The observation that cysteine mutations in particular predispose the dystrophin protein to degradation extends recent observations that show that mis-sense mutations in the N-terminal domains result in similar protein instability [Henderson et al

The observation that cysteine mutations in particular predispose the dystrophin protein to degradation extends recent observations that show that mis-sense mutations in the N-terminal domains result in similar protein instability [Henderson et al., 2010; Singh et al., 2010]. was confirmed by transgenic studies of mice, the principle DMD animal model, expressing full-length dystrophin with consecutive deletions, and by injection into mice of different micro-dystrophin constructs with or without this domain [Rafael et al., 1996; Scott et al., 2002]. These studies showed that removal of the CT domain was essentially without consequence whereas deletions that removed portions of the CR domain resulted in loss of dystrophin function and severe muscle pathology. The importance of the CR region is further demonstrated by the distribution of missense mutations in the dystrophinopathies. missense CORO2A mutations are rare, accounting for 1.4% of all dystrophinopathy mutations and only 0.3% of all DMD mutations in one large cohort [Flanigan et al., 2009]. Among the eleven such mutations identified in the ZZ domain (www.dmd.nl), nine of the patients had a severe DMD phenotype. One of these, p.Cys3340Tyr (NM 004006.2:c.10019G A), involves a mutation in a conserved cysteine residue and was found in a patient with a severe DMD phenotype with reduced expression of both dystrophin (described as 10%C20% of normal) and yielding bacmid DNA for infection Setrobuvir (ANA-598) of Sf9 insect cells. Protein was purified using an anti-FLAG M2 agarose column (Sigma A2220) followed by dialysis. Differential scanning fluorimetry (DSF) was performed as described by Niesen et al [Niesen et al., 2007], in triplicate. differential light scattering was performed on 100ul of protein using a Malvern Instruments Zetasizer gene encoding the C-terminal fragment of the dystrophin protein (encompassing the WW, EF1, EF2, ZZ, and CT domains; Supp. Fig. S2) and introduced three different missense mutations into the ZZ domain by site-directed mutagenesis. In vitro expression of wild-type (ZZ-WT) and mutant constructs was assessed by transfection into 293K cells. Western blot of the supernatant shows amounts of p.Asp3335His close to that of the ZZ-WT construct, whereas amounts of both cysteine mutation constructs are markedly decreased in the supernatant (Fig. 1A). Significant amounts of all the mutant constructs were found in the insoluble (pellet) fraction, confirming expression from the pCMV-delivered transgene, and suggesting that the expressed proteins are not stable. Open in a separate window Figure 1 Mutant and normal-ZZ proteins expression. A: In vitro expression. After transfection with the different pCMV-ZZ constructs, 293 K cells were lysed under denaturing conditions for western blot analysis. Boxed images show the dystrophin expression in the supernatant and in the pellet, and muscles. In order to determine whether the expression pattern is similar in vivo, the same constructs were cloned into an AAV plasmid under control of the MHCK7 promoter to produce an AAV2/8 vector for injection into mice. The difference in expression was even more pronounced in vivo as the cysteine mutant proteins were not detectable by Western blot (Fig. 1B), despite the presence of comparable numbers of vector genomes in transfected muscles by quantitative PCR (data not shown). This difference in expression was confirmed by immunostaining (Fig. 2A); p.Asp3335His is properly Setrobuvir (ANA-598) expressed at the membrane (albeit in diminished amount compared to the WT construct), whereas no expression was detected in the cysteine mutated injected muscles. These results are consistent with the immunohistochemistry described in the published reports of the Setrobuvir (ANA-598) p. Asp3335His and p.Cys3340Tyr patients [Goldberg et al., 1998; Lenk et.