tailieunhanh - Báo cáo khoa học: Subproteomics analysis of Ca2+-binding proteins demonstrates decreased calsequestrin expression in dystrophic mouse skeletal muscle

Duchenne muscular dystrophy represents one of the most common hereditary diseases. Abnormal ion handling is believed to render dystrophin-deficient muscle fibres more susceptible to necrosis. Although a reduced Ca 2+ buffering capacity has been shown to exist in the dystrophic sarco-plasmic reticulum, surprisingly no changes in the abundance of the main luminal Ca 2+ reservoir protein calsequestrin have been observed inmicrosomal preparations. | Eur. J. Biochem. 271 3943-3952 2004 FEBS 2004 doi Subproteomics analysis of Ca2 -binding proteins demonstrates decreased calsequestrin expression in dystrophic mouse skeletal muscle Philip Doran1 Paul Dowling1 James Lohan1 Karen McDonnell1 Stephan Poetsch2 and Kay Ohlendieck1 1Department of Biology National University of Ireland Maynooth County Kildare Ireland 2GE Healthcare Bio-Science Freiburg Germany Duchenne muscular dystrophy represents one of the most common hereditary diseases. Abnormal ion handling is believed to render dystrophin-deficient muscle fibres more susceptible to necrosis. Although a reduced Ca2 buffering capacity has been shown to exist in the dystrophic sarcoplasmic reticulum surprisingly no changes in the abundance of the main luminal Ca2 reservoir protein calsequestrin have been observed in microsomal preparations. To address this unexpected finding and eliminate potential technical artefacts of subcellular fractionation protocols we employed a comparative subproteomics approach with total mouse skeletal muscle extracts. Immunoblotting mass spectrometry and labelling of the entire muscle protein complement with the cationic carbocyanine dye Stains-All was performed in order to evaluate the fate of major Ca2 -binding proteins in dystrophin-deficient skeletal muscle fibres. In contrast to a relatively comparable expression pattern of the main protein population in normal vs. dystrophic fibres our analysis showed that the expression of key Ca2 -binding proteins of the luminal sarcoplasmic reticulum is drastically reduced. This included the main terminal cisternae constituent calsequestrin and the previously implicated Ca2 -shuttle element sarcalumenin. In contrast the Stains-All -positive protein spot representing the cytosolic Ca2 -binding component calmodulin was not changed in dystrophin-deficient fibres. The reduced 2D Stains-All pattern of luminal Ca2 -binding proteins in mdx preparations supports the .