Identification of thiostrepton as a pharmacological approach to rescue misfolded alpha-sarcoglycan mutant proteins from degradation
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ABSTRACT:
PROVIDER: PRJNA490439 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR7824180.fastq.gz | Fastqsanger.gz | |||
| SRR7824181.fastq.gz | Fastqsanger.gz | |||
| SRR7824182.fastq.gz | Fastqsanger.gz | |||
| SRR7824173.fastq.gz | Fastqsanger.gz | |||
| SRR7824183.fastq.gz | Fastqsanger.gz |
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Project description:Limb-girdle muscular dystrophies type 2D (LGMD2D) are characterized by a progressive proximal muscle weakness. LGMD2D is due to mutations in the gene encoding α sarcoglycan (-SG), a dystrophin-associated glycoprotein playing a key role in the maintenance of sarcolemma integrity in striated muscles. Here, we report the development of a new in vitro high-throughput screening assay that allows monitoring the proper localization of the most prevalent mutant form of -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 thiostrepton effect revealed a positive impact on R77C--SG and other missense mutant proteins (R34H, I124T, V247M) localization 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 24h following thiostrepton treatment and a synergic effect with bortezomib, a FDA-approved proteasome inhibitor. This study reports the first in vitro model of LGMD2D compatible with high throughput screening and propose a new therapeutical option for LGMD2D caused by missense mutations of -SG.
2019-09-19 | GSE119841 | GEO
Project description: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.
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Project description:Cells maintain proteostasis by selectively recognizing and targeting misfolded proteins for degradation. In Saccharomyces cerevisiae, the Hsp70 nucleotide exchange factor Fes1 is essential for the degradation of chaperone-associated misfolded proteins by the ubiquitin-proteasome system. Here we show that the FES1 transcript undergoes unique 3' alternative splicing that results in two equally active isoforms with alternative C-termini, Fes1L and Fes1S. Fes1L is actively targeted to the nucleus and represents the first identified nuclear Hsp70 nucleotide exchange factor. In contrast, Fes1S localizes to the cytosol and is essential to maintain proteostasis. In the absence of Fes1S, the heat-shock response is constitutively induced at normally non-stressful conditions. Moreover, cells display severe growth defects when elevated temperatures, amino acid analogues or the ectopic expression of misfolded proteins, induce protein misfolding. Importantly, misfolded proteins are not targeted for degradation by the ubiquitin-proteasome system. These observations support the notion that cytosolic Fes1S maintains proteostasis by supporting the removal of toxic misfolded proteins by proteasomal degradation. This study provides key findings for the understanding of the organization of protein quality control mechanisms in the cytosol and nucleus. 4 strains (WT, fes1Î, fes1ÎS, fes1ÎL) were sequenced in triplicates from independent RNA isolations.
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Project description:Cells maintain proteostasis by selectively recognizing and targeting misfolded proteins for degradation. In Saccharomyces cerevisiae, the Hsp70 nucleotide exchange factor Fes1 is essential for the degradation of chaperone-associated misfolded proteins by the ubiquitin-proteasome system. Here we show that the FES1 transcript undergoes unique 3' alternative splicing that results in two equally active isoforms with alternative C-termini, Fes1L and Fes1S. Fes1L is actively targeted to the nucleus and represents the first identified nuclear Hsp70 nucleotide exchange factor. In contrast, Fes1S localizes to the cytosol and is essential to maintain proteostasis. In the absence of Fes1S, the heat-shock response is constitutively induced at normally non-stressful conditions. Moreover, cells display severe growth defects when elevated temperatures, amino acid analogues or the ectopic expression of misfolded proteins, induce protein misfolding. Importantly, misfolded proteins are not targeted for degradation by the ubiquitin-proteasome system. These observations support the notion that cytosolic Fes1S maintains proteostasis by supporting the removal of toxic misfolded proteins by proteasomal degradation. This study provides key findings for the understanding of the organization of protein quality control mechanisms in the cytosol and nucleus.
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Project description:Cystic Fibrosis (CF) is a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR). Though most people with CF have one or two copies of the ΔF508 mutation, there are hundreds of other distinct CF mutations that vary in their mechanistic effects and response to therapeutics. Endogenous chaperones are known to have divergent effects on the druggability of CF variants. Nevertheless, it remains unclear how this proteostatic modulation is related to the underlying mechanistic effects of distinct classes of CF mutations. Here, we survey the effects of a previously discovered effector (calnexin, CANX) on the expression and pharmacological rescue of 235 CF variants using deep mutational scanning. We find that CANX is generally required for robust plasma membrane expression of the CFTR protein- particularly for CF variants that perturb its second nucleotide binding domain. CANX also appears to be critical for the pharmacological rescue of CF variants with poor basal expression. Though corrector selectivity is generally dictated by the properties of mutations, we find that CANX enhances the sensitivity of CF variants within a domain swapped region of membranes spanning domain 2 to the type III corrector VX-445. Together, our findings suggest CANX modulates the later stages of CFTR assembly and disproportionately affects variants bearing mutations within the C-terminal domains. Interestingly, we find that the proteostatic effects of CANX are generally decoupled from changes in CFTR activity. Together, our findings reveal how the proteostasis machinery may shape the variant-specific effects of corrector molecules.
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