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Phosphorylation alters the mechanical stiffness of a model fragment of the dystrophin homologue utrophin
Duchenne muscular dystrophy is a lethal muscle wasting disease caused by the absence of the protein dystrophin. Utrophin is a dystrophin homologue currently under investigation as a protein replacement therapy for Duchenne muscular dystrophy. Dystrophin is hypothesized to function as a molecular sho...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Society for Biochemistry and Molecular Biology
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9922815/ https://www.ncbi.nlm.nih.gov/pubmed/36587764 http://dx.doi.org/10.1016/j.jbc.2022.102847 |
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author | Ramirez, Maria Paz Rajaganapathy, Sivaraman Hagerty, Anthony R. Hua, Cailong Baxter, Gloria C. Vavra, Joseph Gordon, Wendy R. Muretta, Joseph M. Salapaka, Murti V. Ervasti, James M. |
author_facet | Ramirez, Maria Paz Rajaganapathy, Sivaraman Hagerty, Anthony R. Hua, Cailong Baxter, Gloria C. Vavra, Joseph Gordon, Wendy R. Muretta, Joseph M. Salapaka, Murti V. Ervasti, James M. |
author_sort | Ramirez, Maria Paz |
collection | PubMed |
description | Duchenne muscular dystrophy is a lethal muscle wasting disease caused by the absence of the protein dystrophin. Utrophin is a dystrophin homologue currently under investigation as a protein replacement therapy for Duchenne muscular dystrophy. Dystrophin is hypothesized to function as a molecular shock absorber that mechanically stabilizes the sarcolemma. While utrophin is homologous with dystrophin from a molecular and biochemical perspective, we have recently shown that full-length utrophin expressed in eukaryotic cells is stiffer than what has been reported for dystrophin fragments expressed in bacteria. In this study, we show that differences in expression system impact the mechanical stiffness of a model utrophin fragment encoding the N terminus through spectrin repeat 3 (UtrN-R3). We also demonstrate that UtrN-R3 expressed in eukaryotic cells was phosphorylated while bacterial UtrN-R3 was not detectably phosphorylated. Using atomic force microscopy, we show that phosphorylated UtrN-R3 exhibited significantly higher unfolding forces compared to unphosphorylated UtrN-R3 without altering its actin-binding activity. Consistent with the effect of phosphorylation on mechanical stiffness, mutating the phosphorylated serine residues on insect eukaryotic protein to alanine decreased its stiffness to levels not different from unphosphorylated bacterial protein. Taken together, our data suggest that the mechanical properties of utrophin may be tuned by phosphorylation, with the potential to improve its efficacy as a protein replacement therapy for dystrophinopathies. |
format | Online Article Text |
id | pubmed-9922815 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | American Society for Biochemistry and Molecular Biology |
record_format | MEDLINE/PubMed |
spelling | pubmed-99228152023-02-14 Phosphorylation alters the mechanical stiffness of a model fragment of the dystrophin homologue utrophin Ramirez, Maria Paz Rajaganapathy, Sivaraman Hagerty, Anthony R. Hua, Cailong Baxter, Gloria C. Vavra, Joseph Gordon, Wendy R. Muretta, Joseph M. Salapaka, Murti V. Ervasti, James M. J Biol Chem Research Article Duchenne muscular dystrophy is a lethal muscle wasting disease caused by the absence of the protein dystrophin. Utrophin is a dystrophin homologue currently under investigation as a protein replacement therapy for Duchenne muscular dystrophy. Dystrophin is hypothesized to function as a molecular shock absorber that mechanically stabilizes the sarcolemma. While utrophin is homologous with dystrophin from a molecular and biochemical perspective, we have recently shown that full-length utrophin expressed in eukaryotic cells is stiffer than what has been reported for dystrophin fragments expressed in bacteria. In this study, we show that differences in expression system impact the mechanical stiffness of a model utrophin fragment encoding the N terminus through spectrin repeat 3 (UtrN-R3). We also demonstrate that UtrN-R3 expressed in eukaryotic cells was phosphorylated while bacterial UtrN-R3 was not detectably phosphorylated. Using atomic force microscopy, we show that phosphorylated UtrN-R3 exhibited significantly higher unfolding forces compared to unphosphorylated UtrN-R3 without altering its actin-binding activity. Consistent with the effect of phosphorylation on mechanical stiffness, mutating the phosphorylated serine residues on insect eukaryotic protein to alanine decreased its stiffness to levels not different from unphosphorylated bacterial protein. Taken together, our data suggest that the mechanical properties of utrophin may be tuned by phosphorylation, with the potential to improve its efficacy as a protein replacement therapy for dystrophinopathies. American Society for Biochemistry and Molecular Biology 2022-12-29 /pmc/articles/PMC9922815/ /pubmed/36587764 http://dx.doi.org/10.1016/j.jbc.2022.102847 Text en © 2022 The Authors https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Research Article Ramirez, Maria Paz Rajaganapathy, Sivaraman Hagerty, Anthony R. Hua, Cailong Baxter, Gloria C. Vavra, Joseph Gordon, Wendy R. Muretta, Joseph M. Salapaka, Murti V. Ervasti, James M. Phosphorylation alters the mechanical stiffness of a model fragment of the dystrophin homologue utrophin |
title | Phosphorylation alters the mechanical stiffness of a model fragment of the dystrophin homologue utrophin |
title_full | Phosphorylation alters the mechanical stiffness of a model fragment of the dystrophin homologue utrophin |
title_fullStr | Phosphorylation alters the mechanical stiffness of a model fragment of the dystrophin homologue utrophin |
title_full_unstemmed | Phosphorylation alters the mechanical stiffness of a model fragment of the dystrophin homologue utrophin |
title_short | Phosphorylation alters the mechanical stiffness of a model fragment of the dystrophin homologue utrophin |
title_sort | phosphorylation alters the mechanical stiffness of a model fragment of the dystrophin homologue utrophin |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9922815/ https://www.ncbi.nlm.nih.gov/pubmed/36587764 http://dx.doi.org/10.1016/j.jbc.2022.102847 |
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