Rod Domain Of Dystrophin Research Articles

Coupling of Spectrin Repeat Modules for the Assembly of Nanorods and Presentation of Protein Domains.

Modular protein engineering is a powerful approach for fabricating high-molecular-weight assemblies and biomaterials with nanoscale precision. Herein, we address the challenge of designing an extended nanoscale filamentous architecture inspired by the central rod domain of human dystrophin, which protects sarcolemma during muscle contraction and consists of spectrin repeats composed of three-helical bundles. A module of three tandem spectrin repeats was used as a rigid building block self-assembling via coiled-coil (CC) dimer-forming peptides. CC peptides were precisely integrated to maintain the spectrin α-helix continuity in an appropriate frame to form extended nanorods. An orthogonal set of customizable CC heterodimers was harnessed for modular rigid domain association, which could be additionally regulated by metal ions and chelators. We achieved a robust assembly of rigid rods several micrometers in length, determined by atomic force microscopy and negative stain transmission electron microscopy. Furthermore, these rigid rods can serve as a scaffold for the decoration of diverse proteins or biologically active peptides along their length with adjustable spacing up to tens of nanometers, as confirmed by the DNA-PAINT super-resolution microscopy. This demonstrates the potential of modular bottom-up protein engineering and tunable CCs for the fabrication of functionalized protein biomaterials.

Unusually severe muscular dystrophy upon in-frame deletion of the dystrophin rod domain and lack of compensation by membrane-localized utrophin

Utrophin, a dystrophin homolog, is consistently upregulated in muscles of patients with Duchenne muscular dystrophy (DMD) and is believed to partially compensate for the lack of dystrophin in dystrophic muscle. Even though several animal studies support the idea that utrophin can modulate DMD disease severity, human clinical data are scarce. We describe a patient with the largest reported in-frame deletion in the DMD gene, including exons 10-60 and thus encompassing the entire rod domain. The patient presented with an unusually early and severe progressive weakness, initially suggesting congenital muscular dystrophy. Immunostaining of his muscle biopsy showed that the mutant protein was able to localize at the sarcolemma and stabilize the dystrophin-associated complex. Strikingly, utrophin protein was absent from the sarcolemmal membrane despite the upregulation of utrophin mRNA. Our results suggest that the internally deleted and dysfunctional dystrophin lacking the entire rod domain may exert a dominant-negative effect by preventing upregulated utrophin protein from reaching the sarcolemmal membrane and thus blocking its partial rescue of muscle function. This unique case may set a lower size limit for similar constructs in potential gene therapy approaches. This work was supported by a grant from MDA USA (MDA3896) and by grant number R01AR051999 from NIAMS/NIH to C.G.B.

Molecular Fingerprint of BMD Patients Lacking a Portion in the Rod Domain of Dystrophin.

BMD is characterized by a marked heterogeneity of gene mutations resulting in many abnormal dystrophin proteins with different expression and residual functions. The smaller dystrophin molecules lacking a portion around exon 48 of the rod domain, named the D8 region, are related to milder phenotypes. The study aimed to determine which proteins might contribute to preserving muscle function in these patients. Patients were subdivided, based on the absence or presence of deletions in the D8 region, into two groups, BMD1 and BMD2. Muscle extracts were analyzed by 2-D DIGE, label-free LC-ESI-MS/MS, and Ingenuity pathway analysis (IPA). Increased levels of proteins typical of fast fibers and of proteins involved in the sarcomere reorganization characterize BMD2. IPA of proteomics datasets indicated in BMD2 prevalence of glycolysis and gluconeogenesis and a correct flux through the TCA cycle enabling them to maintain both metabolism and epithelial adherens junction. A 2-D DIGE analysis revealed an increase of acetylated proteoforms of moonlighting proteins aldolase, enolase, and glyceraldehyde-3-phosphate dehydrogenase that can target the nucleus promoting stem cell recruitment and muscle regeneration. In BMD2, immunoblotting indicated higher levels of myogenin and lower levels of PAX7 and SIRT1/2 associated with a set of proteins identified by proteomics as involved in muscle homeostasis maintenance.

Efficacy of Multi-exon Skipping Treatment in Duchenne Muscular Dystrophy Dog Model Neonates

Duchenne muscular dystrophy (DMD) is caused by mutations in DMD, which codes for dystrophin. Because the progressive and irreversible degeneration of muscle occurs from childhood, earlier therapy is required to prevent dystrophic progression. Exon skipping by antisense oligonucleotides called phosphorodiamidate morpholino oligomers (PMOs), which restores the DMD reading frame and dystrophin expression, is a promising candidate for use in neonatal patients, yet the potential remains unclear. Here, we investigate the systemic efficacy and safety of early exon skipping in dystrophic dog neonates. Intravenous treatment of canine X-linked muscular dystrophy in Japan dogs with a 4-PMO cocktail resulted in ∼3%-27% in-frame exon 6-9 skipping and dystrophin restoration across skeletal muscles up to 14% of healthy levels. Histopathology was ameliorated with the reduction of fibrosis and/or necrosis area and centrally nucleated fibers, significantly in the diaphragm. Treatment induced cardiac multi-exon skipping, though dystrophin rescue was not detected. Functionally, treatment led to significant improvement in the standing test. Toxicity was not observed from blood tests. This is the first study to demonstrate successful multi-exon skipping treatment and significant functional improvement in dystrophic dogs. Early treatment was most beneficial for respiratory muscles, with implications for addressing pulmonary malfunction in patients.

MLPA identification of dystrophin mutations and in silico evaluation of the predicted protein in dystrophinopathy cases from India

BackgroundDuchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked recessive disorders caused by mutations in the DMD gene. The aim of this study was to predict the effect of gene mutations on the dystrophin protein and study its impact on clinical phenotype.MethodsIn this study, 415 clinically diagnosed patients were tested for mutations by Multiplex ligation dependent probe amplification (MLPA). Muscle biopsy was performed in 34 patients with negative MLPA. Phenotype-genotype correlation was done using PROVEAN, hydrophobicity and eDystrophin analysis. We have utilized bioinformatics tools in order to evaluate the observed mutations both at the level of primary as well as secondary structure.ResultsMutations were identified in 75.42% cases, of which there were deletions in 91.6% and duplications in 8.30%. As per the reading frame rule, 84.6% out-of frame and 15.3% in-frame mutations were noted. Exon 50 was the most frequently deleted exon and the exon 45–52 region was the hot-spot for deletions in this cohort. There was no correlation noted between age of onset or creatine kinase (CK) values with extent of gene mutation. The PROVEAN analysis showed a deleterious effect in 94.5% cases and a neutral effect in 5.09% cases. Mutations in exon 45–54 (out of frame) and exon 46–54 (in-frame) regions in the central rod domain of dystrophin showed more negative scores compared to other domains in the present study. Hydrophobicity profile analysis showed that the hydrophobic regions I & III were equally affected. Analysis of deletions in hinge III hydrophobic region by the eDystrophin programme also predicted a hybrid repeat seen to be associated with a BMD like disease progression, thus making the hinge III region relatively tolerant to mutations.ConclusionsWe found that, while the predictions made by the software utilized might have overall significance, the results were not convincing on a case by case basis. This reflects the inadequacy of the currently available tools and also underlines the possible inadequacy of MLPA to detect other minor mutations that might enhance or suppress the effect of the primary mutation in this large gene. Next Generation Sequencing or targeted Sanger sequencing on a case by case basis might improve phenotype- genotype correlation.

Immunohistochemistry of sarcolemmal membrane-associated proteins in formalin-fixed and paraffin-embedded skeletal muscle tissue: a promising tool for the diagnostic evaluation of common muscular dystrophies

BackgroundThe analysis of fresh frozen muscle specimens is standard following routine muscle biopsy, but this service is not widely available in countries with limited medical facilities, such as Thailand. Nevertheless, immunohistochemistry (IHC) analysis is essential for the diagnosis of patients with a strong clinical suspicion of muscular dystrophy, in the absence of mutations detected by molecular genetics. As the successful labelling of sarcolemmal membrane-associated proteins in formalin-fixed and paraffin-embedded (FFPE) muscle sections using IHC staining has rarely been described, this study aimed to develop a reproducible IHC method for such an analysis.MethodsThirteen cases were studied from the files of the Department of Pathology, Mahidol University. Diagnoses included three Duchenne muscular dystrophy (DMD), one Becker muscular dystrophy (BMD), one dysferlinopathy, and several not-specified muscular dystrophies. IHC was performed on FFPE sections at different thicknesses (3 μm, 5 μm, and 8 μm) using the heat-mediated antigen retrieval method with citrate/EDTA buffer, followed by an overnight incubation with primary antibodies at room temperature. Antibodies against spectrin, dystrophin (rod domain, C-terminus, and N-terminus), dysferlin, sarcoglycans (α, β, and γ), and β-dystroglycan were used. Frozen sections were tested in parallel for comparative analysis.ResultsAntibodies labelling spectrin, dystrophin (rod domain and C-terminus), dysferlin, sarcoglycans (α, β, and γ), and β-dystroglycan clearly exhibited sarcolemmal staining in FFPE sections. However, staining of FFPE sections using the antibody directed against the N-terminus of dystrophin was unsuccessful. The absence of labeling for dystrophins and dysferlin in FFPE sections was documented in all three DMD patients and the dysferlinopathy patient. The BMD diagnosis could not be made using IHC in FFPE sections alone because of a lack of staining for the dystrophin N-terminus, indicating a limitation of this method.ConclusionsWe developed a reliable and reproducible IHC technique using FFPE muscle. This could become a valuable tool for the diagnosis of some muscular dystrophies, dystrophinopathies, sarcoglycanopathies (LGMD2D, LGMD2E, and LGMD2C), and dysferlinopathy, especially in situations where the analysis of fresh frozen muscle samples is not routinely available.

735. Functional Restoration of Dystrophin Protein in HiPSC-Derived Skeletal Myotubes and Cardiomyocytes After CRISPR/Cas9-Mediated Deletion of 530-725kb of DMD

Duchenne muscular dystrophy (DMD) is typically due to frameshifting mutations in the DMD gene encoding dystrophin. Loss of dystrophin protein results in progressive muscle degeneration and premature death. Approximately 60% of DMD patients have frameshifting mutations in a hotspot region within exons 45-55 in the rod domain of dystrophin. Genotype/phenotype assessments have revealed that in-frame deletion of exons 45-55 leads to the milder, allelic disease, Becker muscular dystrophy. This finding suggests that restoration of the reading frame by targeting exons 45-55 could treat ~60% of DMD patients to greatly reduce disease severity. We have developed a platform using clustered regularly interspaced short palindromic repeats (CRISPR) and- associated protein (Cas9) gene editing to achieve this purpose. We have utilized CRISPR/Cas9-mediated deletion and rejoining of up to 725kb to restore the reading frame in DMD human induced pluripotent stem cells (hiPSCs). This is the largest deletion shown to date in DMD. Clonal hiPSC lines containing the exon 45-55 deletion were differentiated to disease relevant types, such as cardiomyocytes and skeletal muscle myotubes, which had restored dystrophin protein. We demonstrated, for the first time, that the internally deleted dystrophin generated by CRISPR/Cas9 was functional and improved membrane integrity, reduced miR31 expression, and restored the dystrophin glycoprotein complex in vitro and after engraftment of skeletal muscle cells in vivo. This gene editing platform restores the reading frame for the majority of DMD patients and offers potential as an ex vivo correction for stem cell therapy or for use in vivo.

561. CRISPR-Induced Deletion (CinDel) Method Allows Permanent and Efficient Restoration of the DMD Gene Reading Frame in Duchenne Patient Myoblasts and Preserves Truncated Dystrophin Structure

The CRISPR/Cas9 system is a great revolution in biology. This technology allows the modification of genes in vitro and in vivo in a wide variety of living organisms. In most Duchenne Muscular Dystrophy (DMD) patients, expression of dystrophin (DYS) protein is disrupted because exon deletions result in a frame shift. Here we present CRISPR-induced deletion (CinDel), a new gene therapy approach to correct the DMD gene in Duchenne patients with one or more exons deletions. By using adequate pair of gRNAs targeting specifically the exons precede and follow the patient deletion in DMD gene, CinDel induces precise DSB in targeted sequences and allows an additional deletion. The remaining parts of the exons were fusioned by NHEJ to form a hybrid exon and restored the DMD reading frame in 62 % of hybrid exons in vitro in patient myoblasts and in vivo in electroporated muscle hDMD/mdx mice. Moreover, adequate pairs of gRNAs also restored the normal spectrin-like repeat of the dystrophin rod domain; such restoration is not obtained by exon skipping or deletion of complete exons. The expression of an internally deleted dystrophin protein was detected following the formation of myotubes by the unselected treated DMD myoblasts. Given that CinDel induces permanent reparation of the DMD gene this treatment would not have to be repeated as it is the case for exon skipping induced by oligonucleotides.

Efficient Restoration of the Dystrophin Gene Reading Frame and Protein Structure in DMD Myoblasts Using the CinDel Method.

The CRISPR/Cas9 system is a great revolution in biology. This technology allows the modification of genes in vitro and in vivo in a wide variety of living organisms. In most Duchenne muscular dystrophy (DMD) patients, expression of dystrophin (DYS) protein is disrupted because exon deletions result in a frame shift. We present here the CRISPR-induced deletion (CinDel), a new promising genome-editing technology to correct the DMD gene. This strategy is based on the use of two gRNAs targeting specifically exons that precede and follow the patient deletion in the DMD gene. This pair of gRNAs induced a precise large additional deletion leading to fusion of the targeted exons. Using an adequate pair of gRNAs, the deletion of parts of these exons and the intron separating them restored the DMD reading frame in 62% of the hybrid exons in vitro in DMD myoblasts and in vivo in electroporated hDMD/mdx mice. Moreover, adequate pairs of gRNAs also restored the normal spectrin-like repeat of the dystrophin rod domain; such restoration is not obtained by exon skipping or deletion of complete exons. The expression of an internally deleted DYS protein was detected following the formation of myotubes by the unselected, treated DMD myoblasts. Given that CinDel induces permanent reparation of the DMD gene, this treatment would not have to be repeated as it is the case for exon skipping induced by oligonucleotides.

Internal deletion compromises the stability of dystrophin

Duchenne muscular dystrophy (DMD) is a deadly and common childhood disease caused by mutations that disrupt dystrophin protein expression. Several miniaturized dystrophin/utrophin constructs are utilized for gene therapy, and while these constructs have shown promise in mouse models, the functional integrity of these proteins is not well described. Here, we compare the biophysical properties of full-length dystrophin and utrophin with therapeutically relevant miniaturized constructs using an insect cell expression system. Full-length utrophin, like dystrophin, displayed a highly cooperative melting transition well above 37°C. Utrophin constructs involving N-terminal, C-terminal or internal deletions were remarkably stable, showing cooperative melting transitions identical to full-length utrophin. In contrast, large dystrophin deletions from either the N- or C-terminus exhibited variable stability, as evidenced by melting transitions that differed by 20°C. Most importantly, deletions in the large central rod domain of dystrophin resulted in a loss of cooperative unfolding with increased propensity for aggregation. Our results suggest that the functionality of dystrophin therapeutics based on mini- or micro-constructs may be compromised by the presence of non-native protein junctions that result in protein misfolding, instability and aggregation.

The polyproline site in hinge 2 influences the functional capacity of truncated dystrophins.

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.

G.P.13.04 In-frame deletion of the entire dystrophin rod domain prevents compensation by utrophin and causes an unusually severe muscular dystrophy

Clinical data to support the ability of the dystrophin-relative utrophin to ameliorate disease in Duchenne muscular dystrophy (DMD) is scarce, though there is ample evidence for animal data suggesting that forced upregulation of utrophin in dystrophic muscle can restore some function. Similarly, double inactivation of both dystrophin and utrophin in the mouse worsens the phenotype considerably. Here we describe a boy presenting with unusually early, severe weakness suggesting congenital muscular dystrophy.

Mapping of the Lipid-Binding and Stability Properties of the Central Rod Domain of Human Dystrophin

Dystrophin is a cytoskeletal protein that confers resistance to the sarcolemma against the stress of contraction–relaxation cycles by interacting with cytoskeletal and membrane partners. Apart from several proteins, membrane phospholipids are a partner of the central rod domain made up of 24 spectrin-like repeats, separated into sub-domains by four hinges. We previously showed that repeats 1 to 3 bind to membrane anionic phospholipids, while repeats 20 to 24 are not able to do so. We focus here on the phospholipid-binding properties of the major part of the central rod domain, namely, the sub-domain delineated by hinges 2 and 3 comprising 16 repeats ranging from repeat 4 to 19 (R4–19). We designed and produced multirepeat proteins comprising three to five repeats and report their lipid-binding properties as well as their thermal stabilities. When these proteins are mixed with liposomes including the anionic lipid phosphatidylserine, they form stable protein–vesicle complexes as determined by gel-filtration chromatography. The absence of an anionic lipid precludes the formation of such complexes. Spectroscopic analyses by circular dichroism and tryptophan fluorescence show that, while the α-helical secondary structures are not modified by the binding, protein trans conformation leads to the movement of tryptophan residues into more hydrophobic environments. In addition, the decrease in the molar ellipticity ratio at 222/208 nm as observed by circular dichroism indicates that lipid binding reduces the inter-helical interactions of multirepeat proteins, thus suggesting partly “opened” coiled-coil structures. Combining these results with data from our previous studies, we propose a new model of the dystrophin molecule lying along the membrane bilayer, in which the two sub-domains R1–3 and R4–19 interact with lipids and F-actin, while the distal sub-domain R20–24 does not exhibit any interaction. These lipid-binding domains should thus maintain a structural link between cytoskeletal actin and sarcolemma via the membrane phospholipids.

A Two-amino Acid Mutation Encountered in Duchenne Muscular Dystrophy Decreases Stability of the Rod Domain 23 (R23) Spectrin-like Repeat of Dystrophin

Lack of functional dystrophin causes severe Duchenne muscular dystrophy. The subsarcolemmal location of dystrophin, as well as its association with both cytoskeleton and membrane, suggests a role in the mechanical regulation of muscular membrane stress. In particular, phenotype rescue in a Duchenne muscular dystrophy mice model has shown that some parts of the central rod domain of dystrophin, constituted by 24 spectrin-like repeats, are essential. In this study, we made use of rare missense pathogenic mutations in the dystrophin gene and analyzed the biochemical properties of the isolated repeat 23 bearing single or double mutations E2910V and N2912D found in muscle dystrophy with severity grading. No dramatic effect on secondary and tertiary structure of the repeat was found in mutants compared with wild type as revealed by circular dichroism and NMR. Thermal and chemical unfolding data from circular dichroism and tryptophan fluorescence show significant decrease of stability for the mutants, and stopped-flow spectroscopy shows decreased refolding rates. The most deleterious single mutation is the N2912D replacement, although we observe additive effects of the two mutations on repeat stability. Based on three-dimensional structures built by homology molecular modeling, we discuss the modifications of the mutation-induced repeat stability. We conclude that the main forces involved in repeat stability are electrostatic inter-helix interactions that are disrupted following mutations. This study represents the first analysis at the protein level of the consequences of missense mutations in the human dystrophin rod domain. Our results suggest that it may participate in mechanical weakening of dystrophin-deficient muscle.

Sub-domains of the dystrophin rod domain display contrasting lipid-binding and stability properties

Dystrophin is a muscle scaffolding protein that establishes a structural link between the cytoskeleton and the extracellular matrix. Despite the large body of knowledge about the dystrophin gene and its interactions, the functional importance of the large central rod domain remains highly controversial. It is composed of 24 spectrin-like repeats interrupted by four hinges that delineate three sub-domains. We express repeat 1–3 and repeat 20–24 sub-domains, delineated by hinges 1–2 and 3–4 and the single repeats 2 and 23. We determine their lipid-binding properties, thermal and urea stabilities and refolding velocities. By using intrinsic tryptophan fluorescence spectroscopy and size exclusion chromatography, we show that repeat 2 and the repeat 1–3 sub-domain strongly interact with anionic lipids. By contrast, repeat 23 and the repeat 20–24 sub-domain do not interact with lipids. In addition, the repeat 1–3 sub-domain and repeat 2 are dramatically less stable and refold faster than the repeat 20–24 sub-domain and repeat 23. The contrasting properties of the two sub-domains clearly indicate that they make up two units of the rod domain that are not structurally interchangeable, thus providing molecular evidence supporting the observations on the biological function of dystrophin.

Impact de l'étude familiale sur le pronostic et le conseil génétique chez un enfant porteur d'une délétion des exons 50-51 du gène de la dystrophine

Introduction In frame deletions of exons encoding the central rod domain of dystrophin have been associated with a highly variable phenotype, including asymptomatic individuals. The lack of family history impairs accurate genetic counselling. Observation We report on a 4-year-old child suffering from transient episodes of limping at the age of 2 and several episodes of fall since the age of 3. Clinical examination did not show muscle weakness. CPK levels were increased (1300 UI). EMG was normal. Muscle histology showed a rhabdomyolysis without features of muscular dystrophy. Immunolabelling for dystrophin, merosin and dysferlin were normal. Western blot analysis of muscular proteins showed reduced-size dystrophin bands and a slightly reduced intensity for dystrophin, α and γ-sarcoglycan. Multiplex PCR of the dystrophin gene showed an in-frame deletion of exons 50-51, predicted to be associated to a Becker type of dystrophinopathy. Intragenic markers and quantitative PCR suggested maternal inheritance. This was confirmed by testing the maternal grand-parents, revealing that the asymptomatic 69-year-old grand father was a carrier. Three additional healthy males, whose ages ranged from 28 to 55 years and who were asymptomatic, also carried the mutation. The proband became spontaneously asymptomatic and cardiac echography was normal. In light of these data, genetic counselling was more reassuring and the mutation carrier maternal aunt, who was pregnant, decided to continue the pregnancy. Conclusion This case report emphasizes the importance of family molecular analysis, especially in males from the maternal lineage, for genetic counselling of dystrophinopathies associated to atypical features or to an isolate increase of muscular enzymes level in a young boy with no positive family history.

Proximal Dystrophin Gene Deletions and Protein Alterations in Becker Muscular Dystrophy

Alterations in production of cytoskeletal protein dystrophin caused by in-frame gene mutations lead to the Becker muscular dystrophy. In this study we analyzed genotype-phenotype correlation in a group of Becker muscular dystrophy patients with deletions affecting the proximal part of dystrophin gene, encompassing exons 3-13. Four patients with deletions affecting N terminal dystrophin domain had early onset and faster progression of the disease, while three patients with deletions in the proximal part of dystrophin's rod domain had a more benign disease course. Our study suggests that proximal gene deletions in Becker muscular dystrophy have various phenotypic effects depending on the affected domain of protein dystrophin.

Muscular dystrophy with truncated dystrophin in a family of Japanese Spitz dogs

Muscular dystrophy was diagnosed in seven male Japanese Spitz dogs with clinical signs of slowly progressive exercise intolerance, generalized weakness, myalgia, difficulty chewing and dysphagia. Serum creatine kinase (CK) concentrations were markedly elevated. Histopathology showed degeneration and regeneration of muscle, consistent with a dystrophic phenotype. Immunohistochemical staining for dystrophin and related proteins showed no staining with a monoclonal antibody against the rod domain of dystrophin but near-normal staining with an antibody against the C terminus. Immunoblot analysis in two affected dogs showed a truncated dystrophin protein of approximately 70–80 kDa. The severity of disease showed that this fragment was not large enough to protect from the dystrophic process.

Interaction of Dystrophin Rod Domain with Membrane Phospholipids: EVIDENCE OF A CLOSE PROXIMITY BETWEEN TRYPTOPHAN RESIDUES AND LIPIDS

Dystrophin is assumed to act via the central rod domain as a flexible linker between the amino-terminal actin binding domain and carboxyl-terminal proteins associated with the membrane. The rod domain is made up of 24 spectrin-like repeats and has been shown to modify the physical properties of lipid membranes. The nature of this association still remains unclear. Tryptophan residues tend to cluster at or near to the water-lipid interface of the membrane. To assess dystrophin rod domain-membrane interactions, tryptophan residues properties of two recombinant proteins of the rod domain were examined by (1)H NMR and fluorescence techniques in the presence of membrane lipids. F114 (residues 439-553) is a partly folded protein as inferred from (1)H NMR, tryptophan fluorescence emission intensity, and the excited state lifetime. By contrast, F125 (residues 439-564) is a folded compact protein. Tryptophan fluorescence quenching shows that both proteins are characterized by structural fluctuations with their tryptophan residues only slightly buried from the surface. In the presence of negatively charged small vesicles, the fluorescence characteristics of F125 change dramatically, indicating that tryptophan residues are in a more hydrophobic environment. Interestingly, these modifications are not observed with F114. Fluorescence quenching experiments confirm that tryptophan residues are shielded from the solvent in the complex F125 lipids by a close contact with lipids. The use of membrane-bound quenchers allowed us to conclude that dystrophin rod domain lies along the membrane surface and may be involved in a structural array comprising membrane and cytoskeletal proteins as well as membrane lipids.

Spectrin-like repeats from dystrophin and alpha-actinin-2 are not functionally interchangeable.

Mutations in the dystrophin gene result in Duchenne muscular dystrophy (DMD). Dystrophin is a multidomain protein that functions to stabilize the sarcolemmal membrane during muscle contraction. The central rod domain has been proposed to act as a shock absorber, as a force transducer or as a spacer separating important N- and C-terminal domains that interact with actin and the dystrophin-glycoprotein complex (DGC). Structure/function studies demonstrated that deletion of large portions of the rod domain can result in the production of smaller, yet highly functional, dystrophin proteins. In a dramatic example, a 'micro-dystrophin' transgene containing only four dystrophin spectrin-like repeats resulted in complete correction of most of the symptoms associated with dystrophy in the mdx mouse model for DMD. Dystrophin shares considerable homology with the multidomain, actin-crosslinking protein alpha-actinin. To explore the hypothesis that the dystrophin rod domain acts as a spacer region, a chimeric micro-dystrophin transgene containing the four-repeat rod domain of alpha-actinin-2 was expressed in mdx mice. This chimeric transgene was incapable of correcting the morphological pathology of the mdx mouse, but still functioned to assemble the DGC at the membrane and provided some protection from contraction-induced injury. These data demonstrated that different spectrin-like repeats are not equivalent, and reinforced the suggestion that the dystrophin rod domain is not merely a spacer but likely contributes an important mechanical role to overall dystrophin function.