Phosphorylation alters the mechanical stiffness of a model fragment of the dystrophin homologue utrophin.

Maria Paz Ramirez, Sivaraman Rajaganapathy, Anthony R Hagerty, Cailong Hua, Gloria C Baxter, Joseph Vavra, Wendy R Gordon, Joseph M Muretta, Murti V Salapaka, James M Ervasti

The Journal of biological chemistry 2023 Feb

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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. Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.

Citation

Maria Paz Ramirez, Sivaraman Rajaganapathy, Anthony R Hagerty, Cailong Hua, Gloria C Baxter, Joseph Vavra, Wendy R Gordon, Joseph M Muretta, Murti V Salapaka, James M Ervasti. Phosphorylation alters the mechanical stiffness of a model fragment of the dystrophin homologue utrophin. The Journal of biological chemistry. 2023 Feb;299(2):102847