Dataset Information

Disease-proportional proteasomal degradation of missense dystrophins.

ABSTRACT: The 427-kDa protein dystrophin is expressed in striated muscle where it physically links the interior of muscle fibers to the extracellular matrix. A range of mutations in the DMD gene encoding dystrophin lead to a severe muscular dystrophy known as Duchenne (DMD) or a typically milder form known as Becker (BMD). Patients with nonsense mutations in dystrophin are specifically targeted by stop codon read-through drugs, whereas out-of-frame deletions and insertions are targeted by exon-skipping therapies. Both treatment strategies are currently in clinical trials. Dystrophin missense mutations, however, cause a wide range of phenotypic severity in patients. The molecular and cellular consequences of such mutations are not well understood, and there are no therapies specifically targeting this genotype. Here, we have modeled two representative missense mutations, L54R and L172H, causing DMD and BMD, respectively, in full-length dystrophin. In vitro, the mutation associated with the mild phenotype (L172H) caused a minor decrease in tertiary stability, whereas the L54R mutation associated with a severe phenotype had a more dramatic effect. When stably expressed in mammalian muscle cells, the mutations caused steady-state decreases in dystrophin protein levels inversely proportional to the tertiary stability and directly caused by proteasomal degradation. Both proteasome inhibitors and heat shock activators were able to increase mutant dystrophin to WT levels, establishing the new cell lines as a platform to screen for potential therapeutics personalized to patients with destabilized dystrophin.

SUBMITTER: Talsness DM

PROVIDER: S-EPMC4603481 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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Disease-proportional proteasomal degradation of missense dystrophins.

Talsness Dana M DM Belanto Joseph J JJ Ervasti James M JM

Proceedings of the National Academy of Sciences of the United States of America 20150921 40

The 427-kDa protein dystrophin is expressed in striated muscle where it physically links the interior of muscle fibers to the extracellular matrix. A range of mutations in the DMD gene encoding dystrophin lead to a severe muscular dystrophy known as Duchenne (DMD) or a typically milder form known as Becker (BMD). Patients with nonsense mutations in dystrophin are specifically targeted by stop codon read-through drugs, whereas out-of-frame deletions and insertions are targeted by exon-skipping th ...[more]

PMID: 26392559

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Project description:ContextThe pathogenic effect of mutations in the aryl hydrocarbon receptor interacting protein (AIP) gene (AIPmuts) in pituitary adenomas is incompletely understood. We have identified the primary mechanism of loss of function for missense AIPmuts.ObjectiveThis study sought to analyze the mechanism/speed of protein turnover of wild-type and missense AIP variants, correlating protein half-life with clinical parameters.Design and settingHalf-life and protein-protein interaction experiments and cross-sectional analysis of AIPmut positive patients' data were performed in a clinical academic research institution.PatientsData were obtained from our cohort of pituitary adenoma patients and literature-reported cases.InterventionsProtein turnover of endogenous AIP in two cell lines and fifteen AIP variants overexpressed in HEK293 cells was analyzed via cycloheximide chase and proteasome inhibition. Glutathione-S-transferase pull-down and quantitative mass spectrometry identified proteins involved in AIP degradation; results were confirmed by coimmunoprecipitation and gene knockdown. Relevant clinical data was collected.Main outcome measuresHalf-life of wild-type and mutant AIP proteins and its correlation with clinical parameters.ResultsEndogenous AIP half-life was similar in HEK293 and lymphoblastoid cells (43.5 and 32.7 h). AIP variants were divided into stable proteins (median, 77.7 h; interquartile range [IQR], 60.7-92.9 h), and those with short (median, 27 h; IQR, 21.6-28.7 h) or very short (median, 7.7 h; IQR, 5.6-10.5 h) half-life; proteasomal inhibition rescued the rapid degradation of mutant proteins. The experimental half-life significantly correlated with age at diagnosis of acromegaly/gigantism (r = 0.411; P = .002). The FBXO3-containing SKP1-CUL1-F-box protein complex was identified as the E3 ubiquitin-ligase recognizing AIP.ConclusionsAIP is a stable protein, driven to ubiquitination by the SKP1-CUL1-F-box protein complex. Enhanced proteasomal degradation is a novel pathogenic mechanism for AIPmuts, with direct implications for the phenotype.

Dataset Information

Disease-proportional proteasomal degradation of missense dystrophins.

Publications

Disease-proportional proteasomal degradation of missense dystrophins.

Similar Datasets

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