Comparative gel‐based proteomic analysis of chemically crosslinked complexes in dystrophic skeletal muscle

Abstract

Duchenne muscular dystrophy is a highly progressive muscle wasting disease with a complex pathophysiology that is based on primary abnormalities in the dystrophin gene. In order to study potential changes in the oligomerization of high‐molecular‐mass protein complexes in dystrophic skeletal muscle, chemical crosslinking was combined with mass spectrometric analysis. The biochemical stabilization of protein interactions was carried out with the homo‐bifunctional and amine‐reactive agent bis[sulfosuccinimidyl]suberate, followed by protein shift analysis in one‐dimensional gels. The proteomic approach identified 11 and 15 protein species in wild type versus dystrophic microsomal fractions, respectively, as well as eight common proteins, with an electrophoretic mobility shift to very high molecular mass following chemical crosslinking. In dystrophin‐deficient preparations, several protein species with an increased tendency of oligomerisation were identified as components of the sarcolemma and its associated intra‐ and extracellular structures, as well as mitochondria. This included the sarcolemmal proteins myoferlin and caveolin, the cytoskeletal components vimentin and tubulin, extracellular collagen alpha‐1(XII) and the mitochondrial trifunctional enzyme and oxoglutarate dehydrogenase. These changes are probably related to structural and metabolic adaptations, especially cellular repair processes, which agrees with the increased oligomerisation of myosin‐3, myosin‐9 and actin, and their role in cellular regeneration and structural adjustments in dystrophinopathy.

Vimentin regulates Notch signaling strength and arterial remodeling in response to hemodynamic stress

“…Importantly, overexpression of Jagged1 has been shown to rescue the Duchenne Muscular Dystrophy Phenotype, caused by a mutation in the dystrophin gene 74 . Dystrophin links the cytoskeleton to the extracellular matrix and has been implicated in adaptive responses to chronic changes in blood flow 75,76 . A potential link between dystrophin, desmin, vimentin, and Jagged1 should be given further consideration.…”

The Dystrophin Node as Integrator of Cytoskeletal Organization, Lateral Force Transmission, Fiber Stability and Cellular Signaling in Skeletal Muscle

“…A crucial issue with this method is the potential occurrence of random crosslinking events. However, this can be avoided by employing a low ratio of crosslinking agent to protein and using proper conditions in relation to length of incubation time, efficient quenching of the crosslinking reaction, temperature, buffering and pH-value [ 112 ]. Chemical crosslinking has been established as an excellent method in proteomics for studying sensitive protein–protein interactions [ 113 ].…”

“…Figure 4 outlines how a chemical crosslinking mass spectrometry (XL-MS) approach can be employed to study novel protein–protein interaction patterns, which are indirectly linked to dystrophin expression levels [ 112 ]. A variety of highly useful bioinformatic programs are available for the visualization of potential protein–protein interactions [ 120 ], as shown in the below images, which has been carried out with the STRING program [ 121 ].…”

Emerging proteomic biomarkers of X-linked muscular dystrophy

“…Potential compensatory mechanisms that are based on the up-regulation of cytoskeletal elements were confirmed by mass spectrometry. A drastically increased vimentin and desmin concentration seems to be a reliable indicator of cytoskeletal restructuring in Dp427-deficient fibres [51,52,54,55,57,58,63,66,67,77]. The fact that high levels of cellular stress is associated with muscular dystrophy was verified by the proteomic Table 2.…”

“…In contrast, integral membrane proteins, very large protein species and molecules with extreme isoelectric points are often underrepresented in 2Dgel systems. These types of muscle proteins are more appropriately analyzed by one-dimensional gel electrophoretic techniques that can be combined with on-membrane digestion and mass spectrometry [34,43,72,77] or affinity purification and immunoprecipitation approaches [39,41,44,77]. Alternatively, skeletal muscle proteins that are present in crude tissue extracts or subcellular fractions can be conveniently analyzed by bottom-up proteomics, which employs trypsination of total protein complements in combination with liquid chromatography and mass spectrometry [30,45,56,60,62,64,[66][67][68][69]71,[73][74][75][76]78].…”