Inhibition of Proteasome Activity Promotes the Correct Localization of Disease-Causing α-Sarcoglycan Mutants in HEK-293 Cells Constitutively Expressing β-, γ-, and δ-Sarcoglycan

Abstract

Sarcoglycanopathies are progressive muscle-wasting disorders caused by genetic defects of four proteins, ␣-, ␤-, ␥-, and ␦-sarcoglycan, which are elements of a key transmembrane complex of striated muscle. The proper assembly of the sarcoglycan complex represents a critical issue of sarcoglycanopathies, as several mutations severely perturb tetramer formation. Misfolded proteins are generally degraded through the cell's quality-control system; however, this can also lead to the removal of some functional polypeptides. To explore whether it is possible to rescue sarcoglycan mutants by preventing their degradation, we generated a heterologous cell system, based on human embryonic kidney (HEK) 293 cells, constitutively expressing three (␤, ␥, and ␦) of the four sarcoglycans. In these ␤␥␦-HEK cells, the lack of ␣-sarcoglycan prevented complex formation and cell surface localization, wheras the presence of ␣-sarcoglycan allowed maturation and targeting of the tetramer. As in muscles of sarcoglycanopathy patients, transfection of ␤␥␦-HEK cells with disease-causing ␣-sarcoglycan mutants led to dramatic reduction of the mutated proteins and the absence of the complex from the cell surface. Proteasomal inhibition reduced the degradation of mutants and facilitated the assembly and targeting of the sarcoglycan complex to the plasma membrane. These data provide important insights for the potential development of pharmacological therapies for sarcoglycanopathies.

Unique characteristics of Ca 2+ homeostasis of the trans -Golgi compartment

“…Seventytwo hours after transfection, cells were treated as described by Gastaldello et al (43). Proteins were resolved by SDS/PAGE, blotted onto a PVDF membrane, and probed with selected antibodies [rabbit polyclonal anti-SPCA1 (PMR1) (Santa Cruz Biotechnology), rabbit polyclonal anti-GFP (Abcam), and mouse monoclonal anti-β-actin (Sigma).…”

The ubiquitin ligase tripartite-motif-protein 32 is induced in Duchenne muscular dystrophy

“…[26][27][28][29][30][31][32][33] However, degradation of proteins by the ubiquitinproteasome is a complex ATP-dependent multistage process Abbreviations: Del, deletion; Hypercontr., hypercontracted fibers.…”

Overexpression of Galgt2 Reduces Dystrophic Pathology in the Skeletal Muscles of Alpha Sarcoglycan-Deficient Mice

“…For example, Bcl2 overexpression ameliorates disease in dy/dy mice but not in mdx mice, 1 ADAM12 overexpression is effective in mdx mice but not in dy/dy mice, 18,86 myostatin inhibition is effective in boosting muscle mass in mdx mice but not in dy/dy or Sgca Ϫ/Ϫ mice, 2-4,87,88 stimulation of calcineurin signaling is effective in mdx mice but not in dy/dy mice, 89 -91 and integrin ␣7B overexpression ameliorates disease in mdxutrn Ϫ/Ϫ mice but not in Scgd Ϫ/Ϫ mice. 14,92 By contrast, proteosome inhibitors appear to promote membrane localization of sarcoglycan complexes in Sgca-deficient cells 93 and have also been shown to be therapeutic in mdx mice. 94,95 Perhaps the most promising surrogate gene therapy thus far for LGMD2D has been transgenic overexpression of sarcoglycan, which appears to inhibit muscular dystrophy for many months and increase expression of ␤-␦ sarcoglycan on the muscle membrane.…”