We have used time-resolved phosphorescence anisotropy (TPA) of actin to evaluate domains of dystrophin and utrophin, with implications for gene therapy in muscular dystrophy. Dystrophin and its homolog utrophin bind to cytoskeletal actin to form mechanical linkages that prevent muscular damage. Because these proteins are too large for most gene therapy vectors, much effort is currently devoted to smaller constructs. We previously used TPA to show that both dystrophin and utrophin have a paradoxical effect on actin rotational dynamics - restricting amplitude while increasing rate, thus increasing resilience, with utrophin more effective than dystrophin. Here, we have evaluated individual domains of these proteins. We found that a mini-dystrophin, lacking one of the two actin-binding domains, is less effective than dystrophin in regulating actin dynamics, correlating with its moderate effectiveness in rescuing the dystrophic phenotype in mice. In contrast, we found that a micro-utrophin, with more extensive internal deletions, is as effective as full-length dystrophin in the regulation of actin dynamics. Each of utrophin's actin-binding domains promotes resilience in actin, while dystrophin constructs require the presence of both actin-binding domains and the C-terminal domain for full function. This work supports the use of a utrophin template for gene or protein therapy designs. Resilience of the actin-protein complex, measured by TPA, correlates remarkably well with previous reports of functional rescue by dystrophin and utrophin constructs in mdx mice. We propose the use of TPA as an in vitro method to aid in the design and testing of emerging gene therapy constructs.
Bibliographical noteFunding Information:
TPA experiments were performed at the Biophysical Spectroscopy Facility, University of Minnesota, and at Muscular Dystrophy Core Laboratories at the University of Minnesota supported by National Institutes of Health (NIH) grant P30-AR0507220 . Excellent computational resources were provided by the Minnesota Supercomputing Institute. Special thanks to Octavian Cornea for assistance with manuscript preparation and submission and Dawn Lowe for useful discussions. We also thank Hanke Heun-Johnson for her technical assistance in engineering the FLAG-Micro-Utr construct. This study was supported by NIH grants AR032961 , AR057220 Core C , and AG026160 (to D.D.T.); Muscular Dystrophy Association grant 4322 (to D.D.T.); NIH grants AR007612 and AR042423 (to J.M.E.); and NIH grant F30AG034033 (to A.Y.L.).
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- gene therapy
- muscular dystrophy
- time-resolved phosphorescence anisotropy