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

Autor:	Ramirez MP; Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota - Twin Cities, Minneapolis, MN, USA., Rajaganapathy S; Department of Electrical and Computer Engineering, University of Minnesota - Twin Cities, Minneapolis, MN, USA., Hagerty AR; Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota - Twin Cities, Minneapolis, MN, USA., Hua C; Department of Electrical and Computer Engineering, University of Minnesota - Twin Cities, Minneapolis, MN, USA., Baxter GC; Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota - Twin Cities, Minneapolis, MN, USA., Vavra J; Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota - Twin Cities, Minneapolis, MN, USA., Gordon WR; Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota - Twin Cities, Minneapolis, MN, USA., Muretta JM; Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota - Twin Cities, Minneapolis, MN, USA., Salapaka MV; Department of Electrical and Computer Engineering, University of Minnesota - Twin Cities, Minneapolis, MN, USA., Ervasti JM; Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota - Twin Cities, Minneapolis, MN, USA. Electronic address: jervasti@umn.edu.
Jazyk:	angličtina
Zdroj:	The Journal of biological chemistry [J Biol Chem] 2023 Feb; Vol. 299 (2), pp. 102847. Date of Electronic Publication: 2022 Dec 29.
DOI:	10.1016/j.jbc.2022.102847
Abstrakt:	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. Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article. (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)
Databáze:	MEDLINE
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