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

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish (US)
Article number102847
JournalJournal of Biological Chemistry
Volume299
Issue number2
DOIs
StatePublished - Feb 2023

Bibliographical note

Funding Information:
Authors thank Michael Kyba for providing the iC2C12 cell line. M. P. R. M. V. S. and J. M. E. conceptualization; M. P. R. and S. R. methodology; M. P. R. and S. R. visualization; M. P. R. S. R. A. R. H. C. H. and G. C. B. investigation; M. P. R. S. R. A. R. H. C. H. and G. C. B. formal analysis; M. P. R. and J. M. E. writing-original draft; M. P. R. W. R. G. and J. M. E. supervision; M. P. R. and J. M. E. project administration; S. R. data curation; S. R. software; S. R. M. V. S. W. R. G. and J. M. M. writing-reviewing and editing; J. V. validation; M. V. S. resources; J. M. E. funding acquisition. The authors would like to acknowledge funding from NIH (R01AR042423- JME) and the Muscular Dystrophy Association (628925- JME). WRG is a Pew Biomedical Scholar. MPR would like to acknowledge funding from the University of Minnesota Frieda M. Kunze and Doctoral Dissertation Fellowships. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Funding Information:
The authors would like to acknowledge funding from NIH ( R01AR042423- JME ) and the Muscular Dystrophy Association ( 628925- JME ). WRG is a Pew Biomedical Scholar. MPR would like to acknowledge funding from the University of Minnesota Frieda M. Kunze and Doctoral Dissertation Fellowships. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Publisher Copyright:
© 2022 The Authors

Keywords

  • and mechanobiology
  • atomic force microscopy (AFM)
  • dystrophin
  • muscular dystrophy
  • phosphorylation
  • protein stability
  • utrophin

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

Fingerprint

Dive into the research topics of 'Phosphorylation alters the mechanical stiffness of a model fragment of the dystrophin homologue utrophin'. Together they form a unique fingerprint.

Cite this