RESUMO
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 highmolecularmass protein assembly in skeletal muscles. The almost complete loss of the dystrophin isoform Dp427M and concomitant reduction in the dystrophinassociated 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 dystrophinglycoprotein complex, in this study, we used extensive subcellular fractionation and enrichment procedures prior to subproteomic analysis. Mass spectrometry identified high levels of fulllength dystrophin isoform Dp427M, α/ßdystroglycans, α/ß/γ/δsarcoglycans, α1/ß1/ß2syntrophins and α/ßdystrobrevins 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 onmembrane digestion. This involved the subproteomic assessment of various ionregulatory proteins and excitationcontraction coupling components. The comparative profiling of skeletal muscle fractions established a relatively restricted subcellular localization of the dystrophinglycoprotein complex in the muscle fibre periphery by proteomic means and clearly demonstrated the absence of dystrophin from triad junctions by sensitive mass spectrometric analysis.
Assuntos
Proteínas Associadas à Distrofina/isolamento & purificação , Distrofina/isolamento & purificação , Sarcoglicanas/isolamento & purificação , Sarcolema/química , Retículo Sarcoplasmático/química , Animais , Acoplamento Excitação-Contração/fisiologia , Espectrometria de Massas/métodos , Músculo Esquelético/química , Músculo Esquelético/metabolismo , Músculo Esquelético/ultraestrutura , Isoformas de Proteínas/isolamento & purificação , Coelhos , Sarcolema/metabolismo , Sarcolema/ultraestrutura , Retículo Sarcoplasmático/metabolismo , Retículo Sarcoplasmático/ultraestruturaRESUMO
ß2-syntrophin, a dystrophin-associated protein, plays a pivotal role in insulin secretion by pancreatic ß-cells. It contains a PDZ domain (ß2S-PDZ) that, in complex with protein-tyrosine phosphatase ICA512, anchors the dense insulin granules to actin filaments. The phosphorylation state of ß2-syntrophin allosterically regulates the affinity of ß2S-PDZ for ICA512, and the disruption of the complex triggers the mobilization of the insulin granule stores. Here, we investigate the thermal unfolding of ß2S-PDZ at different pH and urea concentrations. Our results indicate that, unlike other PDZ domains, ß2S-PDZ is marginally stable. Thermal denaturation experiments show broad transitions and cold denaturation, and a two-state model fit reveals a significant unfolded fraction under physiological conditions. Furthermore, T(m) and T(max) denaturant-dependent shifts and noncoincidence of melting curves monitored at different wavelengths suggest that two-state and three-state models fail to explain the equilibrium data properly and are in better agreement with a downhill scenario. Its higher stability at pH >9 and the results of molecular dynamics simulations indicate that this behavior of ß2S-PDZ might be related to its charge distribution. All together, our results suggest a link between the conformational plasticity of the native ensemble of this PDZ domain and the regulation of insulin secretion.