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The biochemical and mass spectrometric profiling of the dystrophin complexome from skeletal muscle.

ABSTRACT: The development of advanced mass spectrometric methodology has decisively enhanced the analytical capabilities for studies into the composition and dynamics of multi-subunit protein complexes and their associated components. Large-scale complexome profiling is an approach that combines the systematic isolation and enrichment of protein assemblies with sophisticated mass spectrometry-based identification methods. In skeletal muscles, the membrane cytoskeletal protein dystrophin of 427 kDa forms tight interactions with a variety of sarcolemmal, cytosolic and extracellular proteins, which in turn associate with key components of the extracellular matrix and the intracellular cytoskeleton. A major function of this enormous assembly of proteins, including dystroglycans, sarcoglycans, syntrophins, dystrobrevins, sarcospan, laminin and cortical actin, is postulated to stabilize muscle fibres during the physical tensions of continuous excitation-contraction-relaxation cycles. This article reviews the evidence from recent proteomic studies that have focused on the characterization of the dystrophin-glycoprotein complex and its central role in the establishment of the cytoskeleton-sarcolemma-matrisome axis. Proteomic findings suggest a close linkage of the core dystrophin complex with a variety of protein species, including tubulin, vimentin, desmin, annexin, proteoglycans and collagens. Since the almost complete absence of dystrophin is the underlying cause for X-linked muscular dystrophy, a more detailed understanding of the composition, structure and plasticity of the dystrophin complexome may have considerable biomedical implications.

SUBMITTER: Murphy S

PROVIDER: S-EPMC4688399 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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The biochemical and mass spectrometric profiling of the dystrophin complexome from skeletal muscle.

Murphy Sandra S Ohlendieck Kay K

Computational and structural biotechnology journal 20151126

The development of advanced mass spectrometric methodology has decisively enhanced the analytical capabilities for studies into the composition and dynamics of multi-subunit protein complexes and their associated components. Large-scale complexome profiling is an approach that combines the systematic isolation and enrichment of protein assemblies with sophisticated mass spectrometry-based identification methods. In skeletal muscles, the membrane cytoskeletal protein dystrophin of 427 kDa forms t ...[more]

PMID: 26793286

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Project description:Supramolecular membrane complexes of low abundance are difficult to study by routine bioanalytical techniques. The plasmalemmal complex consisting of sarcoglycans, dystroglycans, dystrobrevins and syntrophins, which is closely associated with the membrane cytoskeletal protein dystrophin, represents such a high?molecular?mass protein assembly in skeletal muscles. The almost complete loss of the dystrophin isoform Dp427?M and concomitant reduction in the dystrophin?associated glycoprotein complex is the underlying cause of the highly progressive neuromuscular disorder named Duchenne muscular dystrophy. This gives the detailed characterization of the dystrophin complex considerable pathophysiological importance. In order to carry out a comprehensive mass spectrometric identification of the dystrophin?glycoprotein complex, in this study, we used extensive subcellular fractionation and enrichment procedures prior to subproteomic analysis. Mass spectrometry identified high levels of full?length dystrophin isoform Dp427?M, ?/??dystroglycans, ?/?/?/??sarcoglycans, ?1/?1/?2?syntrophins and ?/??dystrobrevins in highly purified sarcolemma vesicles. By contrast, lower levels were detected in transverse tubules and no components of the dystrophin complex were identified in triads. For comparative purposes, the presence of organellar marker proteins was studied in crude surface membrane preparations vs. enriched fractions from the sarcolemma, transverse tubules and triad junctions using gradient gel electrophoresis and on?membrane digestion. This involved the subproteomic assessment of various ion?regulatory proteins and excitation?contraction coupling components. The comparative profiling of skeletal muscle fractions established a relatively restricted subcellular localization of the dystrophin?glycoprotein complex in the muscle fibre periphery by proteomic means and clearly demonstrated the absence of dystrophin from triad junctions by sensitive mass spectrometric analysis.

Project description:BackgroundWe are developing a novel therapy for Duchenne muscular dystrophy (DMD), involving the transplantation of autologous, skeletal muscle-derived stem cells that have been genetically corrected to express dystrophin. Dystrophin is normally expressed in activated satellite cells and in differentiated muscle fibres. However, in past preclinical validation studies, dystrophin transgenes have generally been driven by constitutive promoters that would be active at every stage of the myogenic differentiation process, including in proliferating muscle stem cells. It is not known whether artificial dystrophin expression would affect the properties of these cells.AimsOur aims are to determine if mini-dystrophin expression affects the proliferation or myogenic differentiation of DMD skeletal muscle-derived cells.MethodsSkeletal muscle-derived cells from a DMD patient were transduced with lentivirus coding for mini-dystrophins (R3-R13 spectrin-like repeats (ΔR3R13) or hinge2 to spectrin-like repeats R23 (ΔH2R23)) with EGFP (enhanced green fluorescence protein) fused to the C-terminus, driven by a constitutive promoter, spleen focus-forming virus (SFFV). Transduced cells were purified on the basis of GFP expression. Their proliferation and myogenic differentiation were quantified by ethynyl deoxyuridine (EdU) incorporation and fusion index. Furthermore, dystrophin small interfering ribonucleic acids (siRNAs) were transfected to the cells to reverse the effects of the mini-dystrophin. Finally, a phospho-mitogen-activated protein kinase (MAPK) array assay was performed to investigate signalling pathway changes caused by dystrophin expression.ResultsCell proliferation was not affected in cells transduced with ΔR3R13, but was significantly increased in cells transduced with ΔH2R23. The fusion index of myotubes derived from both ΔR3R13- and ΔH2R23 -expressing cells was significantly compromised in comparison to myotubes derived from non-transduced cells. Dystrophin siRNA transfection restored the differentiation of ΔH2R23-expressing cells. The Erk1/2- signalling pathway is altered in cells transduced with mini-dystrophin constructs.ConclusionsEctopic expression of dystrophin in cultured human skeletal muscle-derived cells may affect their proliferation and differentiation capacity. Caution should be taken when considering genetic correction of autologous stem cells to express dystrophin driven by a constitutive promoter.

Project description:Mutations in the genes coding for either dystrophin or dysferlin cause distinct forms of muscular dystrophy. Dystrophin links the cytoskeleton to the sarcolemma through direct interaction with ?-dystroglycan. This link extends to the extracellular matrix by ?-dystroglycan's interaction with ?-dystroglycan, which binds extracellular matrix proteins, including laminin ?2, agrin and perlecan, that possess laminin globular domains. The absence of dystrophin disrupts this link, leading to compromised muscle sarcolemmal integrity. Dysferlin, on the other hand, plays an important role in the Ca2+-dependent membrane repair of damaged sarcolemma in skeletal muscle. Because dysferlin and dystrophin play different roles in maintaining muscle cell integrity, we hypothesized that disrupting sarcolemmal integrity with dystrophin deficiency would exacerbate the pathology in dysferlin-null mice and allow further characterization of the role of dysferlin in skeletal muscle.To test our hypothesis, we generated dystrophin/dysferlin double-knockout (DKO) mice by breeding mdx mice with dysferlin-null mice and analyzed the effects of a combined deficiency of dysferlin and dystrophin on muscle pathology and sarcolemmal integrity.The DKO mice exhibited more severe muscle pathology than either mdx mice or dysferlin-null mice, and, importantly, the onset of the muscle pathology occurred much earlier than it did in dysferlin-deficient mice. The DKO mice showed muscle pathology of various skeletal muscles, including the mandible muscles, as well as a greater number of regenerating muscle fibers, higher serum creatine kinase levels and elevated Evans blue dye uptake into skeletal muscles. Lengthening contractions caused similar force deficits, regardless of dysferlin expression. However, the rate of force recovery within 45 minutes following lengthening contractions was hampered in DKO muscles compared to mdx muscles or dysferlin-null muscles, suggesting that dysferlin is required for the initial recovery from lengthening contraction-induced muscle injury of the dystrophin-glycoprotein complex-compromised muscles.The results of our study suggest that dysferlin-mediated membrane repair helps to limit the dystrophic changes in dystrophin-deficient skeletal muscle. Dystrophin deficiency unmasks the function of dysferlin in membrane repair during lengthening contractions. Dystrophin/dysferlin-deficient mice provide a very useful model with which to evaluate the effectiveness of therapies designed to treat dysferlin deficiency.

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The biochemical and mass spectrometric profiling of the dystrophin complexome from skeletal muscle.

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The biochemical and mass spectrometric profiling of the dystrophin complexome from skeletal muscle.

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