Carmine Ercole - Academia.edu (original) (raw)

Papers by Carmine Ercole

Biopolymers, Dec 1, 2009

Drought influences cereal crop yield and quality. However, little is known about changes in the s... more Drought influences cereal crop yield and quality. However, little is known about changes in the structural and functional properties of wheat starch under soil drought conditions. In this study, two wheat cultivars were subjected to well-watered (WW), moderate soil-drought (MD), and severe soildrought (SD) from 7 tillers in the main stem to maturity. The structural and functional properties of the resultant endosperm starch were investigated. In comparison with WW soil, the MD increased starch accumulation in grains, the proportion of large starch granules, amylose and amylopectin long branch chain contents, and average amylopectin branch chain length, which were accompanied by the increase in activities of granule bound starch synthase and soluble starch synthase. MD treated-starch had a lower gelatinization enthalpy, and swelling power, but a higher gelatinization temperature, retrogradation enthalpy, and retrogradation percentage when compared to WW conditions. The MD also increased starch resistance to acid hydrolysis, amylase hydrolysis, and in vitro digestion. The SD had the opposite effects to the MD in all cases. The results suggest that soil drought more severely affects amylose synthesis than amylopectin synthesis in wheat grains, and moderate soil-drought improves molecular structure and functional properties of the starch. Wheat (Triticum aestivum L.) is an important cereal crop and a staple food for humans and animals worldwide. Starch is the major storage compound in wheat endosperm, accounting for 65-75% of the final dry weight of a grain, and is synthesized in the amyloplast of endosperm cells since 4 days after anthesis (DAA) and the endosperm structure no longer changes after 33 DAA 1,2. Starch is mainly composed of linear amylose and highly branched amylopectin, which assemble to form a semicrystalline granule 3. For linear amylose, the glucose units are joined through α-(1,4)-glycosidic linkages which are mainly catalyzed and elongated by granule-bound starch synthase (GBSS). Amylopectin mainly consists of long chains of α-(1,4)-linked D-glucopyranosyl units with occasional branching α-(1,6)-linkages that form branched structure. The α-(1,6)-glycosidic linkages are catalyzed and elongated by starch branching enzymes (SBE) and soluble starch synthase (SSS), respectively 1,4. Amylose content and amylopectin fine structure greatly influence physicochemical properties that affect grain quality, flour quality, and starch properties 4. Grain quality is a complex trait with various determinants, including physical appearance, nutritional value, and eating and cooking quality. These factors are important for consumers, and are associated with the physicochemical properties of crop starch, including hydration, gelatinization, volume expansion, and digestion properties 5,6. Furthermore, apparent amylose content, pasting viscosity characteristics, gel texture, thermal and retrogradation properties, and amylose and amylopectin fine structures have been established to precisely evaluate the quality of grain and starch-based foods 7,8. It is well known that seed yield and quality are determined both genetically and environmentally 9. Soil water status, especially during the grain development, probably ranks as the most important environmental factor affecting grain yield and quality in cereals 10,11. The arid and semiarid rangelands exist all over the world, such as in the Middle East and North Africa, South and Central Asia, South and North America 10. Various physiological and chemical reactions can be activated when plants are subjected to water stress during various developmental

Bovine seminal RNase (BS RNase) is the only naturally dimeric member of the pancreatic-type RNase... more Bovine seminal RNase (BS RNase) is the only naturally dimeric member of the pancreatic-type RNase superfamily. The enzyme is a mixture of two isoforms: (i) M=M, dimeric through two antiparallel disulfides occurring between the two subunits; (ii) MxM (70% of the total), characterized by the swapping of the N-termini besides the mentioned disulfides (1). When dissolved in 40% acetic acid and subjected to lyophilisation, BS RNase forms a mixture of oligomeric aggregates (2), as does RNase A (3), the proto-type of the super-family. However, while the oligomers of the pancreatic variant have been extensively characterized (3\u20135), the multimers of BS RNase (6) are presently less known. To deepen their characterization we induced BS RNase self-association by using the same conditions used with RNase A, i.e. lyophilisation of enzyme solutions at low pH, or thermally-induced aggregation of concentrated protein solutions in various media. The multimers obtained were analyzed by SEC, cation-exchange chromatography, cross-linking, electrophoresis, proteolysis, and enzymatic assays. The main results obtained are: (a) BS-RNase forms several 3D domain-swapped conformers, in particular at least two tetramers; one is probably a totally N terminal-swapped isoform (4), while the other contains a C terminal swapping. (b) The C-swapped tetramer is less stable than the N-swapped one. (c) BS RNase multimers larger than tetramers appear to be a mixture of various isoforms, similarly to what occurs with RNase A (3). These multimers seem to contain, again, a Cterminus swapping. (4) The validity of the results reported have been strengthened by the studies performed with a K113N-BS RNase mutant. References: 1. Piccoli R., et al. Proc Natl Acad Sci USA 1992; 89: 1870\u20131874. 2. Libonati M. Int J Biochem 1969; 18: 407\u2013417. 3. Libonati M. & Gotte G. Biochem J 2004; 380: 311\u2013327. 4. Liu Y., et al. Proc Natl Acad Sci USA 1998; 95: 3437\u20133442. 5. Liu Y., et al. Nat Str Biol 2001; 8: 211\u2013214. 6. Adinolfi S., et al. FEBS Lett 1996; 398: 326\u2013332

Nutrients, 2019

Sweeteners have become integrating components of the typical western diet, in response to the spr... more Sweeteners have become integrating components of the typical western diet, in response to the spreading of sugar-related pathologies (diabetes, obesity and metabolic syndrome) that have stemmed from the adoption of unbalanced dietary habits. Sweet proteins are a relatively unstudied class of sweet compounds that could serve as innovative sweeteners, but their introduction on the food market has been delayed by some factors, among which is the lack of thorough metabolic and toxicological studies. We have tried to shed light on the potential of a sweet protein, MNEI, as a fructose substitute in beverages in a typical western diet, by studying the metabolic consequences of its consumption on a Wistar rat model of high fat diet-induced obesity. In particular, we investigated the lipid profile, insulin sensitivity and other indicators of metabolic syndrome. We also evaluated systemic inflammation and potential colon damage. MNEI consumption rescued the metabolic derangement elicited by t...

Journal of Solution Chemistry, 2018

The mechanism of conversion of globular native proteins into amyloid fibrils represents one of th... more The mechanism of conversion of globular native proteins into amyloid fibrils represents one of the most attractive research topics in biophysics, because of its involvement in the development of severe pathologies and in various biotechnological processes. Aqueous medium properties, such as pH and ionic strength, as well as interactions with other species in solution, play a key role in tuning the fibrillization process. Here, we describe a comparative study of the influence of different ions from the Hofmeister series on the thermal unfolding and aggregation propensity of MNEI, a model protein, selected because of its tendency to form amyloid aggregates at acidic pH, even at temperatures well below its melting temperature. By selecting a temperature at which only negligible amounts of protein are unfolded, we have focused on the effect of ions on fibril formation. ThT fluorescence experiments indicated that all the salts examined increased the rate and the extent of fibrillization. Moreover, we found that anions, particularly sulfate, strongly influence the process, which instead is only marginally affected by different cations. Finally, a specific link to the chloride concentration was detected.

FEBS Letters, 2013

Bovine seminal ribonuclease (BS-RNase) acquires an interesting anti-tumor activity associated wit... more Bovine seminal ribonuclease (BS-RNase) acquires an interesting anti-tumor activity associated with the swapping on the N-terminal. The first direct experimental evidence on the formation of a C-terminal swapped dimer (C-dimer) obtained from the monomeric derivative of BS-RNase, although under non-native conditions, is here reported. The X-ray model of this dimer reveals a quaternary structure different from that of the C-dimer of RNase A, due to the presence of three mutations in the hinge peptide 111-116. The mutations increase the hinge peptide flexibility and decrease the stability of the C-dimer against dissociation. The biological implications of the structural data are also discussed. Structure summary of protein interactions: BS-RNase and BS-RNase bind by x-ray crystallography (View interaction) BS-RNase and BS-RNase bind by molecular sieving (1, 2) BS-RNase and BS-RNase bind by blue native page (View interaction)

Biopolymers, 2009

The cytotoxic action of bovine seminal ribonuclease (BS-RNase) depends on its noncovalent swapped... more The cytotoxic action of bovine seminal ribonuclease (BS-RNase) depends on its noncovalent swapped dimeric form (NCD-BS), which presents a compact structure that allows the molecule to escape ribonuclease inhibitor (RI). A key role in the acquisition of this structure has been attributed to the concomitant presence of a proline in position 19 and a leucine in position 28. The introduction of Leu28, Cys31, and Cys32 and, in addition, of Pro19 in the sequence of bovine pancreatic ribonuclease (RNase A) has produced two dimeric variants LCC and PLCC, which do exhibit a cytotoxic activity, though at a much lower level than BS-RNase. The crystal structure analysis of the noncovalent swapped form (NCD) of LCC and PLCC, complexed with the substrate analogue 2 0-deoxycytidylyl(3 0 ,5 0)-2 0deoxyguanosine, has revealed that, differently from NCD-BS, the dimers adopt an opened quaternary structure, with the two Leu residues fully exposed to the solvent, that does not hinder the binding of RI. Similar results have been obtained for a third mutant of the pancreatic enzyme, engineered with the hinge peptide sequence of the seminal enzyme (residues 16-22) and the two cysteines in position 31 and 32, but lacking the hydrophobic Leu residue in position 28. The comparison of these three structures with those previously reported for other ribonuclease swapped dimers strongly suggests that, in addition to Pro19 and Leu28, the presence of a glycine at the N-terminal end of the hinge peptide is also important to push the swapped form of RNase A dimer into the compact quaternary organization observed for NCD-BS.

Biochimie, 2012

3D domain swapping is the process by which two or more protein molecules exchange part of their s... more 3D domain swapping is the process by which two or more protein molecules exchange part of their structure to form intertwined dimers or higher oligomers. Bovine pancreatic ribonuclease (RNase A) is able to swap the N-terminal α-helix (residues 1-13) and/or the C-terminal β-strand (residues 116-124), thus forming a variety of oligomers, including two different dimers. Cis-trans isomerization of the Asn113-Pro114 peptide group was observed when the protein formed the C-terminal swapped dimer. To study the effect of the substitution of Pro114 on the swapping process of RNase A, we have prepared and characterized the P114A monomeric and dimeric variants of the enzyme. In contrast with previous reports, the crystal structure and NMR data on the monomer reveals a mixed cis-trans conformation for the Asn113-Ala114 peptide group, whereas the X-ray structure of the C-terminal swapped dimer of the variant is very close to that of the corresponding dimer of RNase A. The mutation at the C-terminus affects the capability of the N-terminal α-helix to swap and the stability of both dimeric forms. The present results underscore the importance of the hydration shell in determining the cross-talk between the chain termini in the swapping process of RNase A.

Acta Crystallographica Section A Foundations of Crystallography, 2005

The sensor proteins for blue-light using the FAD (BLUF) domain belong to the third family of the ... more The sensor proteins for blue-light using the FAD (BLUF) domain belong to the third family of the photoreceptor proteins using a flavin chromophore, where the other two families are phototropins and cryptochromes. We have determined the crystal structure of the Tll0078 protein from Thermosynechococcus elongatus BP-1, which contains a BLUF domain bound to FAD, at 2 Å resolution. The crystals belonged to space group P2 1 2 1 2 1 with cell dimensions of a=89.5 Å, b=109.9 Å, and c=169.9 Å. The asymmetric unit contains 10 monomers of Tll0078 (one decamer). Five Tll0078 monomers are located around the non-crystallographic five-fold axis to form a pentamer, and two pentamers related by twofold noncrystallographic symmetry form a decameric assembly. The monomer consists of two domains, the BLUF domain at the Nterminal region and the C-terminal domain. The overall structure of the BLUF domain consists of a five-stranded mixed-sheet with two helices running parallel to it. The isoalloxazine ring of FAD is accommodated in a pocket formed by several highly-conserved amino acid residues in the BLUF domain.

Biopolymers, Dec 1, 2009

Drought influences cereal crop yield and quality. However, little is known about changes in the s... more Drought influences cereal crop yield and quality. However, little is known about changes in the structural and functional properties of wheat starch under soil drought conditions. In this study, two wheat cultivars were subjected to well-watered (WW), moderate soil-drought (MD), and severe soildrought (SD) from 7 tillers in the main stem to maturity. The structural and functional properties of the resultant endosperm starch were investigated. In comparison with WW soil, the MD increased starch accumulation in grains, the proportion of large starch granules, amylose and amylopectin long branch chain contents, and average amylopectin branch chain length, which were accompanied by the increase in activities of granule bound starch synthase and soluble starch synthase. MD treated-starch had a lower gelatinization enthalpy, and swelling power, but a higher gelatinization temperature, retrogradation enthalpy, and retrogradation percentage when compared to WW conditions. The MD also increased starch resistance to acid hydrolysis, amylase hydrolysis, and in vitro digestion. The SD had the opposite effects to the MD in all cases. The results suggest that soil drought more severely affects amylose synthesis than amylopectin synthesis in wheat grains, and moderate soil-drought improves molecular structure and functional properties of the starch. Wheat (Triticum aestivum L.) is an important cereal crop and a staple food for humans and animals worldwide. Starch is the major storage compound in wheat endosperm, accounting for 65-75% of the final dry weight of a grain, and is synthesized in the amyloplast of endosperm cells since 4 days after anthesis (DAA) and the endosperm structure no longer changes after 33 DAA 1,2. Starch is mainly composed of linear amylose and highly branched amylopectin, which assemble to form a semicrystalline granule 3. For linear amylose, the glucose units are joined through α-(1,4)-glycosidic linkages which are mainly catalyzed and elongated by granule-bound starch synthase (GBSS). Amylopectin mainly consists of long chains of α-(1,4)-linked D-glucopyranosyl units with occasional branching α-(1,6)-linkages that form branched structure. The α-(1,6)-glycosidic linkages are catalyzed and elongated by starch branching enzymes (SBE) and soluble starch synthase (SSS), respectively 1,4. Amylose content and amylopectin fine structure greatly influence physicochemical properties that affect grain quality, flour quality, and starch properties 4. Grain quality is a complex trait with various determinants, including physical appearance, nutritional value, and eating and cooking quality. These factors are important for consumers, and are associated with the physicochemical properties of crop starch, including hydration, gelatinization, volume expansion, and digestion properties 5,6. Furthermore, apparent amylose content, pasting viscosity characteristics, gel texture, thermal and retrogradation properties, and amylose and amylopectin fine structures have been established to precisely evaluate the quality of grain and starch-based foods 7,8. It is well known that seed yield and quality are determined both genetically and environmentally 9. Soil water status, especially during the grain development, probably ranks as the most important environmental factor affecting grain yield and quality in cereals 10,11. The arid and semiarid rangelands exist all over the world, such as in the Middle East and North Africa, South and Central Asia, South and North America 10. Various physiological and chemical reactions can be activated when plants are subjected to water stress during various developmental

Bovine seminal RNase (BS RNase) is the only naturally dimeric member of the pancreatic-type RNase... more Bovine seminal RNase (BS RNase) is the only naturally dimeric member of the pancreatic-type RNase superfamily. The enzyme is a mixture of two isoforms: (i) M=M, dimeric through two antiparallel disulfides occurring between the two subunits; (ii) MxM (70% of the total), characterized by the swapping of the N-termini besides the mentioned disulfides (1). When dissolved in 40% acetic acid and subjected to lyophilisation, BS RNase forms a mixture of oligomeric aggregates (2), as does RNase A (3), the proto-type of the super-family. However, while the oligomers of the pancreatic variant have been extensively characterized (3\u20135), the multimers of BS RNase (6) are presently less known. To deepen their characterization we induced BS RNase self-association by using the same conditions used with RNase A, i.e. lyophilisation of enzyme solutions at low pH, or thermally-induced aggregation of concentrated protein solutions in various media. The multimers obtained were analyzed by SEC, cation-exchange chromatography, cross-linking, electrophoresis, proteolysis, and enzymatic assays. The main results obtained are: (a) BS-RNase forms several 3D domain-swapped conformers, in particular at least two tetramers; one is probably a totally N terminal-swapped isoform (4), while the other contains a C terminal swapping. (b) The C-swapped tetramer is less stable than the N-swapped one. (c) BS RNase multimers larger than tetramers appear to be a mixture of various isoforms, similarly to what occurs with RNase A (3). These multimers seem to contain, again, a Cterminus swapping. (4) The validity of the results reported have been strengthened by the studies performed with a K113N-BS RNase mutant. References: 1. Piccoli R., et al. Proc Natl Acad Sci USA 1992; 89: 1870\u20131874. 2. Libonati M. Int J Biochem 1969; 18: 407\u2013417. 3. Libonati M. & Gotte G. Biochem J 2004; 380: 311\u2013327. 4. Liu Y., et al. Proc Natl Acad Sci USA 1998; 95: 3437\u20133442. 5. Liu Y., et al. Nat Str Biol 2001; 8: 211\u2013214. 6. Adinolfi S., et al. FEBS Lett 1996; 398: 326\u2013332

Nutrients, 2019

Sweeteners have become integrating components of the typical western diet, in response to the spr... more Sweeteners have become integrating components of the typical western diet, in response to the spreading of sugar-related pathologies (diabetes, obesity and metabolic syndrome) that have stemmed from the adoption of unbalanced dietary habits. Sweet proteins are a relatively unstudied class of sweet compounds that could serve as innovative sweeteners, but their introduction on the food market has been delayed by some factors, among which is the lack of thorough metabolic and toxicological studies. We have tried to shed light on the potential of a sweet protein, MNEI, as a fructose substitute in beverages in a typical western diet, by studying the metabolic consequences of its consumption on a Wistar rat model of high fat diet-induced obesity. In particular, we investigated the lipid profile, insulin sensitivity and other indicators of metabolic syndrome. We also evaluated systemic inflammation and potential colon damage. MNEI consumption rescued the metabolic derangement elicited by t...

Journal of Solution Chemistry, 2018

The mechanism of conversion of globular native proteins into amyloid fibrils represents one of th... more The mechanism of conversion of globular native proteins into amyloid fibrils represents one of the most attractive research topics in biophysics, because of its involvement in the development of severe pathologies and in various biotechnological processes. Aqueous medium properties, such as pH and ionic strength, as well as interactions with other species in solution, play a key role in tuning the fibrillization process. Here, we describe a comparative study of the influence of different ions from the Hofmeister series on the thermal unfolding and aggregation propensity of MNEI, a model protein, selected because of its tendency to form amyloid aggregates at acidic pH, even at temperatures well below its melting temperature. By selecting a temperature at which only negligible amounts of protein are unfolded, we have focused on the effect of ions on fibril formation. ThT fluorescence experiments indicated that all the salts examined increased the rate and the extent of fibrillization. Moreover, we found that anions, particularly sulfate, strongly influence the process, which instead is only marginally affected by different cations. Finally, a specific link to the chloride concentration was detected.

FEBS Letters, 2013

Bovine seminal ribonuclease (BS-RNase) acquires an interesting anti-tumor activity associated wit... more Bovine seminal ribonuclease (BS-RNase) acquires an interesting anti-tumor activity associated with the swapping on the N-terminal. The first direct experimental evidence on the formation of a C-terminal swapped dimer (C-dimer) obtained from the monomeric derivative of BS-RNase, although under non-native conditions, is here reported. The X-ray model of this dimer reveals a quaternary structure different from that of the C-dimer of RNase A, due to the presence of three mutations in the hinge peptide 111-116. The mutations increase the hinge peptide flexibility and decrease the stability of the C-dimer against dissociation. The biological implications of the structural data are also discussed. Structure summary of protein interactions: BS-RNase and BS-RNase bind by x-ray crystallography (View interaction) BS-RNase and BS-RNase bind by molecular sieving (1, 2) BS-RNase and BS-RNase bind by blue native page (View interaction)

Biopolymers, 2009

The cytotoxic action of bovine seminal ribonuclease (BS-RNase) depends on its noncovalent swapped... more The cytotoxic action of bovine seminal ribonuclease (BS-RNase) depends on its noncovalent swapped dimeric form (NCD-BS), which presents a compact structure that allows the molecule to escape ribonuclease inhibitor (RI). A key role in the acquisition of this structure has been attributed to the concomitant presence of a proline in position 19 and a leucine in position 28. The introduction of Leu28, Cys31, and Cys32 and, in addition, of Pro19 in the sequence of bovine pancreatic ribonuclease (RNase A) has produced two dimeric variants LCC and PLCC, which do exhibit a cytotoxic activity, though at a much lower level than BS-RNase. The crystal structure analysis of the noncovalent swapped form (NCD) of LCC and PLCC, complexed with the substrate analogue 2 0-deoxycytidylyl(3 0 ,5 0)-2 0deoxyguanosine, has revealed that, differently from NCD-BS, the dimers adopt an opened quaternary structure, with the two Leu residues fully exposed to the solvent, that does not hinder the binding of RI. Similar results have been obtained for a third mutant of the pancreatic enzyme, engineered with the hinge peptide sequence of the seminal enzyme (residues 16-22) and the two cysteines in position 31 and 32, but lacking the hydrophobic Leu residue in position 28. The comparison of these three structures with those previously reported for other ribonuclease swapped dimers strongly suggests that, in addition to Pro19 and Leu28, the presence of a glycine at the N-terminal end of the hinge peptide is also important to push the swapped form of RNase A dimer into the compact quaternary organization observed for NCD-BS.

Biochimie, 2012

3D domain swapping is the process by which two or more protein molecules exchange part of their s... more 3D domain swapping is the process by which two or more protein molecules exchange part of their structure to form intertwined dimers or higher oligomers. Bovine pancreatic ribonuclease (RNase A) is able to swap the N-terminal α-helix (residues 1-13) and/or the C-terminal β-strand (residues 116-124), thus forming a variety of oligomers, including two different dimers. Cis-trans isomerization of the Asn113-Pro114 peptide group was observed when the protein formed the C-terminal swapped dimer. To study the effect of the substitution of Pro114 on the swapping process of RNase A, we have prepared and characterized the P114A monomeric and dimeric variants of the enzyme. In contrast with previous reports, the crystal structure and NMR data on the monomer reveals a mixed cis-trans conformation for the Asn113-Ala114 peptide group, whereas the X-ray structure of the C-terminal swapped dimer of the variant is very close to that of the corresponding dimer of RNase A. The mutation at the C-terminus affects the capability of the N-terminal α-helix to swap and the stability of both dimeric forms. The present results underscore the importance of the hydration shell in determining the cross-talk between the chain termini in the swapping process of RNase A.

Acta Crystallographica Section A Foundations of Crystallography, 2005

The sensor proteins for blue-light using the FAD (BLUF) domain belong to the third family of the ... more The sensor proteins for blue-light using the FAD (BLUF) domain belong to the third family of the photoreceptor proteins using a flavin chromophore, where the other two families are phototropins and cryptochromes. We have determined the crystal structure of the Tll0078 protein from Thermosynechococcus elongatus BP-1, which contains a BLUF domain bound to FAD, at 2 Å resolution. The crystals belonged to space group P2 1 2 1 2 1 with cell dimensions of a=89.5 Å, b=109.9 Å, and c=169.9 Å. The asymmetric unit contains 10 monomers of Tll0078 (one decamer). Five Tll0078 monomers are located around the non-crystallographic five-fold axis to form a pentamer, and two pentamers related by twofold noncrystallographic symmetry form a decameric assembly. The monomer consists of two domains, the BLUF domain at the Nterminal region and the C-terminal domain. The overall structure of the BLUF domain consists of a five-stranded mixed-sheet with two helices running parallel to it. The isoalloxazine ring of FAD is accommodated in a pocket formed by several highly-conserved amino acid residues in the BLUF domain.