A. Ostuni - Academia.edu (original) (raw)
Papers by A. Ostuni
XVI Congress of the Italian Society of Phytochemistry jointly with 2nd International Congress on Edible, Medicinal and Aromatic Plants (ICEMAP 2019), 2019
Annual meeting GIBB 2018., 2018
Journal of Peptide Science, 2006
Introduction of aldehyde groups into protein conjugates enhanced the immune response to a coupled... more Introduction of aldehyde groups into protein conjugates enhanced the immune response to a coupled peptide without the use of strong adjuvants. Synthetic peptides representing the N-terminal (residues 1-16) and internal (residues 53-65) epitopes of toxic shock syndrome toxin-1 (TSST-1) were coupled to carrier protein, and carbonyl tags were introduced by Amadori reaction with glycolaldehyde. Modified and unmodified antigens in alum were used to immunize rabbits and the reactivities of antisera were compared. Aldehyde modification augmented the response detected by ELISA, which included enhanced binding to peptides and to native TSST-1. In western blot, TSST-1 was detected by antiserum elicited to the N-terminal peptide, but not that generated to the peptide representing the internal sequence. The same antiserum also neutralized TSST-1 activity in a lymphocyte proliferation assay. The circular dichroism spectrum of the N-terminal peptide indicated a propensity for helical conformation, similar to the structure at the corresponding sequence of the native protein. These data suggest that aldehyde modification can boost immunogenicity of peptide-based vaccines, generating epitope-specific immune responses against the cognate protein antigens without using potent adjuvants.
Biochemistry, 2006
Lentiviral nucleocapsid proteins are a class of multifunctional proteins that play an essential r... more Lentiviral nucleocapsid proteins are a class of multifunctional proteins that play an essential role in RNA packaging and viral infectivity. They contain two CX 2 CX 4 HX 4 C zinc binding motifs connected by a basic linker of variable length. The 3D structure of a 37-aa peptide corresponding to sequence 22-58 from lentiviral EIAV nucleocapsid protein NCp11, complexed with zinc, has been determined by 2D 1 H NMR spectroscopy, simulated annealing, and molecular dynamics. The solution structure consists of two zinc binding domains held together by a five-residue basic linker Arg 38-Ala-Pro-Lys-Val 42 that allows for spatial proximity between the two finger domains. Observed linker folding is stabilized by H bonded secondary structure elements, resulting in an Ω-shaped central region, asymmetrically centered on the linker. The conformational differences and similarities with other NC zinc binding knuckles have been systematically analyzed. The two CCHC motifs, both characterized by a peculiar Pro-Gly sequence preceding the His residue, although preserving Zn-binding geometry and chirality of other known NC proteins, exhibit local fold differences both between each other and in comparison with other previously characterized retroviral CCHC motifs.
Biochemical and Biophysical Research Communications, 1998
recognition (1-3). Nucleocapsid proteins of retroviruses The metal binding properties of a 18-res... more recognition (1-3). Nucleocapsid proteins of retroviruses The metal binding properties of a 18-residue zinc are basic species of low-molecular weight (from about 6 finger peptide containing a CCHC box which reproto 9 kDa) containing one or two copies of the zinc-binding duces one of the cysteine-rich domains of a putative motif Cys-X 2-Cys-X 4-His-X 4-Cys (called ''CCHC box'') nucleic acid binding protein encoded by the Fw transwhich, at variance with above, bind to single stranded posable element from Drosophila melanogaster were nucleic acids (4-11). Proteins containing the same bindinvestigated through electronic and 1 H NMR spectrosing motif are also encoded by Drosophila melanogaster copy. Dissociation constants of 2({1)110 012 M and transposable elements (4, 12-16). 4({1)110 07 M were determined for the Zn 2/ and Co 2/ A number of investigations have focused on the bindadduct, respectively. These values are similar to those ing properties of synthetic peptides containing zinc for other CCHC-peptides investigated previously, alfinger sequence(s) toward spectroscopically active metthough the length of the spacer between the second als such as Co 2/ , Ni 2/ , Fe 2/ and Cd 2/ and on the struccysteine and the histidine apparently exerts some intural features of the metal adducts (4, 17-23). The fluence on the spectral properties and on the stability metal binding affinities of peptides containing the of the Co 2/-peptide adduct. The 1 H NMR spectrum of CCHH and CCHC box were found to differ to some the present Co 2/-derivative contains a number of well extent (17). Moreover, the peptide domain containing resolved hyperfine-shifted resonances between 350 the CCHC box was shown to fold into a globular strucand 050 ppm which arise from the metal binding resiture (4, 7, 24-26) which differs significantly from the dues and nearby groups. These peaks can in principle antiparallel b hairpin followed by a helix typical of be profitably exploited to monitor protein-nucleic acid ''classical'' CCHH zinc finger species (27).
Inorganica Chimica Acta, 1998
The solution and solid state behavior of the binary system Cu(II)-N-chloroacetylglycine (Cl-acgly... more The solution and solid state behavior of the binary system Cu(II)-N-chloroacetylglycine (Cl-acglyH) and the corresponding ternary systems with 2,2'-bipyridine (bpy) and 1,10-phenanthroline (ophen) were investigated by means of pH-metric and spectrophotometric titrations. The X-ray crystal structure of the complex [Cu(ophen) (Cl-acgly)2] • 2H20 (Cl-acgly = N-chloroacetylglycine monoanion) is also reported. The crystals of the compound C2oCI2CuNaO8H22 are monocli nic, space group P2~ / c, a = 17.574 (3), b = 7.125 (2), c = 25.113 (6) A, /3 = 130.04(2) °, Z= 4, R = 0.077, Rw = 0.088. The structure consists of monomeric [Cu(ophen) (Clgly) 2] units and lattice water molecules. The Cu (II) atom is square planar coordinated by two carboxylic oxygens of two amino acid molecules and two nitrogen atoms of the ophen molecule. Two long contacts involve the uncoordinated carboxylic oxygens. Crystal packing is due to ring-stacking interaction involving ophen molecules and to a strong intramolecular hydrogen bond involving the chlorine atom and the amidic nitrogen of one ligand molecule. In solution the species [CuL2] and [CuALOH] (A = bpy or ophen) prevail in the binary and ternary system, respectively, both arising from carboxylic-oxygen-metal coordination of the amino acid molecule. The coordination of the OH group in the ternary species prevents metal hydrolysis up to pH 11. The possibility of forming hydrogen bond interactions involving the chlorine atom seems the to be determinant factor in stabilizing the ternary complexes.
Journal of Peptide Science, 2013
The interaction between cisplatin and an 18-residue CCHC zinc finger motif derived from a retrovi... more The interaction between cisplatin and an 18-residue CCHC zinc finger motif derived from a retroviral nucleocapsid protein (PyrZf18) has been studied using UV-visible, CD and 1 H NMR spectroscopies and ESI-MS spectrometry. Cisplatin irreversibly blocks the cysteine zinc binding groups in the free peptide and is able to slowly eject zinc from the zinc-peptide complex. The observed end product of the reaction with cisplatin is a complex in which only one ammonia molecule is coordinated to platinum. After an initial binding with two cysteine residues and the formation of the (PyrZf18)platinum-(NH 3) 2 complex, a release of one ammonia molecule occurs because of trans-labilization, and the third cysteine is coordinated, leading to a mixture of isomers and/or conformers of the (PyrZf18)-platinum-NH 3 complex. The results are discussed with respect to the potential antiretroviral activity of platinum(II) compounds and to the possible interaction of cisplatin with the cellular nucleic acid binding proteins.
Journal of Peptide Science, 2014
Experimental vaccination to induce antibodies (Abs) capable of cytokine antagonism shows promise ... more Experimental vaccination to induce antibodies (Abs) capable of cytokine antagonism shows promise as a novel immunotherapy for chronic inflammatory disease. We prepared a hybrid antigen consisting of residues 141-235 of rat TNF-α fused to the C-terminus of glutathione-S-transferase (GST), chemically modified to incorporate aldehyde residues, for development of an auto-vaccine eliciting anti-rTNF-α Abs. In rat immunization the soluble aldehyde-modified fusion protein did not generate observable Ab responses. By contrast, vaccination with the aldehyde-modified fusion protein adsorbed on alum induced anti-TNF-α autoAbs with high titer and neutralizing activity. Induction of adjuvant arthritis in rats pre-immunized with unmodified fusion protein or a control protein in alum resulted in severe inflammation and joint damage, whereas the disease induced in rats immunized with the aldehyde-bearing fusion protein in alum was markedly attenuated. Similar results were obtained in a collageninduced rat arthritis model. Anti-collagen II IgG Ab titers did not deviate significantly in groups pre-immunized with modified fusion protein and control protein, suggesting that anti-TNF vaccination did not skew the immune response related to disease induction. This study demonstrates synergy between particulate alum and protein bound carbonyl residues for enhancement of protein immunogenicity. The antigen-specific co-adjuvant system could prove advantageous for breaking tolerance in emerging auto-vaccination therapies targeting inflammatory cytokines as well as for enhancing a broader category of subunit vaccines. Aldehyde adduction introduces a minimal modification which, together with the established use of alum as a safe adjuvant for human use, could be favorable for further vaccine development.
Frontiers in Physiology
The aim of this pilot study is to evaluate if SARS-CoV-2 infection or vaccination against SARS-Co... more The aim of this pilot study is to evaluate if SARS-CoV-2 infection or vaccination against SARS-CoV-2 infection induce observable metabolic effects in follicular fluid of women who are following in vitro fertilization (IVF) treatments. The possible impact of coronavirus disease 2019 (COVID-19) on fertility and IVF outcome is considered. We have selected for this study: six women vaccinated against SARS-CoV-2 infection, five recovered COVID-19 patients, and we used nine healthy women as the control group. At the time of oocytes retrieval from participants in the study, follicular fluids were collected and metabolomic analysis was performed by 1H NMR spectroscopy in combination with multivariate analysis to interpret the spectral data. The search for antibody positivity in the follicular fluid aspirates was also carried out, together with the western blotting analysis of some inflammatory proteins, interleukin-6, tumor necrosis factor α (TNFα), and the free radical scavenger superoxide...
XVII International Symposium Virus and Virus-Like Diseases of Temperate Fruit Crops, 1998
Research Letters in Biochemistry, 2008
ABCC6 is a member of the adenosine triphosphate-binding cassette (ABC) gene subfamily C that enco... more ABCC6 is a member of the adenosine triphosphate-binding cassette (ABC) gene subfamily C that encodes a protein (MRP6) involved in active transport of intracellular compounds to the extracellular environment. Mutations in ABCC6 cause pseudoxanthoma elasticum (PXE), an autosomal recessive disorder of the connective tissue characterized by progressive calcification of elastic structures in the skin, the eyes, and the cardiovascular system. MRP6 is codified by 31 exons and contains 1503 amino acids. In addition to a full-length transcript of ABCC6, we have identified an alternatively spliced variant of ABCC6 from a cDNA of human liver that lacks exons 19 and 24. The novel isoform was named ABCC6 1924. PCR analysis from cDNA of cell cultures of primary human hepatocites and embryonic kidney confirms the presence of the ABCC61924 isoform. Western blot analysis of the embryonic kidney cells shows a band corresponding to the molecular weight of the truncated protein.
Biochemical and biophysical research communications, Jan 8, 2014
Experimental tools to determine membrane topology of a protein are rather limited in higher eukar... more Experimental tools to determine membrane topology of a protein are rather limited in higher eukaryotic organisms. Here, we report the use of glycosylatable GFP (gGFP) as a sensitive and versatile membrane topology reporter in mammalian cells. gGFP selectively loses its fluorescence upon N-linked glycosylation in the ER lumen. Thus, positive fluorescence signal assigns location of gGFP to the cytosol whereas no fluorescence signal and a glycosylated status of gGFP map the location of gGFP to the ER lumen. By using mammalian gGFP, the membrane topology of disease-associated membrane proteins, URG7, MRP6102, SP-C(Val) and SP-C(Leu) was confirmed. URG7 is partially targeted to the ER, and inserted in Cin form. MRP6102 and SP-C(Leu/Val) are inserted into the membrane in Cout form. A minor population of untargeted SP-C is removed by proteasome dependent quality control system.
The International Journal of Biochemistry & Cell Biology, 1998
Proteins: Structure, Function, and Bioinformatics, 2009
Molecular Membrane Biology, 2004
XVI Congress of the Italian Society of Phytochemistry jointly with 2nd International Congress on Edible, Medicinal and Aromatic Plants (ICEMAP 2019), 2019
Annual meeting GIBB 2018., 2018
Journal of Peptide Science, 2006
Introduction of aldehyde groups into protein conjugates enhanced the immune response to a coupled... more Introduction of aldehyde groups into protein conjugates enhanced the immune response to a coupled peptide without the use of strong adjuvants. Synthetic peptides representing the N-terminal (residues 1-16) and internal (residues 53-65) epitopes of toxic shock syndrome toxin-1 (TSST-1) were coupled to carrier protein, and carbonyl tags were introduced by Amadori reaction with glycolaldehyde. Modified and unmodified antigens in alum were used to immunize rabbits and the reactivities of antisera were compared. Aldehyde modification augmented the response detected by ELISA, which included enhanced binding to peptides and to native TSST-1. In western blot, TSST-1 was detected by antiserum elicited to the N-terminal peptide, but not that generated to the peptide representing the internal sequence. The same antiserum also neutralized TSST-1 activity in a lymphocyte proliferation assay. The circular dichroism spectrum of the N-terminal peptide indicated a propensity for helical conformation, similar to the structure at the corresponding sequence of the native protein. These data suggest that aldehyde modification can boost immunogenicity of peptide-based vaccines, generating epitope-specific immune responses against the cognate protein antigens without using potent adjuvants.
Biochemistry, 2006
Lentiviral nucleocapsid proteins are a class of multifunctional proteins that play an essential r... more Lentiviral nucleocapsid proteins are a class of multifunctional proteins that play an essential role in RNA packaging and viral infectivity. They contain two CX 2 CX 4 HX 4 C zinc binding motifs connected by a basic linker of variable length. The 3D structure of a 37-aa peptide corresponding to sequence 22-58 from lentiviral EIAV nucleocapsid protein NCp11, complexed with zinc, has been determined by 2D 1 H NMR spectroscopy, simulated annealing, and molecular dynamics. The solution structure consists of two zinc binding domains held together by a five-residue basic linker Arg 38-Ala-Pro-Lys-Val 42 that allows for spatial proximity between the two finger domains. Observed linker folding is stabilized by H bonded secondary structure elements, resulting in an Ω-shaped central region, asymmetrically centered on the linker. The conformational differences and similarities with other NC zinc binding knuckles have been systematically analyzed. The two CCHC motifs, both characterized by a peculiar Pro-Gly sequence preceding the His residue, although preserving Zn-binding geometry and chirality of other known NC proteins, exhibit local fold differences both between each other and in comparison with other previously characterized retroviral CCHC motifs.
Biochemical and Biophysical Research Communications, 1998
recognition (1-3). Nucleocapsid proteins of retroviruses The metal binding properties of a 18-res... more recognition (1-3). Nucleocapsid proteins of retroviruses The metal binding properties of a 18-residue zinc are basic species of low-molecular weight (from about 6 finger peptide containing a CCHC box which reproto 9 kDa) containing one or two copies of the zinc-binding duces one of the cysteine-rich domains of a putative motif Cys-X 2-Cys-X 4-His-X 4-Cys (called ''CCHC box'') nucleic acid binding protein encoded by the Fw transwhich, at variance with above, bind to single stranded posable element from Drosophila melanogaster were nucleic acids (4-11). Proteins containing the same bindinvestigated through electronic and 1 H NMR spectrosing motif are also encoded by Drosophila melanogaster copy. Dissociation constants of 2({1)110 012 M and transposable elements (4, 12-16). 4({1)110 07 M were determined for the Zn 2/ and Co 2/ A number of investigations have focused on the bindadduct, respectively. These values are similar to those ing properties of synthetic peptides containing zinc for other CCHC-peptides investigated previously, alfinger sequence(s) toward spectroscopically active metthough the length of the spacer between the second als such as Co 2/ , Ni 2/ , Fe 2/ and Cd 2/ and on the struccysteine and the histidine apparently exerts some intural features of the metal adducts (4, 17-23). The fluence on the spectral properties and on the stability metal binding affinities of peptides containing the of the Co 2/-peptide adduct. The 1 H NMR spectrum of CCHH and CCHC box were found to differ to some the present Co 2/-derivative contains a number of well extent (17). Moreover, the peptide domain containing resolved hyperfine-shifted resonances between 350 the CCHC box was shown to fold into a globular strucand 050 ppm which arise from the metal binding resiture (4, 7, 24-26) which differs significantly from the dues and nearby groups. These peaks can in principle antiparallel b hairpin followed by a helix typical of be profitably exploited to monitor protein-nucleic acid ''classical'' CCHH zinc finger species (27).
Inorganica Chimica Acta, 1998
The solution and solid state behavior of the binary system Cu(II)-N-chloroacetylglycine (Cl-acgly... more The solution and solid state behavior of the binary system Cu(II)-N-chloroacetylglycine (Cl-acglyH) and the corresponding ternary systems with 2,2'-bipyridine (bpy) and 1,10-phenanthroline (ophen) were investigated by means of pH-metric and spectrophotometric titrations. The X-ray crystal structure of the complex [Cu(ophen) (Cl-acgly)2] • 2H20 (Cl-acgly = N-chloroacetylglycine monoanion) is also reported. The crystals of the compound C2oCI2CuNaO8H22 are monocli nic, space group P2~ / c, a = 17.574 (3), b = 7.125 (2), c = 25.113 (6) A, /3 = 130.04(2) °, Z= 4, R = 0.077, Rw = 0.088. The structure consists of monomeric [Cu(ophen) (Clgly) 2] units and lattice water molecules. The Cu (II) atom is square planar coordinated by two carboxylic oxygens of two amino acid molecules and two nitrogen atoms of the ophen molecule. Two long contacts involve the uncoordinated carboxylic oxygens. Crystal packing is due to ring-stacking interaction involving ophen molecules and to a strong intramolecular hydrogen bond involving the chlorine atom and the amidic nitrogen of one ligand molecule. In solution the species [CuL2] and [CuALOH] (A = bpy or ophen) prevail in the binary and ternary system, respectively, both arising from carboxylic-oxygen-metal coordination of the amino acid molecule. The coordination of the OH group in the ternary species prevents metal hydrolysis up to pH 11. The possibility of forming hydrogen bond interactions involving the chlorine atom seems the to be determinant factor in stabilizing the ternary complexes.
Journal of Peptide Science, 2013
The interaction between cisplatin and an 18-residue CCHC zinc finger motif derived from a retrovi... more The interaction between cisplatin and an 18-residue CCHC zinc finger motif derived from a retroviral nucleocapsid protein (PyrZf18) has been studied using UV-visible, CD and 1 H NMR spectroscopies and ESI-MS spectrometry. Cisplatin irreversibly blocks the cysteine zinc binding groups in the free peptide and is able to slowly eject zinc from the zinc-peptide complex. The observed end product of the reaction with cisplatin is a complex in which only one ammonia molecule is coordinated to platinum. After an initial binding with two cysteine residues and the formation of the (PyrZf18)platinum-(NH 3) 2 complex, a release of one ammonia molecule occurs because of trans-labilization, and the third cysteine is coordinated, leading to a mixture of isomers and/or conformers of the (PyrZf18)-platinum-NH 3 complex. The results are discussed with respect to the potential antiretroviral activity of platinum(II) compounds and to the possible interaction of cisplatin with the cellular nucleic acid binding proteins.
Journal of Peptide Science, 2014
Experimental vaccination to induce antibodies (Abs) capable of cytokine antagonism shows promise ... more Experimental vaccination to induce antibodies (Abs) capable of cytokine antagonism shows promise as a novel immunotherapy for chronic inflammatory disease. We prepared a hybrid antigen consisting of residues 141-235 of rat TNF-α fused to the C-terminus of glutathione-S-transferase (GST), chemically modified to incorporate aldehyde residues, for development of an auto-vaccine eliciting anti-rTNF-α Abs. In rat immunization the soluble aldehyde-modified fusion protein did not generate observable Ab responses. By contrast, vaccination with the aldehyde-modified fusion protein adsorbed on alum induced anti-TNF-α autoAbs with high titer and neutralizing activity. Induction of adjuvant arthritis in rats pre-immunized with unmodified fusion protein or a control protein in alum resulted in severe inflammation and joint damage, whereas the disease induced in rats immunized with the aldehyde-bearing fusion protein in alum was markedly attenuated. Similar results were obtained in a collageninduced rat arthritis model. Anti-collagen II IgG Ab titers did not deviate significantly in groups pre-immunized with modified fusion protein and control protein, suggesting that anti-TNF vaccination did not skew the immune response related to disease induction. This study demonstrates synergy between particulate alum and protein bound carbonyl residues for enhancement of protein immunogenicity. The antigen-specific co-adjuvant system could prove advantageous for breaking tolerance in emerging auto-vaccination therapies targeting inflammatory cytokines as well as for enhancing a broader category of subunit vaccines. Aldehyde adduction introduces a minimal modification which, together with the established use of alum as a safe adjuvant for human use, could be favorable for further vaccine development.
Frontiers in Physiology
The aim of this pilot study is to evaluate if SARS-CoV-2 infection or vaccination against SARS-Co... more The aim of this pilot study is to evaluate if SARS-CoV-2 infection or vaccination against SARS-CoV-2 infection induce observable metabolic effects in follicular fluid of women who are following in vitro fertilization (IVF) treatments. The possible impact of coronavirus disease 2019 (COVID-19) on fertility and IVF outcome is considered. We have selected for this study: six women vaccinated against SARS-CoV-2 infection, five recovered COVID-19 patients, and we used nine healthy women as the control group. At the time of oocytes retrieval from participants in the study, follicular fluids were collected and metabolomic analysis was performed by 1H NMR spectroscopy in combination with multivariate analysis to interpret the spectral data. The search for antibody positivity in the follicular fluid aspirates was also carried out, together with the western blotting analysis of some inflammatory proteins, interleukin-6, tumor necrosis factor α (TNFα), and the free radical scavenger superoxide...
XVII International Symposium Virus and Virus-Like Diseases of Temperate Fruit Crops, 1998
Research Letters in Biochemistry, 2008
ABCC6 is a member of the adenosine triphosphate-binding cassette (ABC) gene subfamily C that enco... more ABCC6 is a member of the adenosine triphosphate-binding cassette (ABC) gene subfamily C that encodes a protein (MRP6) involved in active transport of intracellular compounds to the extracellular environment. Mutations in ABCC6 cause pseudoxanthoma elasticum (PXE), an autosomal recessive disorder of the connective tissue characterized by progressive calcification of elastic structures in the skin, the eyes, and the cardiovascular system. MRP6 is codified by 31 exons and contains 1503 amino acids. In addition to a full-length transcript of ABCC6, we have identified an alternatively spliced variant of ABCC6 from a cDNA of human liver that lacks exons 19 and 24. The novel isoform was named ABCC6 1924. PCR analysis from cDNA of cell cultures of primary human hepatocites and embryonic kidney confirms the presence of the ABCC61924 isoform. Western blot analysis of the embryonic kidney cells shows a band corresponding to the molecular weight of the truncated protein.
Biochemical and biophysical research communications, Jan 8, 2014
Experimental tools to determine membrane topology of a protein are rather limited in higher eukar... more Experimental tools to determine membrane topology of a protein are rather limited in higher eukaryotic organisms. Here, we report the use of glycosylatable GFP (gGFP) as a sensitive and versatile membrane topology reporter in mammalian cells. gGFP selectively loses its fluorescence upon N-linked glycosylation in the ER lumen. Thus, positive fluorescence signal assigns location of gGFP to the cytosol whereas no fluorescence signal and a glycosylated status of gGFP map the location of gGFP to the ER lumen. By using mammalian gGFP, the membrane topology of disease-associated membrane proteins, URG7, MRP6102, SP-C(Val) and SP-C(Leu) was confirmed. URG7 is partially targeted to the ER, and inserted in Cin form. MRP6102 and SP-C(Leu/Val) are inserted into the membrane in Cout form. A minor population of untargeted SP-C is removed by proteasome dependent quality control system.
The International Journal of Biochemistry & Cell Biology, 1998
Proteins: Structure, Function, and Bioinformatics, 2009
Molecular Membrane Biology, 2004