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Identification of thiostrepton as a pharmacological approach to rescue misfolded alpha-sarcoglycan mutant proteins from degradation.

ABSTRACT: Limb-girdle muscular dystrophy type 2D (LGMD2D) is characterized by a progressive proximal muscle weakness. LGMD2D is caused by mutations in the gene encoding α-sarcoglycan (α-SG), a dystrophin-associated glycoprotein that plays a key role in the maintenance of sarcolemma integrity in striated muscles. We report here on the development of a new in vitro high-throughput screening assay that allows the monitoring of the proper localization of the most prevalent mutant form of α-SG (R77C substitution). Using this assay, we screened a library of 2560 FDA-approved drugs and bioactive compounds and identified thiostrepton, a cyclic antibiotic, as a potential drug to repurpose for LGMD2D treatment. Characterization of the thiostrepton effect revealed a positive impact on R77C-α-SG and other missense mutant protein localization (R34H, I124T, V247M) in fibroblasts overexpressing these proteins. Finally, further investigations of the molecular mechanisms of action of the compound revealed an inhibition of the chymotrypsin-like activity of the proteasome 24 h after thiostrepton treatment and a synergistic effect with bortezomib, an FDA-approved proteasome inhibitor. This study reports on the first in vitro model for LGMD2D that is compatible with high-throughput screening and proposes a new therapeutic option for LGMD2D caused by missense mutations of α-SG.

SUBMITTER: Hoch L

PROVIDER: S-EPMC6502821 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Identification of thiostrepton as a pharmacological approach to rescue misfolded alpha-sarcoglycan mutant proteins from degradation.

Hoch Lucile L Henriques Sara F SF Bruge Celine C Marsolier Justine J Benabides Manon M Bourg Nathalie N Tournois Johana J Mahé Gurvan G Morizur Lise L Jarrige Margot M Bigot Anne A Richard Isabelle I Nissan Xavier X

Scientific reports 20190506 1

Limb-girdle muscular dystrophy type 2D (LGMD2D) is characterized by a progressive proximal muscle weakness. LGMD2D is caused by mutations in the gene encoding α-sarcoglycan (α-SG), a dystrophin-associated glycoprotein that plays a key role in the maintenance of sarcolemma integrity in striated muscles. We report here on the development of a new in vitro high-throughput screening assay that allows the monitoring of the proper localization of the most prevalent mutant form of α-SG (R77C substituti ...[more]

PMID: 31061434

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Project description:BackgroundProteasome functional insufficiency is implicated in a large subset of cardiovascular diseases and may play an important role in their pathogenesis. The regulation of proteasome function is poorly understood, hindering the development of effective strategies to improve proteasome function.Methods and resultsProtein kinase G (PKG) was manipulated genetically and pharmacologically in cultured cardiomyocytes. Activation of PKG increased proteasome peptidase activities, facilitated proteasome-mediated degradation of surrogate (enhanced green fluorescence protein modified by carboxyl fusion of degron CL1) and bona fide (CryAB(R120G)) misfolded proteins, and attenuated CryAB(R120G) overexpression-induced accumulation of ubiquitinated proteins and cellular injury. PKG inhibition elicited the opposite responses. Differences in the abundance of the key 26S proteasome subunits Rpt6 and β5 between the PKG-manipulated and control groups were not statistically significant, but the isoelectric points were shifted by PKG activation. In transgenic mice expressing a surrogate substrate (GFPdgn), PKG activation by sildenafil increased myocardial proteasome activities and significantly decreased myocardial GFPdgn protein levels. Sildenafil treatment significantly increased myocardial PKG activity and significantly reduced myocardial accumulation of CryAB(R120G), ubiquitin conjugates, and aberrant protein aggregates in mice with CryAB(R120G)-based desmin-related cardiomyopathy. No discernible effect on bona fide native substrates of the ubiquitin-proteasome system was observed from PKG manipulation in vitro or in vivo.ConclusionsPKG positively regulates proteasome activities and proteasome-mediated degradation of misfolded proteins, likely through posttranslational modifications to proteasome subunits. This may be a new mechanism underlying the benefit of PKG stimulation in treating cardiac diseases. Stimulation of PKG by measures such as sildenafil administration is potentially a new therapeutic strategy to treat cardiac proteinopathies.

Dataset Information

Identification of thiostrepton as a pharmacological approach to rescue misfolded alpha-sarcoglycan mutant proteins from degradation.

Publications

Identification of thiostrepton as a pharmacological approach to rescue misfolded alpha-sarcoglycan mutant proteins from degradation.

Similar Datasets

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