The Polyproline Site in Hinge 2 Influences the Functional Capacity of Truncated Dystrophins

Abstract

Mutations in dystrophin can lead to Duchenne muscular dystrophy or the more mild form of the disease, Becker muscular dystrophy. The hinge 3 region in the rod domain of dystrophin is particularly prone to deletion mutations. In-frame deletions of hinge 3 are predicted to lead to BMD, however the severity of disease can vary considerably. Here we performed extensive structure-function analyses of truncated dystrophins with modified hinges and spectrin-like repeats in mdx mice. We found that the polyproline site in hinge 2 profoundly influences the functional capacity of a microdystrophinΔR4-R23/ΔCT with a large deletion in the hinge 3 region. Inclusion of polyproline in microdystrophinΔR4-R23/ΔCT led to small myofibers (12% smaller than wild-type), Achilles myotendinous disruption, ringed fibers, and aberrant neuromuscular junctions in the mdx gastrocnemius muscles. Replacing hinge 2 of microdystrophinΔR4-R23/ΔCT with hinge 3 significantly improved the functional capacity to prevent muscle degeneration, increase muscle fiber area, and maintain the junctions. We conclude that the rigid α-helical structure of the polyproline site significantly impairs the functional capacity of truncated dystrophins to maintain appropriate connections between the cytoskeleton and extracellular matrix.

In-frame Dystrophin Following Exon 51-Skipping Improves Muscle Pathology and Function in the Exon 52–Deficient mdx Mouse

“…27 Based on the molecular structure of dystrophin, the small segment of the hinge 3 might act as a "turn, " which is bound to the helix of the 20th repeat. 27,38 Our results suggest that the hinge 3 region is more essential in the short microdystrophin (167 kDa) than in the almost full-length dystrophin lacking hinge 3 due to exon 51-skipping (420 kDa). 38,39 In-frame dystrophin expression in skeletal muscle at 20% of normal levels produced moderate/mild BMD phenotypes.…”

“…[4][5][6] The molecular structure of dystrophin is composed of the actin-binding domain 1 at the N-terminus (ABD1), the central rod domain containing 24 spectrin-like repeats (R1-24), four hinge domains, a 20-amino acid insertion between spectrin-like repeats 15 and 16 (segment 5), the cysteine-rich domain, and the C-terminal domain (Figure 1b). 27,38 Until now, the in-frame dystrophin formed following exon 23-skipping, which lacks half of the 6th spectrin-like repeat and part of the 7th, ameliorated the muscle pathology and function in mdx mice with a point mutation in exon 23 (refs. 15,16).…”

“…20,22 Recently, it was reported that hinge 3 is more important than hinge 2 in preventing muscle degeneration and promoting muscle maturation in the microdystrophin ΔR4-R23 /mdx transgenic mice. 38 The in-frame dystrophin produced following exon-51-skipping is predicted to lack most of the hinge 3 region (Figure 1b); hinges 1, 2, 3, and 4 consist of 75, 50, 43, and 72 amino acid residues, respectively. 27,38 The small segment of hinge 3 that remains following exon 51-skipping consists of 13 amino acid residues with two prolines and would not be predicted to function as a hinge (Figure 1c).…”

“…38 The in-frame dystrophin produced following exon-51-skipping is predicted to lack most of the hinge 3 region (Figure 1b); hinges 1, 2, 3, and 4 consist of 75, 50, 43, and 72 amino acid residues, respectively. 27,38 The small segment of hinge 3 that remains following exon 51-skipping consists of 13 amino acid residues with two prolines and would not be predicted to function as a hinge (Figure 1c). This remaining fragment is very similar to segment 5, which consists of 20-amino acid residues with one proline and is located between the 15th and 16th spectrin-like repeats.…”

A Simplified Immune Suppression Scheme Leads to Persistent Micro-dystrophin Expression in Duchenne Muscular Dystrophy Dogs

“…The proviral cis plasmid (YL196) for the R16-17/H3/DC lDys vector was based on the canine dystrophin gene. This microgene is modeled according to a previously described R16-17/DC micro-dystrophin gene (Lai et al, 2009;Banks et al, 2010;Li et al, 2011). It contains the dystrophin N-terminal domain, hinge 1, spectrin-like repeat 1, 16 and 17 (R1, R16, and R17), hinge 3, R24, hinge 4, and the cysteine-rich (CR) domain.…”