Giuseppe Grasso | Università di Catania (original) (raw)

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Papers by Giuseppe Grasso

Research paper thumbnail of Zn 2+ ’s Ability to Alter the Distribution of Cu 2+ among the Available Binding Sites of Aβ(1–16)-Polyethylenglycol-ylated Peptide: Implications in Alzheimer’s Disease

Inorganic Chemistry, 2011

Research paper thumbnail of Copper(I) and Copper(II) Inhibit Aβ Peptides Proteolysis by Insulin-Degrading Enzyme Differently: Implications for Metallostasis Alteration in Alzheimer’s Disease

Chemistry - A European Journal, 2011

Research paper thumbnail of Zinc(II) Interactions with Brain-Derived Neurotrophic Factor N-Terminal Peptide Fragments: Inorganic Features and Biological Perspectives

Inorganic Chemistry, 2013

Brain-derived neurotrophic factor (BDNF) is a neurotrophin essential for neuronal differentiation... more Brain-derived neurotrophic factor (BDNF) is a neurotrophin essential for neuronal differentiation, growth, and survival; it is involved in memory formation and higher cognitive functions. The N-terminal domain of BDNF is crucial for the binding selectivity and activation of its specific TrkB receptor. Zn(2+) ion binding may influence BDNF activity. Zn(2+) complexes with the peptide fragment BDNF(1-12) encompassing the sequence 1-12 of the N-terminal domain of BDNF were studied by means of potentiometry, electrospray mass spectrometry, NMR, and density functional theory (DFT) approaches. The predominant Zn(2+) complex species, at physiological pH, is [ZnL] in which the metal ion is bound to an amino, an imidazole, and two water molecules (NH2, N(Im), and 2O(water)) in a tetrahedral environment. DFT-based geometry optimization of the zinc coordination environment showed a hydrogen bond between the carboxylate and a water molecule bound to zinc in [ZnL]. The coordination features of the acetylated form [AcBDNF(1-12)] and of a single mutated peptide [BDNF(1-12)D3N] were also characterized, highlighting the role of the imidazole side chain as the first anchoring site and ruling out the direct involvement of the aspartate residue in the metal binding. Zn(2+) addition to the cell culture medium induces an increase in the proliferative activity of the BDNF(1-12) peptide and of the whole protein on the SHSY5Y neuroblastoma cell line. The effect of Zn(2+) is opposite to that previously observed for Cu(2+) addition, which determines a decrease in the proliferative activity for both peptide and protein, suggesting that these metals might discriminate and modulate differently the activity of BDNF.

Research paper thumbnail of Adhesion properties on nanometric scale of silicon oxide and silicon nitride surfaces modified by 1-octadecene

Research paper thumbnail of Ultrasensitive Detection of DNA by PNA and Nanoparticle-Enhanced Surface Plasmon Resonance Imaging

Research paper thumbnail of The Collagen Binding Domain of Gelatinase A Modulates Degradation of Collagen IV by Gelatinase B

Journal of Molecular Biology, 2009

Research paper thumbnail of In Situ AP/MALDI-MS characterization of anchored matrix metalloproteinases

Journal of Mass Spectrometry, 2006

Several different procedures are available for the immobilization of proteins on solid supports, ... more Several different procedures are available for the immobilization of proteins on solid supports, as many advantages derive from this approach, such as the possibility to develop new protein solid-state assays. Enzymes that are anchored on gold surfaces can interact with several different molecules in a tag-free environment, opening the way to surface plasmon resonance (SPR) investigations. Nevertheless, it is often important to know the identity of the affinity-retained analyte, and mass spectrometric analysis, via its unique molecular mass identification, represents a very valuable complementary method.There are many pieces of evidence to suggest that matrix metalloproteinases (MMPs) are involved in normal and pathological processes, including embryogenesis, wound healing, inflammation, arthritis and cancer, but presumably also exhibiting other functions. The search for new inhibitors of MMPs has prompted research towards the development of new solid-state assays for the rapid evaluation of MMP activity. We have already reported the possibility of measuring the activity of MMP-1 anchored on solid support by coupling SPR with ESI-MS analysis. In this work, we show the in situ atmospheric pressure (AP) MALDI-MS characterization of MMPs anchored on a gold chip with known surface coverage. The study extends the MS analysis to different proteins, and sequence coverage is reported for different digestion and MS procedures. Copyright © 2006 John Wiley & Sons, Ltd.

Research paper thumbnail of Somatostatin: A Novel Substrate and a Modulator of Insulin-Degrading Enzyme Activity

Journal of Molecular Biology, 2009

Research paper thumbnail of A new methodology for monitoring the activity of cdMMP-12 anchored and freeze-dried on Au (111

Journal of The American Society for Mass Spectrometry, 2007

Matrix metalloproteinases (MMPs) are cell-secreted soluble and membrane-tethered enzymes that are... more Matrix metalloproteinases (MMPs) are cell-secreted soluble and membrane-tethered enzymes that are capable of degrading extracellular matrix proteins, but also can process a number of bioactive molecules. They are involved in the cleavage of cell surface receptors, but are also thought to play a major role on cell behaviors as well as in diverse physiological and pathological processes, including embryonic development, wound repair, inflammatory diseases, and cancer. For these reasons, it is obvious that a control over MMPs activity is highly desirable. Consequently, the frantic search for new inhibitors has been coupled to the development of high-throughput methods able to rapidly screen the effect of possible MMP inhibitors on the activity of these enzymes. In this scenario, solid-state—based methods play a major role because of their compatibility with array formats that are able to extract more information from smaller sample volumes and offer some important advantages that are not available in the standard solution assays. In this work, the catalytic domain of MMP-12 was immobilized on a gold substrate and the surface coverage was measured by FT-SPR experiments. A new experimental procedure was developed to freeze-dry the anchored molecules and their activity was measured by ESI-MS. The kinetics parameters obtained for the immobilized enzyme are in good accordance with those reported for similar systems in solution. Inhibition of the immobilized molecules was also carried out, demonstrating the applicability of the method for rapid screening of MMP inhibitors.

Research paper thumbnail of AP/MALDI-MS complete characterization of the proteolytic fragments produced by the interaction of insulin degrading enzyme with bovine insulin

Journal of Mass Spectrometry, 2007

The prominent role that insulin degrading enzyme (IDE) has in the clearance of insulin as well as... more The prominent role that insulin degrading enzyme (IDE) has in the clearance of insulin as well as of other molecules such as amyloid-β has recently drawn much interest in the scientific community toward this protease. In order to give an insight into the manner of interaction of IDE with its substrates, several papers have focused on the structure of the IDE/insulin complex. In this scenario, although the cleavage sites involved in the interaction of insulin with IDE are known, a convenient experimental method that is able to identify in a complete and unambiguous way, all the peptide fragments generated by such interaction has yet to be found. MS-based experiments have often represented to be invaluable tools for the assessment of the cleavage sites, but the reported MS-spectra always show a partial coverage of all the peptide fragments generated by the enzyme interaction, lacking a complete characterization.In this work, we report a new experimental procedure by which an unambiguous as well as complete assignment of all the peptide fragments generated by the interaction of insulin with IDE is described. Atmospheric pressure/matrix-assisted laser desorption ionization (AP/MALDI) mass spectra are reported and the data recorded, together with the introduction of a reduction/alkylation step, allows us to fully characterize the cleavage sites of the bovine insulin interacting with IDE. Different experimental conditions are screened and some insights into the IDE/insulin system regarding preference of the cleavage and its dependence on particular experimental conditions used are also given. Investigation on the tendency that different insulin fragments have toward aggregation is also carried out.Good reproducibility, global and unambiguous assignment, low time-consuming experimental procedure, and requirements of enzyme in small amounts are some of the advantages of the proposed AP/MALDI based approach. Copyright © 2007 John Wiley & Sons, Ltd.

Research paper thumbnail of Activity of anchored human matrix metalloproteinase-1 catalytic domain on Au (111) surfaces monitored by ESI-MS

Journal of Mass Spectrometry, 2005

Matrix metalloproteinases (MMPs) are a family of Zn-dependent endo-peptidases known for their abi... more Matrix metalloproteinases (MMPs) are a family of Zn-dependent endo-peptidases known for their ability to cleave several components of the extracellular matrix, but which can also cleave many non-matrix proteins. There are many evidences that MMPs are involved in physiological and pathological processes, and a huge effort has been put in the development of possible inhibitors that could reduce the activity of MMPs, as it is clear that the ability to monitor and control such activity plays a pivotal role in the search for potential drugs aimed at finding a cure for several diseases such as pulmonary emphysema, rheumatoid arthritis, fibrotic disorders and cancer.A powerful method currently available to study enzyme–inhibitor interactions is based on the use of the surface plasmon resonance (SPR) technique. When MMP interactions are studied, a procedure by which inhibitors are normally anchored on sensor chips and SPR technique is used in order to study their interaction with MMPs molecules is usually followed. This is because it is currently believed that MMPs cannot be anchored on the sensor-chip surface without losing their activity. However, this approach gives rise to problems, as the anchoring of low-molecular-weight inhibitors on gold surfaces easily affects their ability to interact with MMPs. For this reason, the anchoring of MMPs is highly desirable.A new experimental protocol that couples the Fourier transform-SPR (FT-SPR) technique with electrospray ionization-mass spectroscopy (ESI-MS) is described here for the evaluation of the activity of MMP-1 catalytic domain (cdMMP-1) anchored on gold surfaces. The cdMMP-1 surface coverage is calculated by using FT-SPR and the enzyme activity is estimated by ESI-MS. The proposed method is label-free. Copyright © 2005 John Wiley & Sons, Ltd.

Research paper thumbnail of How the binding and degrading capabilities of insulin degrading enzyme are affected by ubiquitin

Biochimica Et Biophysica Acta-proteins and Proteomics, 2008

Insulin degrading enzyme (IDE) is known to play a pivotal role on amyloidogenic peptide degradati... more Insulin degrading enzyme (IDE) is known to play a pivotal role on amyloidogenic peptide degradation but little is known about the changes in the proteolytic activity of the enzyme upon modification of external factors. Particularly, although it has been reported that altered ubiquitin concentration and/or hyperinsulinaemia increase the risk of developing Alzheimer's disease (AD), the molecular mechanism involved is unclear. In this work, we study the role that ubiquitin plays on IDE capability of binding and degrading insulin molecules and the obtained results indicate that ubiquitin has an allosteric role for IDE and high ubiquitin levels impair IDE activity.

Research paper thumbnail of The use of atmospheric pressure laser desorption mass spectrometry for the study of iron-gall ink

Applied Physics A-materials Science & Processing, 2007

The study of ancient works of art is a very challenging task, mainly due to the impossibility of ... more The study of ancient works of art is a very challenging task, mainly due to the impossibility of applying experimental approaches that could damage anyhow the object from which analytical information has to be obtained. Spatially resolved analytical methods have significantly enhanced our capacity to study ancient material since they cause minimal and at times no damage to the studied object. Unfortunately, only a few analytical techniques operating within the requested spatial resolution are applicable for the investigation of the organic components of artistic and archaeological objects. In this work, iron-gall ink was prepared according to ancient recipes and its composition was evaluated by AP/MALDI-MS. The latter is demonstrated to be a very valuable tool for the study of the organic components of ancient works of art as it combines the advantages of the vacuum MALDI-MS analytical approach to the possibility of analysing samples in air. In situ analyses were also carried out by analysing strokes of ink directly from paper and parchment.

Research paper thumbnail of Microfluidic networks for surface plasmon resonance imaging real-time kinetics experiments

Microchemical Journal, 2009

Research paper thumbnail of Real-Time Binding Kinetics Monitored with Surface Plasmon Resonance Imaging in a Diffusion-Free Environment

The Open Spectroscopy Journal, 2008

Research paper thumbnail of The clearance of misfolded proteins in neurodegenerative diseases by zinc metalloproteases: An inorganic perspective Contents

Research paper thumbnail of Zn 2+ ’s Ability to Alter the Distribution of Cu 2+ among the Available Binding Sites of Aβ(1–16)-Polyethylenglycol-ylated Peptide: Implications in Alzheimer’s Disease

Inorganic Chemistry, 2011

Research paper thumbnail of Copper(I) and Copper(II) Inhibit Aβ Peptides Proteolysis by Insulin-Degrading Enzyme Differently: Implications for Metallostasis Alteration in Alzheimer’s Disease

Chemistry - A European Journal, 2011

Research paper thumbnail of Zinc(II) Interactions with Brain-Derived Neurotrophic Factor N-Terminal Peptide Fragments: Inorganic Features and Biological Perspectives

Inorganic Chemistry, 2013

Brain-derived neurotrophic factor (BDNF) is a neurotrophin essential for neuronal differentiation... more Brain-derived neurotrophic factor (BDNF) is a neurotrophin essential for neuronal differentiation, growth, and survival; it is involved in memory formation and higher cognitive functions. The N-terminal domain of BDNF is crucial for the binding selectivity and activation of its specific TrkB receptor. Zn(2+) ion binding may influence BDNF activity. Zn(2+) complexes with the peptide fragment BDNF(1-12) encompassing the sequence 1-12 of the N-terminal domain of BDNF were studied by means of potentiometry, electrospray mass spectrometry, NMR, and density functional theory (DFT) approaches. The predominant Zn(2+) complex species, at physiological pH, is [ZnL] in which the metal ion is bound to an amino, an imidazole, and two water molecules (NH2, N(Im), and 2O(water)) in a tetrahedral environment. DFT-based geometry optimization of the zinc coordination environment showed a hydrogen bond between the carboxylate and a water molecule bound to zinc in [ZnL]. The coordination features of the acetylated form [AcBDNF(1-12)] and of a single mutated peptide [BDNF(1-12)D3N] were also characterized, highlighting the role of the imidazole side chain as the first anchoring site and ruling out the direct involvement of the aspartate residue in the metal binding. Zn(2+) addition to the cell culture medium induces an increase in the proliferative activity of the BDNF(1-12) peptide and of the whole protein on the SHSY5Y neuroblastoma cell line. The effect of Zn(2+) is opposite to that previously observed for Cu(2+) addition, which determines a decrease in the proliferative activity for both peptide and protein, suggesting that these metals might discriminate and modulate differently the activity of BDNF.

Research paper thumbnail of Adhesion properties on nanometric scale of silicon oxide and silicon nitride surfaces modified by 1-octadecene

Research paper thumbnail of Ultrasensitive Detection of DNA by PNA and Nanoparticle-Enhanced Surface Plasmon Resonance Imaging

Research paper thumbnail of The Collagen Binding Domain of Gelatinase A Modulates Degradation of Collagen IV by Gelatinase B

Journal of Molecular Biology, 2009

Research paper thumbnail of In Situ AP/MALDI-MS characterization of anchored matrix metalloproteinases

Journal of Mass Spectrometry, 2006

Several different procedures are available for the immobilization of proteins on solid supports, ... more Several different procedures are available for the immobilization of proteins on solid supports, as many advantages derive from this approach, such as the possibility to develop new protein solid-state assays. Enzymes that are anchored on gold surfaces can interact with several different molecules in a tag-free environment, opening the way to surface plasmon resonance (SPR) investigations. Nevertheless, it is often important to know the identity of the affinity-retained analyte, and mass spectrometric analysis, via its unique molecular mass identification, represents a very valuable complementary method.There are many pieces of evidence to suggest that matrix metalloproteinases (MMPs) are involved in normal and pathological processes, including embryogenesis, wound healing, inflammation, arthritis and cancer, but presumably also exhibiting other functions. The search for new inhibitors of MMPs has prompted research towards the development of new solid-state assays for the rapid evaluation of MMP activity. We have already reported the possibility of measuring the activity of MMP-1 anchored on solid support by coupling SPR with ESI-MS analysis. In this work, we show the in situ atmospheric pressure (AP) MALDI-MS characterization of MMPs anchored on a gold chip with known surface coverage. The study extends the MS analysis to different proteins, and sequence coverage is reported for different digestion and MS procedures. Copyright © 2006 John Wiley & Sons, Ltd.

Research paper thumbnail of Somatostatin: A Novel Substrate and a Modulator of Insulin-Degrading Enzyme Activity

Journal of Molecular Biology, 2009

Research paper thumbnail of A new methodology for monitoring the activity of cdMMP-12 anchored and freeze-dried on Au (111

Journal of The American Society for Mass Spectrometry, 2007

Matrix metalloproteinases (MMPs) are cell-secreted soluble and membrane-tethered enzymes that are... more Matrix metalloproteinases (MMPs) are cell-secreted soluble and membrane-tethered enzymes that are capable of degrading extracellular matrix proteins, but also can process a number of bioactive molecules. They are involved in the cleavage of cell surface receptors, but are also thought to play a major role on cell behaviors as well as in diverse physiological and pathological processes, including embryonic development, wound repair, inflammatory diseases, and cancer. For these reasons, it is obvious that a control over MMPs activity is highly desirable. Consequently, the frantic search for new inhibitors has been coupled to the development of high-throughput methods able to rapidly screen the effect of possible MMP inhibitors on the activity of these enzymes. In this scenario, solid-state—based methods play a major role because of their compatibility with array formats that are able to extract more information from smaller sample volumes and offer some important advantages that are not available in the standard solution assays. In this work, the catalytic domain of MMP-12 was immobilized on a gold substrate and the surface coverage was measured by FT-SPR experiments. A new experimental procedure was developed to freeze-dry the anchored molecules and their activity was measured by ESI-MS. The kinetics parameters obtained for the immobilized enzyme are in good accordance with those reported for similar systems in solution. Inhibition of the immobilized molecules was also carried out, demonstrating the applicability of the method for rapid screening of MMP inhibitors.

Research paper thumbnail of AP/MALDI-MS complete characterization of the proteolytic fragments produced by the interaction of insulin degrading enzyme with bovine insulin

Journal of Mass Spectrometry, 2007

The prominent role that insulin degrading enzyme (IDE) has in the clearance of insulin as well as... more The prominent role that insulin degrading enzyme (IDE) has in the clearance of insulin as well as of other molecules such as amyloid-β has recently drawn much interest in the scientific community toward this protease. In order to give an insight into the manner of interaction of IDE with its substrates, several papers have focused on the structure of the IDE/insulin complex. In this scenario, although the cleavage sites involved in the interaction of insulin with IDE are known, a convenient experimental method that is able to identify in a complete and unambiguous way, all the peptide fragments generated by such interaction has yet to be found. MS-based experiments have often represented to be invaluable tools for the assessment of the cleavage sites, but the reported MS-spectra always show a partial coverage of all the peptide fragments generated by the enzyme interaction, lacking a complete characterization.In this work, we report a new experimental procedure by which an unambiguous as well as complete assignment of all the peptide fragments generated by the interaction of insulin with IDE is described. Atmospheric pressure/matrix-assisted laser desorption ionization (AP/MALDI) mass spectra are reported and the data recorded, together with the introduction of a reduction/alkylation step, allows us to fully characterize the cleavage sites of the bovine insulin interacting with IDE. Different experimental conditions are screened and some insights into the IDE/insulin system regarding preference of the cleavage and its dependence on particular experimental conditions used are also given. Investigation on the tendency that different insulin fragments have toward aggregation is also carried out.Good reproducibility, global and unambiguous assignment, low time-consuming experimental procedure, and requirements of enzyme in small amounts are some of the advantages of the proposed AP/MALDI based approach. Copyright © 2007 John Wiley & Sons, Ltd.

Research paper thumbnail of Activity of anchored human matrix metalloproteinase-1 catalytic domain on Au (111) surfaces monitored by ESI-MS

Journal of Mass Spectrometry, 2005

Matrix metalloproteinases (MMPs) are a family of Zn-dependent endo-peptidases known for their abi... more Matrix metalloproteinases (MMPs) are a family of Zn-dependent endo-peptidases known for their ability to cleave several components of the extracellular matrix, but which can also cleave many non-matrix proteins. There are many evidences that MMPs are involved in physiological and pathological processes, and a huge effort has been put in the development of possible inhibitors that could reduce the activity of MMPs, as it is clear that the ability to monitor and control such activity plays a pivotal role in the search for potential drugs aimed at finding a cure for several diseases such as pulmonary emphysema, rheumatoid arthritis, fibrotic disorders and cancer.A powerful method currently available to study enzyme–inhibitor interactions is based on the use of the surface plasmon resonance (SPR) technique. When MMP interactions are studied, a procedure by which inhibitors are normally anchored on sensor chips and SPR technique is used in order to study their interaction with MMPs molecules is usually followed. This is because it is currently believed that MMPs cannot be anchored on the sensor-chip surface without losing their activity. However, this approach gives rise to problems, as the anchoring of low-molecular-weight inhibitors on gold surfaces easily affects their ability to interact with MMPs. For this reason, the anchoring of MMPs is highly desirable.A new experimental protocol that couples the Fourier transform-SPR (FT-SPR) technique with electrospray ionization-mass spectroscopy (ESI-MS) is described here for the evaluation of the activity of MMP-1 catalytic domain (cdMMP-1) anchored on gold surfaces. The cdMMP-1 surface coverage is calculated by using FT-SPR and the enzyme activity is estimated by ESI-MS. The proposed method is label-free. Copyright © 2005 John Wiley & Sons, Ltd.

Research paper thumbnail of How the binding and degrading capabilities of insulin degrading enzyme are affected by ubiquitin

Biochimica Et Biophysica Acta-proteins and Proteomics, 2008

Insulin degrading enzyme (IDE) is known to play a pivotal role on amyloidogenic peptide degradati... more Insulin degrading enzyme (IDE) is known to play a pivotal role on amyloidogenic peptide degradation but little is known about the changes in the proteolytic activity of the enzyme upon modification of external factors. Particularly, although it has been reported that altered ubiquitin concentration and/or hyperinsulinaemia increase the risk of developing Alzheimer's disease (AD), the molecular mechanism involved is unclear. In this work, we study the role that ubiquitin plays on IDE capability of binding and degrading insulin molecules and the obtained results indicate that ubiquitin has an allosteric role for IDE and high ubiquitin levels impair IDE activity.

Research paper thumbnail of The use of atmospheric pressure laser desorption mass spectrometry for the study of iron-gall ink

Applied Physics A-materials Science & Processing, 2007

The study of ancient works of art is a very challenging task, mainly due to the impossibility of ... more The study of ancient works of art is a very challenging task, mainly due to the impossibility of applying experimental approaches that could damage anyhow the object from which analytical information has to be obtained. Spatially resolved analytical methods have significantly enhanced our capacity to study ancient material since they cause minimal and at times no damage to the studied object. Unfortunately, only a few analytical techniques operating within the requested spatial resolution are applicable for the investigation of the organic components of artistic and archaeological objects. In this work, iron-gall ink was prepared according to ancient recipes and its composition was evaluated by AP/MALDI-MS. The latter is demonstrated to be a very valuable tool for the study of the organic components of ancient works of art as it combines the advantages of the vacuum MALDI-MS analytical approach to the possibility of analysing samples in air. In situ analyses were also carried out by analysing strokes of ink directly from paper and parchment.

Research paper thumbnail of Microfluidic networks for surface plasmon resonance imaging real-time kinetics experiments

Microchemical Journal, 2009

Research paper thumbnail of Real-Time Binding Kinetics Monitored with Surface Plasmon Resonance Imaging in a Diffusion-Free Environment

The Open Spectroscopy Journal, 2008

Research paper thumbnail of The clearance of misfolded proteins in neurodegenerative diseases by zinc metalloproteases: An inorganic perspective Contents