The Experts below are selected from a list of 1342398 Experts worldwide ranked by ideXlab platform
Jean-marc Chobert - One of the best experts on this subject based on the ideXlab platform.
-
Selective Introduction of Sulfhydryl Groups into Recombinant Proteins for Study of Protein-Protein Interactions
Chromatographia, 2013Co-Authors: Yulia Stroylova, Thomas Haertlé, Jean-marc Chobert, Yvan Choiset, Tatyana Konnova, Yuriy F. Zuev, Vladimir I. MuronetzAbstract:In the present work, we proposed to create special sorbents for the study of protein-protein interactions, based on the fixation of cysteine-inserted Beta-Casein mutants with thiol-Sepharose resin. As a model system, we used bovine Beta-Casein, which belongs to the family of intrinsically unstructured proteins. Insertion of distal cysteines into the unfolded protein was not found to significantly change Beta-Casein properties. An amphiphilic Beta-Casein molecule has one hydrophilic domain and one hydrophobic domain placed on the N- and C-terminus, thus enabling one to exploit its capacity to engage in different types of intermolecular interactions. Two different casein-Sepharose sorbents incorporating either C-4 or C-208 Beta-Casein mutants bound to thiol-Sepharose were produced, exposing the hydrophobic domain in the case of the C-4 and the hydrophilic domain in the case of the C-208 mutant, respectively. The results obtained using the proposed sorbents with native Beta-Casein, another partially unfolded protein prion, and an oligomeric globular glyceraldehyde-3-phosphate dehydrogenase were found to be consistent with the data obtained by ELISA on free protein-protein complexes. Thus, Sepharose modified with various proteins is suitable for isolation of proteins interacting with the chromatographic phase bound partners from multicomponent systems such as milk. The obtained results allow the proposing of a fast and convenient method to be used for isolation of proteins, determination of protein-interacting partners, and the study of multi-protein complexes.
-
Modification of IgE binding to beta- and alpha(S1)-caseins by proteolytic activity of Lactobacillus helveticus A75
Journal of Food Biochemistry, 2013Co-Authors: Aynur Ahmadova Fehruz, Jean-marc Chobert, Shady El-ghaish, Yvan Choiset, Hanitra Rabesona, Martine Drouet, Akif A. Kuliev, Thomas HaertléAbstract:Proteolytic activity of Lactobacillus helveticus A75 and effects of caseins hydrolysis by this strain on their immuno-reactivity were studied. Proteases of L. helveticus A75 hydrolyzed both alpha(S1)- and Beta-Caseins and were inhibited by serine-and metalloproteases inhibitors. Polymerase chain reaction amplification revealed the presence of prtH gene, which was 99% identical to prtH gene of L. helveticus CNRZ 32. Effect of proteolysis of alpha(S1)- and Beta-Caseins by L. helveticus A75 on their immunoglobulin E (IgE) binding ability was studied by an enzyme-linked immunosorbent assay with the pool of eight sera from cow milk allergy patients. The IgE binding ability of hydrolyzed proteins was lower than that of intact proteins, which indicates that hydrolysis of alpha(S1)- and Beta-Caseins by proteases of L. helveticus A75 decreases their recognition by specific IgE. Half maximal inhibitory concentration values were 2.01 and 2.28 mu g/mL for alpha(S1)- and Beta-Casein, respectively, and increased until 9.08 and 13.65 mu g/mL, respectively, after their hydrolysis by L. helveticus A75.
-
Characterization of new strain Lactobacillus paracasei I-N-10 with proteolytic activity: Potential role in decrease in Beta-Casein immuno-reactivity
European Food Research and Technology, 2012Co-Authors: Imen Hadji Sfaxi, Thomas Haertlé, Aynur Ahmadova Fehruz, Shady El-ghaish, Hanitra Rabesona, Jean-marc ChobertAbstract:The proteolytic activity of thirty-three Lisolates from Mongolian tarag was tested on skimmed milk. The strain displaying the highest proteolytic activity was purified and presented by 16S rDNA sequencing 99.9 % homology with Lactobacillus paracasei 1-4-2A. It was named L. paracasei I-N-10. Proteases of L. paracasei I-N-10 hydrolyze predominately Beta-Casein and in some level alpha(S2)-casein; hydrolysis of alpha(S1)-casein was not observed. Proteolytic activity was optimal at 42 A degrees C and neutral pH. Proteases of L. paracasei I-N-10 were inhibited by serine- and metalloproteases inhibitors. PCR amplification revealed the presence of prtP gene, which was identical to prtP gene of L. paracasei genus. Mass spectrometry analysis of Beta-Casein hydrolysate allowed to characterize 7 peptides resulting from proteolysis by L. paracasei I-N-10. The isolated strain was able to cleave Beta-Casein in different sites including 2 of the major linear epitopes implicated in its allergenicity. Being sensitive to main antibiotics classes, L. paracasei I-N-10 could be considered as safe and used as starter culture with a potential role in decreasing Beta-Casein immuno-reactivity.
-
Aggregation and structural changes of alpha(S1)-, beta- andkappa-caseins induced by homocysteinylation
Biochimica et Biophysica Acta Proteins and Proteomics, 2011Co-Authors: Yulia Stroylova, Jaroslaw Zimny, Reza Yousefi, Jean-marc Chobert, Hieronim Jakubowski, Vladimir I. Muronetz, Thomas HaertléAbstract:Elevated homocysteine levels are resulting in N-homocysteinylation of lysyl residues in proteins and they correlate with a number of human pathologies. However, the role of N-homocysteinylation of lysyl residues is still poorly known. In order to study the features of homocysteinylation of intrinsically unstructured proteins (IUP) bovine caseins were used as a model. alpha(S1)-, beta- and kappa-caseins, showing different aggregations and micelle formation, were modified with homocysteine-thiolactone and their physico-chemical properties were studied. Efficiency of homocysteine incorporation was estimated to be about 1.5.2.1 and 1.3 homocysteyl residues per one beta-, alpha(S1)-, and kappa-casein molecule, respectively. Use of intrinsic and extrinsic fluorescent markers such as Trp, thioflavin T and ANS, reveal structural changes of casein structures after homocysteinylation reflected by an increase in beta-sheet content, which in some cases may be characteristic of amyloid-like transformations. CD spectra also show an increase in beta-sheet content of homocysteinylated caseins. Casein homocysteinylation leads in all cases to aggregation. The sizes of aggregates and aggregation rates were dependent on homocysteine thiolactone concentration and temperature. DES and microscopic studies have revealed the formation of large aggregates of about 1-3 mu m. Homocysteinylation of alpha(S1)- and Beta-Caseins results in formation of regular spheres. Homocysteinylated kappa-casein forms thin unbranched fibrils about 400-800 nm long. In case of kappa-casein amyloidogenic effect of homocysteinylation was confirmed by Congo red spectra. Taken together, data indicate that N-homocysteinylation provokes significant changes in properties of native caseins. A comparison of amyloidogenic transformation of 3 different casein types, belonging to the IUP protein family, shows that the efficiency of amyloidogenic transformation upon homocysteinylation depends on micellization capacity, additional disulphide bonds and other structural features. (C) 2011 Elsevier B.V. All rights reserved.
-
Comparative analysis of Beta-Casein proteolysis by PrtP proteinase from Lactobacillus paracasei subsp paracasei BGHN14, PrtR proteinase from Lactobacillus rhamnosus BGT10 and PrtH proteinase from Lactobacillus helveticus BGRA43
International Dairy Journal, 2011Co-Authors: Jelena Lozo, Thomas Haertlé, Jean-marc Chobert, Ivana Strahinic, Michèle Dalgalarrondo, Ljubiša TopisirovićAbstract:Lactic acid bacteria contain different cell-envelope proteinases responsible for hydrolysis of caseins. Isolates of Lactobacillus paracasei subsp. paracasei BGHN14 and Lactobacillus rhamnosus BGT10 harbour PrtP and PrtR proteinases, respectively, whereas the PrtH proteinase of Lactobacillus helveticus BGRA43 is similar to that of Lb. helveticus CNRZ32. Mass spectrometry analysis of the major peptides isolated by reversed phase-high performance liquid chromatography allowed the identification of 25, 22 and 17 peptides after Beta-Casein hydrolysis by strains BGHN14, BGRA43 and BGT10, respectively. Regardless of the type of proteinase, Beta-Casein was hydrolyzed preferentially after hydrophobic residues and glutamines ( Q). PrtP and PrtR proteinases preferentially targeted the C-terminus of Beta-Casein. In the case of PrtH proteinase, most of the peptides obtained were cleaved from N-terminus of this casein. Nine identical peptides were identified after hydrolysis with PrtP and PrtR, three after proteolysis with PrtR and PrtH, and two after hydrolysis with all three proteinases studied. (C) 2011 Elsevier Ltd. All rights reserved.
Patrizia Restani - One of the best experts on this subject based on the ideXlab platform.
-
Proteolysis of Beta-Casein as a marker of Grana Padano cheese ripening.
Journal of dairy science, 2001Co-Authors: A. Gaiaschi, Barbara Beretta, Claudio Poiesi, A. Conti, M.g. Giuffrida, Corrado L. Galli, Patrizia RestaniAbstract:Proteolysis has a critical role in defining the typical organoleptic characteristics of Grana Padano, a well-known Italian cheese. During the ripening process, hydrolysis of Beta-Casein produces different fragments, the most abundant and widely studied of which are gamma-caseins, three polypeptides containing the HOOC-terminal portion of Beta-Casein. By sodium dodecyl sulfate-PAGE and a specific anti-Beta-Casein monoclonal antibody, two Beta-Casein-derived bands were identified in Grana Padano cheese: betaa and betab. Thanks to the identification of the amino acid sequences, it was shown that: a) betaa contains gamma1-casein [Beta-Casein (29-209)] and the correlated peptide [Beta-Casein (30-209)]; b) betab contains gamma2-casein [Beta-Casein (106-209)] and gamma3-casein [Beta-Casein (108-209)]. The production of betaa and betab by the three enzymes most involved in cheese proteolysis (pepsin, chymosin, and plasmin) was evaluated by performing in vitro digestions. A significant correlation between abundance of some polypeptides and ripening process was shown.
-
Proteolysis of αs-Casein as a Marker of Grana Padano Cheese Ripening
Journal of Dairy Science, 2000Co-Authors: A. Gaiaschi, Barbara Beretta, Claudio Poiesi, A. Conti, M.g. Giuffrida, Corrado L. Galli, Patrizia RestaniAbstract:Proteolysis has a critical role in defining the typical organoleptic characteristics of Grana Padano, a well-known Italian cheese. During the ripening process, hydrolysis of Beta-Casein produces different fragments, the most abundant and widely studied of which are gamma-caseins, three polypeptides containing the HOOC-terminal portion of Beta-Casein. By sodium dodecyl sulfate-PAGE and a specific anti-Beta-Casein monoclonal antibody, two Beta-Casein-derived bands were identified in Grana Padano cheese: betaa and betab. Thanks to the identification of the amino acid sequences, it was shown that: a) betaa contains gamma1-casein [Beta-Casein (29-209)] and the correlated peptide [Beta-Casein (30-209)]; b) betab contains gamma2-casein [Beta-Casein (106-209)] and gamma3-casein [Beta-Casein (108-209)]. The production of betaa and betab by the three enzymes most involved in cheese proteolysis (pepsin, chymosin, and plasmin) was evaluated by performing in vitro digestions. A significant correlation between abundance of some polypeptides and ripening process was shown.
Thomas Haertlé - One of the best experts on this subject based on the ideXlab platform.
-
Micellar properties of Beta-Casein-cationic surfactant solutions
Chemical Monthly Monatshefte für Chemie, 2013Co-Authors: Fahimeh Mehranfar, Abdol-khalegh Bordbar, Thomas Haertlé, A.a. Moosavi-movahedi, Nahid FarziAbstract:The micellar properties of Beta-Casein in solution with a homologous series of cationic surfactants have been investigated using turbidity, fluorescence spectroscopy, and quenching fluorescence measurements. Turbidity measurements indicated that monomeric cationic surfactants initially bind cooperatively with a site on the Beta-Casein micelle in negatively charged amino acids. This leads to the formation of low-soluble Beta-Casein micelle-monomeric surfactants complexes. Further addition of surfactant leads to the formation of more highly soluble complexes of Beta-Casein micelle-cationic surfactant. The fluorescence experiments were also used to study the formation of Beta-Casein-cationic surfactant complexes and to determine critical micelle concentration of surfactants and Beta-Casein. The mean aggregation number of Beta-Casein and cationic surfactant micelles at various experimental conditions and the dependence of aggregation phenomenon on the chain length of cationic surfactants were investigated. The mean aggregation number for Beta-Casein micelle has been determined to be 45.70 using quenching fluorescence.
-
Modification of IgE binding to beta- and alpha(S1)-caseins by proteolytic activity of Lactobacillus helveticus A75
Journal of Food Biochemistry, 2013Co-Authors: Aynur Ahmadova Fehruz, Jean-marc Chobert, Shady El-ghaish, Yvan Choiset, Hanitra Rabesona, Martine Drouet, Akif A. Kuliev, Thomas HaertléAbstract:Proteolytic activity of Lactobacillus helveticus A75 and effects of caseins hydrolysis by this strain on their immuno-reactivity were studied. Proteases of L. helveticus A75 hydrolyzed both alpha(S1)- and Beta-Caseins and were inhibited by serine-and metalloproteases inhibitors. Polymerase chain reaction amplification revealed the presence of prtH gene, which was 99% identical to prtH gene of L. helveticus CNRZ 32. Effect of proteolysis of alpha(S1)- and Beta-Caseins by L. helveticus A75 on their immunoglobulin E (IgE) binding ability was studied by an enzyme-linked immunosorbent assay with the pool of eight sera from cow milk allergy patients. The IgE binding ability of hydrolyzed proteins was lower than that of intact proteins, which indicates that hydrolysis of alpha(S1)- and Beta-Caseins by proteases of L. helveticus A75 decreases their recognition by specific IgE. Half maximal inhibitory concentration values were 2.01 and 2.28 mu g/mL for alpha(S1)- and Beta-Casein, respectively, and increased until 9.08 and 13.65 mu g/mL, respectively, after their hydrolysis by L. helveticus A75.
-
Selective Introduction of Sulfhydryl Groups into Recombinant Proteins for Study of Protein-Protein Interactions
Chromatographia, 2013Co-Authors: Yulia Stroylova, Thomas Haertlé, Jean-marc Chobert, Yvan Choiset, Tatyana Konnova, Yuriy F. Zuev, Vladimir I. MuronetzAbstract:In the present work, we proposed to create special sorbents for the study of protein-protein interactions, based on the fixation of cysteine-inserted Beta-Casein mutants with thiol-Sepharose resin. As a model system, we used bovine Beta-Casein, which belongs to the family of intrinsically unstructured proteins. Insertion of distal cysteines into the unfolded protein was not found to significantly change Beta-Casein properties. An amphiphilic Beta-Casein molecule has one hydrophilic domain and one hydrophobic domain placed on the N- and C-terminus, thus enabling one to exploit its capacity to engage in different types of intermolecular interactions. Two different casein-Sepharose sorbents incorporating either C-4 or C-208 Beta-Casein mutants bound to thiol-Sepharose were produced, exposing the hydrophobic domain in the case of the C-4 and the hydrophilic domain in the case of the C-208 mutant, respectively. The results obtained using the proposed sorbents with native Beta-Casein, another partially unfolded protein prion, and an oligomeric globular glyceraldehyde-3-phosphate dehydrogenase were found to be consistent with the data obtained by ELISA on free protein-protein complexes. Thus, Sepharose modified with various proteins is suitable for isolation of proteins interacting with the chromatographic phase bound partners from multicomponent systems such as milk. The obtained results allow the proposing of a fast and convenient method to be used for isolation of proteins, determination of protein-interacting partners, and the study of multi-protein complexes.
-
Chaperone-Like Activity of Beta-Casein and Thermal Stability of Alcohol Dehydrogenase
Bioorganicheskaya Khimiya Russian Journal of Bioorganic Chemistry, 2012Co-Authors: N L Zakharchenko, Thomas Haertlé, T A Konnova, N E Gogoleva, D. A. Faizullin, Yu. F. ZuevAbstract:Correlation between structural peculiarities of Beta-Casein and its chaperon-like activity was investigated using the recombinant forms of the protein containing the cysteine residues in the polypeptide chain. Aggregation of native and modified forms of Beta-Casein was studied, as well as their chaperon-like activity towards alcohol dehydrogenase thermal aggregation. It has been shown that dimeric and oligomeric forms of Beta-Casein, which are formed due to intermolecular disulfide bonds, significantly differ in their physico-chemical and chaperon-like properties from monomeric forms. The thermal stability of alcohol dehydrogenase has been found to depend on the Beta-Casein concentration.
-
Characterization of new strain Lactobacillus paracasei I-N-10 with proteolytic activity: Potential role in decrease in Beta-Casein immuno-reactivity
European Food Research and Technology, 2012Co-Authors: Imen Hadji Sfaxi, Thomas Haertlé, Aynur Ahmadova Fehruz, Shady El-ghaish, Hanitra Rabesona, Jean-marc ChobertAbstract:The proteolytic activity of thirty-three Lisolates from Mongolian tarag was tested on skimmed milk. The strain displaying the highest proteolytic activity was purified and presented by 16S rDNA sequencing 99.9 % homology with Lactobacillus paracasei 1-4-2A. It was named L. paracasei I-N-10. Proteases of L. paracasei I-N-10 hydrolyze predominately Beta-Casein and in some level alpha(S2)-casein; hydrolysis of alpha(S1)-casein was not observed. Proteolytic activity was optimal at 42 A degrees C and neutral pH. Proteases of L. paracasei I-N-10 were inhibited by serine- and metalloproteases inhibitors. PCR amplification revealed the presence of prtP gene, which was identical to prtP gene of L. paracasei genus. Mass spectrometry analysis of Beta-Casein hydrolysate allowed to characterize 7 peptides resulting from proteolysis by L. paracasei I-N-10. The isolated strain was able to cleave Beta-Casein in different sites including 2 of the major linear epitopes implicated in its allergenicity. Being sensitive to main antibiotics classes, L. paracasei I-N-10 could be considered as safe and used as starter culture with a potential role in decreasing Beta-Casein immuno-reactivity.
R D Burgoyne - One of the best experts on this subject based on the ideXlab platform.
-
Characterization of the effects of Ca2+ depletion on the synthesis, phosphorylation and secretion of caseins in lactating mammary epithelial cells.
The Biochemical journal, 1996Co-Authors: J S Duncan, R D BurgoyneAbstract:We have examined the effects of depleting lumenal Ca2+ on the synthesis, phosphorylation and secretion of caseins in lactating mouse mammary cells by using inhibitors of the endoplasmic reticulum Ca(2+)-ATPase or the ionophore ionomycin in the absence of external Ca2+. Treatment with these drugs resulted in a transient increase in the cytosolic Ca2+ concentration due to Ca2+ mobilization. Protein synthesis over a 1 h period was substantially inhibited by Ca2+ depletion, but in a pulse-chase protocol secretion of pre-synthesized proteins was unaffected by Ca2+ depletion. Analysis of polysome profiles showed that Ca2+ depletion resulted in a loss of polysomes, consistent with an inhibition of initiation of protein synthesis. Neither treatment with Ca(2+)-ATPase inhibitors to deplete endoplasmic reticulum Ca2+ nor treatment with ionomycin/EGTA had any effect on an early phase of phosphorylation of alpha- or beta/gamma-caseins, but Ca2+ depletion resulted in a decrease in a late phase of casein phosphorylation. These results indicate that lumenal Ca2+ is required to maintain protein synthesis in lactating mammary cells but is not required for protein secretion, and that Ca2+ accumulation in the Golgi cisternae is required for a late but not for an early phase of casein phosphorylation.
-
Differential effect of brefeldin A on phosphorylation of the caseins in lactating mouse mammary epithelial cells.
Journal of cell science, 1993Co-Authors: M D Turner, S E Handel, C J Wilde, R D BurgoyneAbstract:The major milk proteins, the caseins, contain multiple phosphorylation sites. Phosphorylation of the caseins is necessary to allow Ca2+ binding and aggregation of the caseins to form micelles. We have followed the phosphorylation of the caseins in isolated acini from lactating mouse mammary gland. Incubation of mammary cells with [32P]orthophosphate revealed that phosphorylation of newly synthesised caseins was complete within 20 minutes of synthesis. Extensive secretion of alpha-, beta- and gamma-caseins occurred over a 2 hour period. Activation of the regulated secretory pathway using ionomycin over the last hour resulted in a preferential increase in secretion of alpha- and gamma-caseins. Brefeldin A (BFA) inhibited protein secretion and synthesis in mammary cells in prolonged incubations. An examination of short-term treatments with BFA on 32P incorporation into the caseins revealed a differential effect of BFA in which the drug inhibited phosphorylation of beta- and gamma- but not alpha-caseins. These results suggest that phosphorylation of alpha-casein normally occurs in Golgi cisternae whereas that of beta- and gamma-caseins occurs in the trans-Golgi network. Phosphorylation of specific secretory proteins may, therefore, occur in different Golgi compartments.
A. Gaiaschi - One of the best experts on this subject based on the ideXlab platform.
-
Proteolysis of Beta-Casein as a marker of Grana Padano cheese ripening.
Journal of dairy science, 2001Co-Authors: A. Gaiaschi, Barbara Beretta, Claudio Poiesi, A. Conti, M.g. Giuffrida, Corrado L. Galli, Patrizia RestaniAbstract:Proteolysis has a critical role in defining the typical organoleptic characteristics of Grana Padano, a well-known Italian cheese. During the ripening process, hydrolysis of Beta-Casein produces different fragments, the most abundant and widely studied of which are gamma-caseins, three polypeptides containing the HOOC-terminal portion of Beta-Casein. By sodium dodecyl sulfate-PAGE and a specific anti-Beta-Casein monoclonal antibody, two Beta-Casein-derived bands were identified in Grana Padano cheese: betaa and betab. Thanks to the identification of the amino acid sequences, it was shown that: a) betaa contains gamma1-casein [Beta-Casein (29-209)] and the correlated peptide [Beta-Casein (30-209)]; b) betab contains gamma2-casein [Beta-Casein (106-209)] and gamma3-casein [Beta-Casein (108-209)]. The production of betaa and betab by the three enzymes most involved in cheese proteolysis (pepsin, chymosin, and plasmin) was evaluated by performing in vitro digestions. A significant correlation between abundance of some polypeptides and ripening process was shown.
-
Proteolysis of αs-Casein as a Marker of Grana Padano Cheese Ripening
Journal of Dairy Science, 2000Co-Authors: A. Gaiaschi, Barbara Beretta, Claudio Poiesi, A. Conti, M.g. Giuffrida, Corrado L. Galli, Patrizia RestaniAbstract:Proteolysis has a critical role in defining the typical organoleptic characteristics of Grana Padano, a well-known Italian cheese. During the ripening process, hydrolysis of Beta-Casein produces different fragments, the most abundant and widely studied of which are gamma-caseins, three polypeptides containing the HOOC-terminal portion of Beta-Casein. By sodium dodecyl sulfate-PAGE and a specific anti-Beta-Casein monoclonal antibody, two Beta-Casein-derived bands were identified in Grana Padano cheese: betaa and betab. Thanks to the identification of the amino acid sequences, it was shown that: a) betaa contains gamma1-casein [Beta-Casein (29-209)] and the correlated peptide [Beta-Casein (30-209)]; b) betab contains gamma2-casein [Beta-Casein (106-209)] and gamma3-casein [Beta-Casein (108-209)]. The production of betaa and betab by the three enzymes most involved in cheese proteolysis (pepsin, chymosin, and plasmin) was evaluated by performing in vitro digestions. A significant correlation between abundance of some polypeptides and ripening process was shown.