Enhancement of Phosphorylation and Transport Activity of the Neuronal Glutamate Transporter Excitatory Amino Acid Transporter 3 by C3bot and a 26mer C3bot Peptide
In primary murine hippocampal neurons we investigated the regulation of EAAT3-mediated glutamate transport by the Clostridium botulinum C3 transferase C3bot and a 26mer peptide derived from full length protein. Incubation with either enzyme-competent C3bot or enzyme-deficient C3bot156–181 peptide re...
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Frontiers Media S.A.
2022-06-01
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| Series: | Frontiers in Cellular Neuroscience |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fncel.2022.860823/full |
| _version_ | 1828793783341285376 |
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| author | Johannes Piepgras Astrid Rohrbeck Ingo Just Stefan Bittner Gudrun Ahnert-Hilger Markus Höltje |
| author_facet | Johannes Piepgras Astrid Rohrbeck Ingo Just Stefan Bittner Gudrun Ahnert-Hilger Markus Höltje |
| author_sort | Johannes Piepgras |
| collection | DOAJ |
| description | In primary murine hippocampal neurons we investigated the regulation of EAAT3-mediated glutamate transport by the Clostridium botulinum C3 transferase C3bot and a 26mer peptide derived from full length protein. Incubation with either enzyme-competent C3bot or enzyme-deficient C3bot156–181 peptide resulted in the upregulation of glutamate uptake by up to 22% compared to untreated cells. A similar enhancement of glutamate transport was also achieved by the classical phorbol-ester-mediated activation of protein kinase C subtypes. Yet comparable, effects elicited by C3 preparations seemed not to rely on PKCα, γ, ε, or ζ activation. Blocking of tyrosine phosphorylation by tyrosine kinase inhibitors prevented the observed effect mediated by C3bot and C3bot 26mer. By using biochemical and molecular biological assays we could rule out that the observed C3bot and C3bot 26mer-mediated effects solely resulted from enhanced transporter expression or translocation to the neuronal surface but was rather mediated by transporter phosphorylation at tyrosine residues that was found to be significantly enhanced following incubation with either full length protein or the 26mer C3 peptide. |
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| id | doaj.art-9d3102a50b474ca286dc79b4ba87a2a7 |
| institution | Directory Open Access Journal |
| issn | 1662-5102 |
| language | English |
| last_indexed | 2024-12-12T03:33:42Z |
| publishDate | 2022-06-01 |
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| record_format | Article |
| series | Frontiers in Cellular Neuroscience |
| spelling | doaj.art-9d3102a50b474ca286dc79b4ba87a2a72022-12-22T00:39:51ZengFrontiers Media S.A.Frontiers in Cellular Neuroscience1662-51022022-06-011610.3389/fncel.2022.860823860823Enhancement of Phosphorylation and Transport Activity of the Neuronal Glutamate Transporter Excitatory Amino Acid Transporter 3 by C3bot and a 26mer C3bot PeptideJohannes Piepgras0Astrid Rohrbeck1Ingo Just2Stefan Bittner3Gudrun Ahnert-Hilger4Markus Höltje5Department of Neurology, Focus Program Translational Neuroscience and Immunotherapy, Rhine-Main Neuroscience Network, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, GermanyInstitute of Toxicology, Hannover Medical School, Hanover, GermanyInstitute of Toxicology, Hannover Medical School, Hanover, GermanyDepartment of Neurology, Focus Program Translational Neuroscience and Immunotherapy, Rhine-Main Neuroscience Network, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, GermanyDepartment of Neurobiology, Max Planck Institute for Biophysical Chemistry, University of Göttingen, Göttingen, GermanyInstitut für Integrative Neuroanatomie, Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin, GermanyIn primary murine hippocampal neurons we investigated the regulation of EAAT3-mediated glutamate transport by the Clostridium botulinum C3 transferase C3bot and a 26mer peptide derived from full length protein. Incubation with either enzyme-competent C3bot or enzyme-deficient C3bot156–181 peptide resulted in the upregulation of glutamate uptake by up to 22% compared to untreated cells. A similar enhancement of glutamate transport was also achieved by the classical phorbol-ester-mediated activation of protein kinase C subtypes. Yet comparable, effects elicited by C3 preparations seemed not to rely on PKCα, γ, ε, or ζ activation. Blocking of tyrosine phosphorylation by tyrosine kinase inhibitors prevented the observed effect mediated by C3bot and C3bot 26mer. By using biochemical and molecular biological assays we could rule out that the observed C3bot and C3bot 26mer-mediated effects solely resulted from enhanced transporter expression or translocation to the neuronal surface but was rather mediated by transporter phosphorylation at tyrosine residues that was found to be significantly enhanced following incubation with either full length protein or the 26mer C3 peptide.https://www.frontiersin.org/articles/10.3389/fncel.2022.860823/fullC3 transferaseglutamate transporterEAAT3phosphorylationuptake |
| spellingShingle | Johannes Piepgras Astrid Rohrbeck Ingo Just Stefan Bittner Gudrun Ahnert-Hilger Markus Höltje Enhancement of Phosphorylation and Transport Activity of the Neuronal Glutamate Transporter Excitatory Amino Acid Transporter 3 by C3bot and a 26mer C3bot Peptide Frontiers in Cellular Neuroscience C3 transferase glutamate transporter EAAT3 phosphorylation uptake |
| title | Enhancement of Phosphorylation and Transport Activity of the Neuronal Glutamate Transporter Excitatory Amino Acid Transporter 3 by C3bot and a 26mer C3bot Peptide |
| title_full | Enhancement of Phosphorylation and Transport Activity of the Neuronal Glutamate Transporter Excitatory Amino Acid Transporter 3 by C3bot and a 26mer C3bot Peptide |
| title_fullStr | Enhancement of Phosphorylation and Transport Activity of the Neuronal Glutamate Transporter Excitatory Amino Acid Transporter 3 by C3bot and a 26mer C3bot Peptide |
| title_full_unstemmed | Enhancement of Phosphorylation and Transport Activity of the Neuronal Glutamate Transporter Excitatory Amino Acid Transporter 3 by C3bot and a 26mer C3bot Peptide |
| title_short | Enhancement of Phosphorylation and Transport Activity of the Neuronal Glutamate Transporter Excitatory Amino Acid Transporter 3 by C3bot and a 26mer C3bot Peptide |
| title_sort | enhancement of phosphorylation and transport activity of the neuronal glutamate transporter excitatory amino acid transporter 3 by c3bot and a 26mer c3bot peptide |
| topic | C3 transferase glutamate transporter EAAT3 phosphorylation uptake |
| url | https://www.frontiersin.org/articles/10.3389/fncel.2022.860823/full |
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