Abstract
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.
Highlights
Out-of-frame mutations in the DMD gene cause Duchenne muscular dystrophy (DMD), characterized by lack of dystrophin expression and severe phenotype; while in-frame DMD mutations cause Becker muscular dystrophy (BMD), with reduced protein expression and milder phenotype
Disease onset, and rate of progression has been observed in BMD patients; this, rather than to the variable amount of dystrophin produced [5], seems more related to the marked heterogeneity of gene mutations that results in many abnormal dystrophin proteins with different expression patterns and residual function [6,7]
Searching for possible key elements that could help to elucidate which proteins might contribute to improving muscle function in BMD patients, we reanalysed some of the BMD muscle extracts from our recent study on proteomic characterization of BMD and DMD muscle biopsies [11], subdividing them into two groups, BMD1 and BMD2, based on the presence or absence of the D8 antibody reactivity, respectively
Summary
Out-of-frame mutations in the DMD gene cause Duchenne muscular dystrophy (DMD), characterized by lack of dystrophin expression and severe phenotype; while in-frame DMD mutations cause Becker muscular dystrophy (BMD), with reduced protein expression and milder phenotype. Using a homemade anti-dystrophin monoclonal antibody, called D8, and commercial antidystrophin monoclonal antibodies in our routine diagnostic workup, we found that almost one-third of our BMD patients lacked positivity the D8 antibody. The latter was obtained utilizing the fusion peptide containing the portion of dystrophin corresponding to amino acids 2334–2626 (D8 region) of the original dystrophin sequence [10]. BMD patients were characterized by reduced and uneven dystrophin expression at the muscle fiber surface, assessed by immunohistochemistry, and a reduced amount of dystrophin with expected, or slightly increased MW, detected by SDS-PAGE and immunoblotting, as reported in the literature [8,9]. Searching for possible key elements that could help to elucidate which proteins might contribute to improving muscle function in BMD patients, we reanalysed some of the BMD muscle extracts from our recent study on proteomic characterization of BMD and DMD muscle biopsies [11], subdividing them into two groups, BMD1 and BMD2, based on the presence or absence of the D8 antibody reactivity, respectively
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