidic pH conditions. About 90% of this activity is inhibited by the oligopeptide pepstatin, which specifically inhibits the activity of aspartyl proteases such as pepsin, cathepsins D and E or renin. We have purified enzymes responsible for this proteolytic activity by a single-step affinity chromatography on pepstatin±agarose. Analysis of the purified fraction by 1D SDS gel electrophoresis revealed six bands ranging from 35 to 52 kDa. After electrotransfer to poly(vinylidene difluoride) membranes, all bands were successfully subjected to N-terminal microsequencing. On 2D gels, the purified protein bands split into 19 spots which, after renewed microsequencing, were identified as isoelectric variants of the six proteins already described. The N-termini obtained for these proteins could be correlated to genomic DNA sequences determined in the course of the C. elegans genome sequencing project. All these sequences were predicted to code for expressed proteins as collected in the WORMPEP database. Five of the six coding sequences identified in this study were found to contain the typical active-site consensus sequence of aspartyl proteases and displayed an overall amino acid identity between 25 and 66% as compared to aspartyl proteases from other organisms. In addition to the five aspartyl proteases detected at the protein level, we have identified the coding sequences for seven other enzymes of this protease family by a similarity search in the genomic DNA of C. elegans which has recently been completely sequenced.
Spinal muscular atrophy (SMA) type 1 is a severe infantile autosomal-recessive neuromuscular diso... more Spinal muscular atrophy (SMA) type 1 is a severe infantile autosomal-recessive neuromuscular disorder caused by a survival motor neuron 1 gene (SMN1) mutation and characterized by progressive muscle weakness. Without supportive care, SMA type 1 is rapidly fatal. The antisense oligonucleotide nusinersen has recently improved the natural course of this disease. Here, we investigated, with a functional proteomic approach, cerebrospinal fluid (CSF) protein profiles from SMA type 1 patients who underwent nusinersen administration to clarify the biochemical response to the treatment and to monitor disease progression based on therapy. Six months after starting treatment (12 mg/5 mL × four doses of loading regimen administered at days 0, 14, 28, and 63), we observed a generalized reversion trend of the CSF protein pattern from our patient cohort to that of control donors. Notably, a marked up-regulation of apolipoprotein A1 and apolipoprotein E and a consistent variation in transthyretin p...
We identified, by two-dimensional electrophoretic analysis and microsequencing, a protein of Chla... more We identified, by two-dimensional electrophoretic analysis and microsequencing, a protein of Chlamydia trachomatis elementary bodies which corresponds to the polypeptide (pgp3) encoded by open reading frame 3 (ORF3). Amino acid analysis showed that the first residue (Gly) found in the native protein is the one encoded by the second ORF3 codon, implying a typical bacterial removal of the first Met residue. Relatively large amounts of recombinant pgp3 (r-pgp3) in a stable, water-soluble form were obtained by overexpressing ORF3 in Escherichia coli and purifying the product from periplasmic extracts under nondenaturing conditions. These r-pgp3 preparations allowed specific detection of anti-pgp3 antibodies by enzyme-linked immunosorbent assay. Analysis of a group of 170 sera from healthy blood donors and from patients who were seropositive or -negative for C. trachomatis and Chlamydia pneumoniae showed that an immune response to pgp3 occurs in the majority (ca. 81%) of patients with se...
Inflammation research : official journal of the European Histamine Research Society ... [et al.], 2017
Mutations in the cyclin-dependent kinase-like 5 gene cause a clinical variant of Rett syndrome (C... more Mutations in the cyclin-dependent kinase-like 5 gene cause a clinical variant of Rett syndrome (CDKL5-RTT). A role for the acute-phase response (APR) is emerging in typical RTT caused by methyl-CpG-binding protein 2 gene mutations (MECP2-RTT). No information is, to date, available on the inflammatory protein response in CDKL5-RTT. We evaluated, for the first time, the APR protein response in CDKL5-RTT. Protein patterns in albumin- and IgG-depleted plasma proteome from CDKL5-RTT patients were evaluated by two-dimensional gel electrophoresis/mass spectrometry. The resulting data were related to circulating cytokines and compared to healthy controls or MECP2-RTT patients. The effects of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) were evaluated. CDKL5-RTT mutations resulted in a subclinical attenuated inflammation, specifically characterized by an overexpression of the complement component C3 and CD5 antigen-like, both strictly related to the inflammatory response. Cytokine dysregu...
Pollen tubes are the vehicle for sperm cell delivery to the embryo sac during fertilisation of An... more Pollen tubes are the vehicle for sperm cell delivery to the embryo sac during fertilisation of Angiosperms. They provide an intriguing model for unravelling mechanisms of growing to extremes. The asymmetric distribution of lipids and proteins in the pollen tube plasma membrane modulates ion fluxes and actin dynamics and is maintained by a delicate equilibrium between exocytosis and endocytosis. The structural constraints regulating polarised secretion and asymmetric protein distribution on the plasma membrane are mostly unknown. To address this problem, we investigated whether ordered membrane microdomains, namely membrane rafts, might contribute to sperm cell delivery. Detergent insoluble membranes, rich in sterols and sphingolipids, were isolated from tobacco pollen tubes. MALDI TOF/MS analysis revealed that actin, prohibitins and proteins involved in methylation reactions and in phosphoinositide pattern regulation are specifically present in pollen tube detergent insoluble membra...
Proteomic approach is an effective method to study changes in human plasma proteome. Coagulopathi... more Proteomic approach is an effective method to study changes in human plasma proteome. Coagulopathies are commonly encountered in victims of viper envenomation which were treated with an administration of immunoglobulin. Unfortunately, this treatment shows significant risk to the patient due to an anaphylactic reaction. Since Echis carinatus Venom (EV) toxins mainly acts both directly and indirectly on fibrinogen, we planned to establish a suitable analysis of its beta (FIBB) e gamma (FIBG) chains. This study will help us to understand the mechanism of envenomation and to find alternative treatments other than the common treatment with the administration of IgG. We evaluated the EV proteolytic activity on whole human plasma proteome from the blood of an healthy volunteer. Two-dimensional electrophoresis (2-DE) using mini-gel was performed to analyse EV effects on the differents fibrinogen chains. Changes in whole plasma proteome were focused on fibrinogen beta and gamma chains after E...
Previous studies by us and other groups characterized protein expression variation following long... more Previous studies by us and other groups characterized protein expression variation following long-term moderate training, whereas the effects of single bursts of exercise are less known. Making use of a proteomic approach, we investigated the effects of acute swimming exercise (ASE) on protein expression and carbonylation patterns in two hind limb muscles: the Extensor Digitorum Longus (EDL) and the Soleus, mostly composed of fast-twitch and slow-twitch fibres, respectively. Carbonylation is one of the most common oxidative modifications of proteins and a marker of oxidative stress. In fact, several studies suggest that physical activity and the consequent increase in oxygen consumption can lead to increase in reactive oxygen and nitrogen species (RONS) production, hence the interest in examining the impact of RONS on skeletal muscle proteins following ASE. Results indicate that protein expression is unaffected by ASE in both muscle types. Unexpectedly, the protein carbonylation level was reduced following ASE. In particular, the analysis found 31 and 5 spots, in Soleus and EDL muscles respectively, whose carbonylation is reduced after ASE. Lipid peroxidation levels in Soleus were markedly reduced as well. Most of the decarbonylated proteins are involved either in the regulation of muscle contractions or in the regulation of energy metabolism. A number of hypotheses may be advanced to account for such results, which will be addressed in future studies.
The Saccharomyces cerevisiae gene SCO1 has been shown to play an essential role in copper deliver... more The Saccharomyces cerevisiae gene SCO1 has been shown to play an essential role in copper delivery to cytochrome c oxidase. Biochemical studies demonstrated specific transfer of copper from Cox17p to Sco1p, and physical interactions between the Sco1p and Cox2p. Deletion of SCO1 yeast gene results in a respiratory deficient phenotype. This study aims to gain a more detailed insight on the effects of SCO1 deletion on S. cerevisiae metabolism. We compared, using a proteomic approach, the protein pattern of SCO1 null mutant strain and wild-type BY4741 strain grown on fermentable and on nonfermentable carbon sources. The analysis showed that on nonfermentable medium, the SCO1 mutant displayed a protein profile similar to that of actively fermenting yeast cells. Indeed, on 3% glycerol, this mutant displayed an increase of some glycolytic and fermentative enzymes such as glyceraldehyde-3-phosphate dehydrogenase 1, enolase 2, pyruvate decarboxylase 1, and alcohol dehydrogenase 1. These data were supported by immunoblotting and enzyme activity assay. Moreover, the ethanol assay and the oxygen consumption measurement demonstrated a fermentative activity in SCO1 mutant on respiratory medium. Our results suggest that on nonfermentable carbon source, the lack of Sco1p causes a metabolic shift from respiration to fermentation.
Crude homogenates of the nematode Caenorhabditis elegans exhibit maximal proteolytic activity und... more Crude homogenates of the nematode Caenorhabditis elegans exhibit maximal proteolytic activity under acidic pH conditions. About 90% of this activity is inhibited by the oligopeptide pepstatin, which specifically inhibits the activity of aspartyl proteases such as pepsin, cathepsins D and E or renin. We have purified enzymes responsible for this proteolytic activity by a single-step affinity chromatography on pepstatin±agarose. Analysis of the purified fraction by 1D SDS gel electrophoresis revealed six bands ranging from 35 to 52 kDa. After electrotransfer to poly(vinylidene difluoride) membranes, all bands were successfully subjected to N-terminal microsequencing. On 2D gels, the purified protein bands split into 19 spots which, after renewed microsequencing, were identified as isoelectric variants of the six proteins already described. The N-termini obtained for these proteins could be correlated to genomic DNA sequences determined in the course of the C. elegans genome sequencing project. All these sequences were predicted to code for expressed proteins as collected in the WORMPEP database. Five of the six coding sequences identified in this study were found to contain the typical active-site consensus sequence of aspartyl proteases and displayed an overall amino acid identity between 25 and 66% as compared to aspartyl proteases from other organisms. In addition to the five aspartyl proteases detected at the protein level, we have identified the coding sequences for seven other enzymes of this protease family by a similarity search in the genomic DNA of C. elegans which has recently been completely sequenced.
The Saccharomyces cerevisiae gene SCO1 has been shown to play an essential role in copper deliver... more The Saccharomyces cerevisiae gene SCO1 has been shown to play an essential role in copper delivery to cytochrome c oxidase. Biochemical studies demonstrated specific transfer of copper from Cox17p to Sco1p, and physical interactions between the Sco1p and Cox2p. Deletion of SCO1 yeast gene results in a respiratory deficient phenotype. This study aims to gain a more detailed insight on the effects of SCO1 deletion on S. cerevisiae metabolism. We compared, using a proteomic approach, the protein pattern of SCO1 null mutant strain and wild-type BY4741 strain grown on fermentable and on nonfermentable carbon sources. The analysis showed that on nonfermentable medium, the SCO1 mutant displayed a protein profile similar to that of actively fermenting yeast cells. Indeed, on 3% glycerol, this mutant displayed an increase of some glycolytic and fermentative enzymes such as glyceraldehyde-3-phosphate dehydrogenase 1, enolase 2, pyruvate decarboxylase 1, and alcohol dehydrogenase 1. These data were supported by immunoblotting and enzyme activity assay. Moreover, the ethanol assay and the oxygen consumption measurement demonstrated a fermentative activity in SCO1 mutant on respiratory medium. Our results suggest that on nonfermentable carbon source, the lack of Sco1p causes a metabolic shift from respiration to fermentation.
Cardiovascular diseases represent the main cause of mortality in the industrialized world and the... more Cardiovascular diseases represent the main cause of mortality in the industrialized world and the identification of effective preventive strategies is of fundamental importance. Sulforaphane, an isothiocyanate from cruciferous vegetables, has been shown to up-regulate phase II enzymes in cardiomyocytes and counteract oxidative stressinduced apoptosis. Aim of the present study was the identification and characterization of novel sulforaphane targets in cardiomyocytes applying a proteomic approach. Two-dimensional gel electrophoresis and mass spectrometry were used to generate protein profiles of primary neonatal rat cardiomyocytes treated and untreated with 5 µM sulforaphane for 1-48 h. According to image analysis, 64 protein spots were found as differentially expressed and their functional correlations were investigated using the MetaCore program. We mainly focused on 3 proteins: macrophage migration inhibitory factor (MIF), CLP36 or Elfin, and glyoxalase 1, due to their possible involvement in cardioprotection. Validation of the time-dependent differential expression of these proteins was performed by western blotting. In particular, to gain insight into the cardioprotective role of the modulation of glyoxalase 1 by sulforaphane, further experiments were performed using methylglyoxal to mimic glycative stress. Sulforaphane was able to counteract methylglyoxal-induced apoptosis, ROS production, and glycative stress, likely through glyoxalase 1 upregulation. In this study, we reported for the first time new molecular targets of sulforaphane, such as MIF, CLP36 and glyoxalase 1. In particular, we gave new insights into the anti-glycative role of sulforaphane in cardiomyocytes, confirming its pleiotropic behavior in counteracting cardiovascular diseases.
Cardiovascular diseases represent the main cause of mortality in the industrialized world and the... more Cardiovascular diseases represent the main cause of mortality in the industrialized world and the identification of effective preventive strategies is of fundamental importance. Sulforaphane, an isothiocyanate from cruciferous vegetables, has been shown to up-regulate phase II enzymes in cardiomyocytes and counteract oxidative stressinduced apoptosis. Aim of the present study was the identification and characterization of novel sulforaphane targets in cardiomyocytes applying a proteomic approach. Two-dimensional gel electrophoresis and mass spectrometry were used to generate protein profiles of primary neonatal rat cardiomyocytes treated and untreated with 5 µM sulforaphane for 1-48 h. According to image analysis, 64 protein spots were found as differentially expressed and their functional correlations were investigated using the MetaCore program. We mainly focused on 3 proteins: macrophage migration inhibitory factor (MIF), CLP36 or Elfin, and glyoxalase 1, due to their possible involvement in cardioprotection. Validation of the time-dependent differential expression of these proteins was performed by western blotting. In particular, to gain insight into the cardioprotective role of the modulation of glyoxalase 1 by sulforaphane, further experiments were performed using methylglyoxal to mimic glycative stress. Sulforaphane was able to counteract methylglyoxal-induced apoptosis, ROS production, and glycative stress, likely through glyoxalase 1 upregulation. In this study, we reported for the first time new molecular targets of sulforaphane, such as MIF, CLP36 and glyoxalase 1. In particular, we gave new insights into the anti-glycative role of sulforaphane in cardiomyocytes, confirming its pleiotropic behavior in counteracting cardiovascular diseases.
In Nigeria, Mucuna pruriens seeds are locally prescribed as an oral prophylactic for snake bite a... more In Nigeria, Mucuna pruriens seeds are locally prescribed as an oral prophylactic for snake bite and it is claimed that when two seeds are swallowed they protect the individual for a year against snake bites. In order to understand the Mucuna pruriens antisnake properties, the proteins from the acqueous extract of seeds were purified by three chromatographic steps: ConA affinity chromatography, tandem anionic-cationic exchange and gel filtration, obtaining a fraction conventionally called gpMucB. This purified fraction was analysed by SDS-PAGE obtaining 3 bands with apparent masses ranging from 20 to 24 kDa, and by MALDI-TOF which showed two main peaks of 21 and 23 kDa and another small peak of 19 kDa. On the other hand, gel filtration analysis of the native protein indicated a molecular mass of about 70 kDa suggesting that in its native form, gpMucB is most likely an oligomeric multiform protein. Infrared spectroscopy of gpMucB indicated that the protein is particularly thermostable both at neutral and acidic pHs and that it is an all beta protein. All data suggest that gpMucB belongs to the Kunitz-type trypsin inhibitor family explaining the direct anti-snake venom activity of Mucuna pruriens seeds.
In Nigeria, Mucuna pruriens seeds are locally prescribed as an oral prophylactic for snake bite a... more In Nigeria, Mucuna pruriens seeds are locally prescribed as an oral prophylactic for snake bite and it is claimed that when two seeds are swallowed they protect the individual for a year against snake bites. In order to understand the Mucuna pruriens antisnake properties, the proteins from the acqueous extract of seeds were purified by three chromatographic steps: ConA affinity chromatography, tandem anionic-cationic exchange and gel filtration, obtaining a fraction conventionally called gpMucB. This purified fraction was analysed by SDS-PAGE obtaining 3 bands with apparent masses ranging from 20 to 24 kDa, and by MALDI-TOF which showed two main peaks of 21 and 23 kDa and another small peak of 19 kDa. On the other hand, gel filtration analysis of the native protein indicated a molecular mass of about 70 kDa suggesting that in its native form, gpMucB is most likely an oligomeric multiform protein. Infrared spectroscopy of gpMucB indicated that the protein is particularly thermostable both at neutral and acidic pHs and that it is an all beta protein. All data suggest that gpMucB belongs to the Kunitz-type trypsin inhibitor family explaining the direct anti-snake venom activity of Mucuna pruriens seeds.
Tyrosine (Tyr) phosphorylation is implicated in the modification of several erythrocyte functions... more Tyrosine (Tyr) phosphorylation is implicated in the modification of several erythrocyte functions, such as metabolic pathways and membrane transport, as well as in signal transduction systems. Here we describe the map of Tyr-phosphorylated soluble proteins of newborn red blood cells (RBC) using an in vitro model simulating RBC reoxygenation at birth after an intrauterine hypoxic event. We tested the hypothesis that a hypoxic environment and subsequent reoxygenation promote posttranslational changes in the RBC protein map of newborns, in addition to desferrioxamine (DFO)-chelatable iron (DCI) release and methemoglobin (MetHb) formation. Umbilical cord blood RBC were incubated under hypoxic conditions for 16 h at 37°C, and subsequently for 8 h under aerobic conditions. Control erythrocytes were incubated under aerobic conditions at 37°C for the period of the experiment, i.e. for 24 h. Tyr-phosphorylation proteins were assessed using advanced high-resolution twodimensional electrophoresis, 2-D immunoblot analysis with antiphosphotyrosine (anti-pTyr) antibodies, and computer-aided electrophoretogram analysis. Higher DCI release and MetHb formation were observed in newborn RBC incubated under hypoxic conditions than in those incubated aerobically. Different immunoreactivity patterns with anti-pTyr antibodies were also observed between newborn RBC incubated under hypoxic conditions and controls. A hypoxic environment is a factor promoting DCI release, a well-known condition of oxidative stress. This is the first map of Tyr-phosphorylated soluble proteins of newborn RBC obtained using an in vitro model simulating RBC reoxygenation at birth after an intrauterine hypoxic event. Our results suggest that hypoxia increases Tyr-phosphorylation of antioxidant proteins, protecting RBC against oxidative stress.
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Papers by Luca Bini