Missense mutations in the dystrophin protein can cause Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD) through an undefined pathomechanism. In vitro studies suggest that missense mutations in the N-terminal actinbinding domain (ABD1) cause protein instability, and cultured myoblast studies reveal decreased expression levels that can be restored to wild-type with proteasome inhibitors. To further elucidate the pathophysiology of missense dystrophin in vivo, we generated two transgenic mdx mouse lines expressing L54R or L172H mutant dystrophin, which correspond to missense mutations identified in human patients with DMD or BMD, respectively. Our biochemical, histologic and physiologic analysis of the L54R and L172H mice show decreased levels of dystrophin which are proportional to the phenotypic severity. Proteasome inhibitors were ineffective in both the L54R and L172H mice, yet mice homozygous for the L172H transgene were able to express even higher levels of dystrophin which caused further improvements in muscle histology and physiology. Given that missense dystrophin is likely being degraded by the proteasome but whole body proteasome inhibition was not possible, we screened for ubiquitin-conjugating enzymes involved in targeting dystrophin to the proteasome. A myoblast cell line expressing L54R mutant dystrophin was screened with an siRNA library targeting E1, E2 and E3 ligases which identified Amn1, FBXO33, Zfand5 and Trim75. Our study establishes new mouse models of dystrophinopathy and identifies candidate E3 ligases that may specifically regulate dystrophin protein turnover in vivo.
Bibliographical noteFunding Information:
The study was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases grant RO1 AR042423 to J.M.E. and R01 AR055685 to M.K. J.L.M., J.J.B. and J.T.O. were supported by the National Institutes of Health Training Program in Muscle Research (AR007612). J.J.B. was also supported by a University of Minnesota Doctoral Dissertation Fellowship. J.T.O. and D.M.N. were each supported by fellowships from the National Institute on Aging Training Program for Functional Proteomics of Aging (T32 AG029796). D.M.T. was supported by an American Heart Association Predoctoral Fellowship (12PRE12040402). C.M.C. was supported by Muscular Dystrophy Association grant MDA349549.