The Experts below are selected from a list of 51 Experts worldwide ranked by ideXlab platform
Richard Desplantes - One of the best experts on this subject based on the ideXlab platform.
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affinity biosensors using recombinant native membrane proteins displayed on exosomes application to botulinum neurotoxin b receptor
Scientific Reports, 2017Co-Authors: Richard Desplantes, Christian Leveque, Benjamin Muller, Manuela Lotierzo, Geraldine Ferracci, Michel R Popoff, Michael SeagarAbstract:The development of simple molecular assays with membrane protein receptors in a native conformation still represents a challenging task. Exosomes are extracellular vesicles which, due to their stability and small size, are suited for analysis in various assay formats. Here, we describe a novel approach to sort recombinant fully native and functional membrane proteins to exosomes using a targeting peptide. Specific binding of high affinity ligands to the Potassium Channel Kv1.2, the G-protein coupled receptor CXCR4, and the botulinum neurotoxin type B (BoNT/B) receptor, indicated their correct assembly and outside out orientation in exosomes. We then developed, using a label-free optical biosensor, a new method to determine the kinetic constants of BoNT/B holotoxin binding to its receptor synaptotagmin2/GT1b ganglioside (k on = 2.3 ×10 5 M −1 .s −1 , k off = 1.3 10 −4 s −1), yielding an affinity constant (K D = 0.6 nM) similar to values determined from native tissue. In addition, the recombinant binding domain of BoNT/B, a potential vector for neuronal delivery, bound quasi-irreversibly to synaptotagmin 2/GT1b exosomes. Engineered exosomes provide thus a novel means to study membrane proteins for biotechnology and clinical applications. Determination of the binding kinetics of analytes is potentially extremely useful for drug development and assay validation. In principle, purified endogenous or recombinant membrane proteins can be integrated into a lipid environment, such as proteoliposomes, supported lipid bilayers or nanodiscs. However, this approach is not ideal, due to the fact that membrane proteins are difficult to produce, prone to denaturation and do not always retain their functional integrity in artificial lipid bilayers 1, 2. Exosomes are extracellular vesicles, produced by many cell types and can be detected or purified using anti-bodies 3, 4. Furthermore, the ability of exosomes to carry molecules from native or engineered parental cells and fuse with recipient cells to deliver their cargo, confers considerable biomedical potential 5, 6. Unlike intra-cellular vesicles, exosomes display homogeneous topology in which plasma membrane proteins have the same outside-out orientation as at the surface of intact cells. Moreover, exosomes can potentially provide the means of expressing recombinant proteins in small membrane vesicles that can be used as vaccine strategies 7, 8. Our principal aim was to develop a general approach to address recombinant membrane proteins to exosomes, using a specific targeting sequence to direct exosomal sorting of a set of membrane drug targets, with a particular focus on the botulinum neurotoxin/B (BoNT/B) receptor. BoNT serotypes A, B and E (BoNT/A, BoNT/B, BoNT/E), produced by the bacteria Clostridium botulinum are the main cause of human botulism and are produced as a complex of holotoxin (MW 150 000 Da) and several nontoxic proteins. Although BoNTs are among the most neurotoxic substances known, their ability to reversibly block cholinergic nerves has provided the basis 1 INSERM
Michael Seagar - One of the best experts on this subject based on the ideXlab platform.
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affinity biosensors using recombinant native membrane proteins displayed on exosomes application to botulinum neurotoxin b receptor
Scientific Reports, 2017Co-Authors: Richard Desplantes, Christian Leveque, Benjamin Muller, Manuela Lotierzo, Geraldine Ferracci, Michel R Popoff, Michael SeagarAbstract:The development of simple molecular assays with membrane protein receptors in a native conformation still represents a challenging task. Exosomes are extracellular vesicles which, due to their stability and small size, are suited for analysis in various assay formats. Here, we describe a novel approach to sort recombinant fully native and functional membrane proteins to exosomes using a targeting peptide. Specific binding of high affinity ligands to the Potassium Channel Kv1.2, the G-protein coupled receptor CXCR4, and the botulinum neurotoxin type B (BoNT/B) receptor, indicated their correct assembly and outside out orientation in exosomes. We then developed, using a label-free optical biosensor, a new method to determine the kinetic constants of BoNT/B holotoxin binding to its receptor synaptotagmin2/GT1b ganglioside (k on = 2.3 ×10 5 M −1 .s −1 , k off = 1.3 10 −4 s −1), yielding an affinity constant (K D = 0.6 nM) similar to values determined from native tissue. In addition, the recombinant binding domain of BoNT/B, a potential vector for neuronal delivery, bound quasi-irreversibly to synaptotagmin 2/GT1b exosomes. Engineered exosomes provide thus a novel means to study membrane proteins for biotechnology and clinical applications. Determination of the binding kinetics of analytes is potentially extremely useful for drug development and assay validation. In principle, purified endogenous or recombinant membrane proteins can be integrated into a lipid environment, such as proteoliposomes, supported lipid bilayers or nanodiscs. However, this approach is not ideal, due to the fact that membrane proteins are difficult to produce, prone to denaturation and do not always retain their functional integrity in artificial lipid bilayers 1, 2. Exosomes are extracellular vesicles, produced by many cell types and can be detected or purified using anti-bodies 3, 4. Furthermore, the ability of exosomes to carry molecules from native or engineered parental cells and fuse with recipient cells to deliver their cargo, confers considerable biomedical potential 5, 6. Unlike intra-cellular vesicles, exosomes display homogeneous topology in which plasma membrane proteins have the same outside-out orientation as at the surface of intact cells. Moreover, exosomes can potentially provide the means of expressing recombinant proteins in small membrane vesicles that can be used as vaccine strategies 7, 8. Our principal aim was to develop a general approach to address recombinant membrane proteins to exosomes, using a specific targeting sequence to direct exosomal sorting of a set of membrane drug targets, with a particular focus on the botulinum neurotoxin/B (BoNT/B) receptor. BoNT serotypes A, B and E (BoNT/A, BoNT/B, BoNT/E), produced by the bacteria Clostridium botulinum are the main cause of human botulism and are produced as a complex of holotoxin (MW 150 000 Da) and several nontoxic proteins. Although BoNTs are among the most neurotoxic substances known, their ability to reversibly block cholinergic nerves has provided the basis 1 INSERM
Geraldine Ferracci - One of the best experts on this subject based on the ideXlab platform.
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affinity biosensors using recombinant native membrane proteins displayed on exosomes application to botulinum neurotoxin b receptor
Scientific Reports, 2017Co-Authors: Richard Desplantes, Christian Leveque, Benjamin Muller, Manuela Lotierzo, Geraldine Ferracci, Michel R Popoff, Michael SeagarAbstract:The development of simple molecular assays with membrane protein receptors in a native conformation still represents a challenging task. Exosomes are extracellular vesicles which, due to their stability and small size, are suited for analysis in various assay formats. Here, we describe a novel approach to sort recombinant fully native and functional membrane proteins to exosomes using a targeting peptide. Specific binding of high affinity ligands to the Potassium Channel Kv1.2, the G-protein coupled receptor CXCR4, and the botulinum neurotoxin type B (BoNT/B) receptor, indicated their correct assembly and outside out orientation in exosomes. We then developed, using a label-free optical biosensor, a new method to determine the kinetic constants of BoNT/B holotoxin binding to its receptor synaptotagmin2/GT1b ganglioside (k on = 2.3 ×10 5 M −1 .s −1 , k off = 1.3 10 −4 s −1), yielding an affinity constant (K D = 0.6 nM) similar to values determined from native tissue. In addition, the recombinant binding domain of BoNT/B, a potential vector for neuronal delivery, bound quasi-irreversibly to synaptotagmin 2/GT1b exosomes. Engineered exosomes provide thus a novel means to study membrane proteins for biotechnology and clinical applications. Determination of the binding kinetics of analytes is potentially extremely useful for drug development and assay validation. In principle, purified endogenous or recombinant membrane proteins can be integrated into a lipid environment, such as proteoliposomes, supported lipid bilayers or nanodiscs. However, this approach is not ideal, due to the fact that membrane proteins are difficult to produce, prone to denaturation and do not always retain their functional integrity in artificial lipid bilayers 1, 2. Exosomes are extracellular vesicles, produced by many cell types and can be detected or purified using anti-bodies 3, 4. Furthermore, the ability of exosomes to carry molecules from native or engineered parental cells and fuse with recipient cells to deliver their cargo, confers considerable biomedical potential 5, 6. Unlike intra-cellular vesicles, exosomes display homogeneous topology in which plasma membrane proteins have the same outside-out orientation as at the surface of intact cells. Moreover, exosomes can potentially provide the means of expressing recombinant proteins in small membrane vesicles that can be used as vaccine strategies 7, 8. Our principal aim was to develop a general approach to address recombinant membrane proteins to exosomes, using a specific targeting sequence to direct exosomal sorting of a set of membrane drug targets, with a particular focus on the botulinum neurotoxin/B (BoNT/B) receptor. BoNT serotypes A, B and E (BoNT/A, BoNT/B, BoNT/E), produced by the bacteria Clostridium botulinum are the main cause of human botulism and are produced as a complex of holotoxin (MW 150 000 Da) and several nontoxic proteins. Although BoNTs are among the most neurotoxic substances known, their ability to reversibly block cholinergic nerves has provided the basis 1 INSERM
Christian Leveque - One of the best experts on this subject based on the ideXlab platform.
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affinity biosensors using recombinant native membrane proteins displayed on exosomes application to botulinum neurotoxin b receptor
Scientific Reports, 2017Co-Authors: Richard Desplantes, Christian Leveque, Benjamin Muller, Manuela Lotierzo, Geraldine Ferracci, Michel R Popoff, Michael SeagarAbstract:The development of simple molecular assays with membrane protein receptors in a native conformation still represents a challenging task. Exosomes are extracellular vesicles which, due to their stability and small size, are suited for analysis in various assay formats. Here, we describe a novel approach to sort recombinant fully native and functional membrane proteins to exosomes using a targeting peptide. Specific binding of high affinity ligands to the Potassium Channel Kv1.2, the G-protein coupled receptor CXCR4, and the botulinum neurotoxin type B (BoNT/B) receptor, indicated their correct assembly and outside out orientation in exosomes. We then developed, using a label-free optical biosensor, a new method to determine the kinetic constants of BoNT/B holotoxin binding to its receptor synaptotagmin2/GT1b ganglioside (k on = 2.3 ×10 5 M −1 .s −1 , k off = 1.3 10 −4 s −1), yielding an affinity constant (K D = 0.6 nM) similar to values determined from native tissue. In addition, the recombinant binding domain of BoNT/B, a potential vector for neuronal delivery, bound quasi-irreversibly to synaptotagmin 2/GT1b exosomes. Engineered exosomes provide thus a novel means to study membrane proteins for biotechnology and clinical applications. Determination of the binding kinetics of analytes is potentially extremely useful for drug development and assay validation. In principle, purified endogenous or recombinant membrane proteins can be integrated into a lipid environment, such as proteoliposomes, supported lipid bilayers or nanodiscs. However, this approach is not ideal, due to the fact that membrane proteins are difficult to produce, prone to denaturation and do not always retain their functional integrity in artificial lipid bilayers 1, 2. Exosomes are extracellular vesicles, produced by many cell types and can be detected or purified using anti-bodies 3, 4. Furthermore, the ability of exosomes to carry molecules from native or engineered parental cells and fuse with recipient cells to deliver their cargo, confers considerable biomedical potential 5, 6. Unlike intra-cellular vesicles, exosomes display homogeneous topology in which plasma membrane proteins have the same outside-out orientation as at the surface of intact cells. Moreover, exosomes can potentially provide the means of expressing recombinant proteins in small membrane vesicles that can be used as vaccine strategies 7, 8. Our principal aim was to develop a general approach to address recombinant membrane proteins to exosomes, using a specific targeting sequence to direct exosomal sorting of a set of membrane drug targets, with a particular focus on the botulinum neurotoxin/B (BoNT/B) receptor. BoNT serotypes A, B and E (BoNT/A, BoNT/B, BoNT/E), produced by the bacteria Clostridium botulinum are the main cause of human botulism and are produced as a complex of holotoxin (MW 150 000 Da) and several nontoxic proteins. Although BoNTs are among the most neurotoxic substances known, their ability to reversibly block cholinergic nerves has provided the basis 1 INSERM
Anthony D. Morielli - One of the best experts on this subject based on the ideXlab platform.
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homeostatic regulation of Kv1.2 Potassium Channel trafficking by cyclic amp
Journal of Biological Chemistry, 2008Co-Authors: Emilee Colleen Connors, Bryan A Ballif, Anthony D. MorielliAbstract:The Shaker family Potassium Channel, Kv1.2, is a key determinant of membrane excitability in neurons and cardiovascular tissue. Kv1.2 is subject to multiple forms of regulation and therefore integrates cellular signals involved in the homeostasis of excitability. The cyclic AMP/protein kinase A (PKA) pathway enhances Kv1.2 ionic current; however, the mechanisms for this are not fully known. Here we show that cAMP maintains Kv1.2 homeostasis through opposing effects on Channel trafficking. We found that Kv1.2 is regulated by two distinct cAMP pathways, one PKA-dependent and the other PKA-independent. PKA inhibitors elevate Kv1.2 surface levels, suggesting that basal levels of cAMP control steady-state turnover of the Channel. Elevation of cAMP above basal levels also increases the amount of Kv1.2 at the cell surface. This effect is not blocked by PKA inhibitors, but is blocked by inhibition of Kv1.2 endocytosis. We conclude that Kv1.2 levels at the cell surface are kept in dynamic balance by opposing effects of cAMP.
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An essential role for cortactin in the modulation of the Potassium Channel Kv1.2
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Michael R. Williams, Jonathan C. Markey, Megan A. Doczi, Anthony D. MorielliAbstract:Ion Channels are key determinants of membrane excitability. The actin cytoskeleton has a central role in morphology, migration, intracellular transport, and signaling. In this article, we show that the actin-binding protein cortactin regulates the Potassium Channel Kv1.2 and thereby provides a direct link between actin dynamics and membrane excitability. In previous reports, we showed that the tyrosine phosphorylation-mediated suppression of Kv1.2 ionic current occurs by endocytosis of the Channel protein. Pull-down assays using recombinant-purified cortactin and Kv1.2 demonstrated that their interaction is direct and reduced by tyrosine phosphorylation of Kv1.2. This finding suggests a link between cortactin and Kv1.2 endocytosis. Here, we confirm that relationship and identify the molecular mechanisms involved. We use FRET to demonstrate that Kv1.2 and cortactin interact in vivo. By manipulating the cortactin-binding site within Kv1.2, we confirm that cortactin proximity influences Channel function. We used flow cytometry in conjunction with cortactin gene replacement to identify C-terminal tyrosines, the fourth repeat actin-binding domain, and the N-terminal Arp2/3-binding region, as critical to Kv1.2 regulation. Surprisingly, cortactin's dynamin-binding Src homology 3 domain is not required for Kv1.2 endocytosis, despite that process being dynamin-dependent. These findings predict that cortactin-mediated actin remodeling in excitable cells is not only important for cell structure, but may directly impact membrane excitability.