Missense mutations in dystrophin that trigger muscular dystrophy decrease protein stability and lead to cross-β aggregates

Abstract

A deficiency of functional dystrophin protein in muscle cells causes muscular dystrophy (MD). More than 50% of missense mutations that trigger the disease occur in the N-terminal actin binding domain (N-ABD or ABD1). We examined the effect of four diseasecausing mutations-L54R, A168D, A171P, and Y231N-on the structural and biophysical properties of isolated N-ABD. Our results indicate that N-ABD is a monomeric, well-folded α-helical protein in solution, as is evident from its α-helical circular dichroism spectrum, blue shift of the native state tryptophan fluorescence, well-dispersed amide crosspeaks in 2D NMR 15 N-1 H HSQC fingerprint region, and rotational correlation time calculated from NMR longitudinal ðT 1 Þ and transverse ðT 2 Þ relaxation experiments. Compared to WT, three mutants-L54R, A168D, and A171P-show a decreased α-helicity and do not show a cooperative sigmoidal melt with temperature, indicating that these mutations exist in a wide range of conformations or in a "molten globule" state. In contrast, Y231N has an α-helical content similar to WT and shows a cooperative sigmoidal temperature melt but with a decreased stability. All four mutants experience serious misfolding and aggregation. FT-IR, circular dichroism, increase in thioflavin T fluorescence, and the congo red spectral shift and birefringence show that these aggregates contain intermolecular cross-β structure similar to that found in amyloid diseases. These results indicate that disease-causing mutants affect N-ABD structure by decreasing its thermodynamic stability and increasing its misfolding, thereby decreasing the net functional dystrophin concentration.actin binding domain | Becker muscular dystrophy | calponin homology domain | Duchenne muscular dystrophy | protein aggregation

Disease-proportional proteasomal degradation of missense dystrophins

“…Previous studies have demonstrated that several missense mutations in ABD1 of dystrophin cause protein instability in vitro (18,19). Here, we were able to distinguish between a missense mutation associated with DMD (L54R) from one associated with BMD (L172H) by monitoring protein tertiary structure in vitro (Fig.…”

“…In vitro biophysical analyses of the full-length proteins instead revealed that each of the mutations rendered dystrophin more insoluble and less stable during thermal denaturation. Missense mutations analyzed in purified ABD1 fragments also exhibited significant aggregation as assessed by thioflavin T fluorescence, Congo red staining, and electron microscopy (19).…”

Novel Mutation in Spectrin-like Repeat 1 of Dystrophin Central Domain Causes Protein Misfolding and Mild Becker Muscular Dystrophy

“…Mutated dystrophin is expressed in skeletal muscle of cases with missense point mutations, but to date there are very few publications that evaluate potential genotype-phenotype correlations. Previous studies of point mutations within the actin-binding domain of patients with either DMD or BMD phenotypes have concluded that the dystrophin produced is mostly misfolded, rather unstable and prone to aggregation (39,40). We recently showed that a double point mutation in the central rod domain repeat R23 of a DMD patient leads to a substantially lower thermal and chemical stability as well as slower refolding of the mutated protein compared with the wild-type protein (25).…”

“…In-frame deletions constitute the majority of the mutations, and it appears that mutations of the N terminus of dystrophin affecting the actin-binding domain 1 (39,40) or the Cys-rich domain (41) are more severe than the others. However, deletions around exons 45-55 are in a large number of cases accompanied by mild skeletal muscle disease but a possibly severe cardiomyopathy.…”

CUGC for Duchenne muscular dystrophy (DMD)

“…Missense variants can result in DMD rather than BMD if the variant affects the actin binding domain [2,21] or the β-dystroglycan binding domain of dystrophin [1]. There are other rare cases of in-frame variants resulting in DMD rather than BMD, e.g.…”