Disease-proportional proteasomal degradation of missense dystrophins

Abstract

The 427-kDa protein dystrophin is expressed in striated muscle where it physically links the interior of muscle fibers to the extracellular matrix. A range of mutations in the DMD gene encoding dystrophin lead to a severe muscular dystrophy known as Duchenne (DMD) or a typically milder form known as Becker (BMD). Patients with nonsense mutations in dystrophin are specifically targeted by stop codon read-through drugs, whereas out-of-frame deletions and insertions are targeted by exon-skipping therapies. Both treatment strategies are currently in clinical trials. Dystrophin missense mutations, however, cause a wide range of phenotypic severity in patients. The molecular and cellular consequences of such mutations are not well understood, and there are no therapies specifically targeting this genotype. Here, we have modeled two representative missense mutations, L54R and L172H, causing DMD and BMD, respectively, in full-length dystrophin. In vitro, the mutation associated with the mild phenotype (L172H) caused a minor decrease in tertiary stability, whereas the L54R mutation associated with a severe phenotype had a more dramatic effect. When stably expressed in mammalian muscle cells, the mutations caused steadystate decreases in dystrophin protein levels inversely proportional to the tertiary stability and directly caused by proteasomal degradation. Both proteasome inhibitors and heat shock activators were able to increase mutant dystrophin to WT levels, establishing the new cell lines as a platform to screen for potential therapeutics personalized to patients with destabilized dystrophin.Duchenne muscular dystrophy | missense mutation | protein folding | proteasome inhibitor | heat shock activator

Functional correction of dystrophin actin binding domain mutations by genome editing

“…We observed less dystrophin expression in ΔEx7-11 iPSC-derived cardiomyocytes compared with control iPSC-derived cardiomyocytes and the other corrected lines, ΔEx3-9 and ΔEx6-9. Previously, it was shown that missense mutations associated with the DMD phenotype caused steady-state decreases in dystrophin protein levels inversely proportional to the tertiary stability and directly caused by proteasomal degradation (41). Consistent with this report, we observed increased dystrophin protein levels when ΔEx7-11 iPSC-derived cardiomyocytes were treated with the proteasome inhibitor MG-132, as measured by Western blot analysis ( Figure 4F and Supplemental Figure 1C).…”

Dynamics of Dystrophin’s Actin-Binding Domain

“…Indeed, when many of the same missense mutations were examined in full-length Dys, changes in binding affinity were modest or statistically insignificant (48). This suggests that for DMD and BMD, missense mutations that perturb the core fold of ABD1 CH domains are promoting disease via a misfolding-induced degradation mechanism (49). For some missense mutations-perhaps those that are more superficially located within the ABD1, or those that do not misfold the ABD1 but perturb stability-a functional impact like that discussed above for ABDs in general may become relevant.…”

Identification of a novel DMD duplication identified by a combination of MLPA and targeted exome sequencing

“…As a significant component of the dystrophin-glycoprotein complex (DGC), dystrophin, plays a key role in the contraction of sarcomeres [13]. Dystrophin is a structural protein of 427-kD, consisting of a N-terminal actin binding domain (ABD1), a central rod region, a cysteine-rich (CR) globular domain, and a C-terminal tail (CT) [13]. The C-terminus is associated with glycoproteins and the N-terminus is associated with actin or actin-like proteins [1].…”