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Caspase-dependent regulation of the ubiquitin-proteasome system through direct substrate targeting.

ABSTRACT: Drosophila inhibitor of apoptosis (IAP) 1 (DIAP1) is an E3 ubiquitin ligase that regulates apoptosis in flies, in large part through direct inhibition and/or ubiquitinylation of caspases. IAP antagonists, such as Reaper, Hid, and Grim, are thought to induce cell death by displacing active caspases from baculovirus IAP repeat domains in DIAP1, but can themselves become targets of DIAP1-mediated ubiquitinylation. Herein, we demonstrate that Grim self-associates in cells and is ubiquitinylated by DIAP1 at Lys136 in an UbcD1-dependent manner, resulting in its rapid turnover. K48-linked ubiquitin chains are added almost exclusively to BIR2-bound Grim as a result of its structural proximity to DIAP1's RING domain. However, active caspases can simultaneously cleave Grim at Asp132, removing the lysine necessary for ubiquitinylation as well as any existing ubiquitin conjugates. Cleavage therefore enhances the stability of Grim and initiates a feed-forward caspase amplification loop, resulting in greater cell death. In summary, Grim is a caspase substrate whose cleavage promotes apoptosis by limiting, in a target-specific fashion, its ubiquitinylation and turnover by the proteasome.

SUBMITTER: Yeh TC

PROVIDER: S-EPMC3761634 | biostudies-other | 2013 Aug

REPOSITORIES: biostudies-other

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Caspase-dependent regulation of the ubiquitin-proteasome system through direct substrate targeting.

Yeh Ting-Chun TC Bratton Shawn B SB

Proceedings of the National Academy of Sciences of the United States of America 20130812 35

Drosophila inhibitor of apoptosis (IAP) 1 (DIAP1) is an E3 ubiquitin ligase that regulates apoptosis in flies, in large part through direct inhibition and/or ubiquitinylation of caspases. IAP antagonists, such as Reaper, Hid, and Grim, are thought to induce cell death by displacing active caspases from baculovirus IAP repeat domains in DIAP1, but can themselves become targets of DIAP1-mediated ubiquitinylation. Herein, we demonstrate that Grim self-associates in cells and is ubiquitinylated by D ...[more]

PMID: 23940367

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Project description:BackgroundMuscle atrophy, leading to muscular dysfunction and weakness, is an adverse outcome of sustained period of glucocorticoids usage. However, the molecular mechanism underlying this detrimental condition is currently unclear. Pyruvate dehydrogenase kinase 4 (PDK4), a central regulator of cellular energy metabolism, is highly expressed in skeletal muscle and has been implicated in the pathogenesis of several diseases. The current study was designed to investigated and delineate the role of PDK4 in the context of muscle atrophy, which could be identified as a potential therapeutic avenue to protect against dexamethasone-induced muscle wasting.MethodsThe dexamethasone-induced muscle atrophy in C2C12 myotubes was evaluated at the molecular level by expression of key genes and proteins involved in myogenesis, using immunoblotting and qPCR analyses. Muscle dysfunction was studied in vivo in wild-type and PDK4 knockout mice treated with dexamethasone (25 mg/kg body weight, i.p., 10 days). Body weight, grip strength, muscle weight and muscle histology were assessed. The expression of myogenesis markers were analysed using qPCR, immunoblotting and immunoprecipitation. The study was extended to in vitro human skeletal muscle atrophy analysis.ResultsKnockdown of PDK4 was found to prevent glucocorticoid-induced muscle atrophy and dysfunction in C2C12 myotubes, which was indicated by induction of myogenin (0.3271 ± 0.102 vs 2.163 ± 0.192, ****P < 0.0001) and myosin heavy chain (0.3901 ± 0.047 vs. 0.7222 ± 0.082, **P < 0.01) protein levels and reduction of muscle atrophy F-box (10.77 ± 2.674 vs. 1.518 ± 0.172, **P < 0.01) expression. In dexamethasone-induced muscle atrophy model, mice with genetic ablation of PDK4 revealed increased muscle strength (162.1 ± 22.75 vs. 200.1 ± 37.09 g, ***P < 0.001) and muscle fibres (54.20 ± 11.85% vs. 84.07 ± 28.41%, ****P < 0.0001). To explore the mechanism, we performed coimmunoprecipitation and liquid chromatography-mass spectrometry analysis and found that myogenin is novel substrate of PDK4. PDK4 phosphorylates myogenin at S43/T57 amino acid residues, which facilitates the recruitment of muscle atrophy F-box to myogenin and leads to its subsequent ubiquitination and degradation. Finally, overexpression of non-phosphorylatable myogenin mutant using intramuscular injection prevented dexamethasone-induced muscle atrophy and preserved muscle fibres.ConclusionsWe have demonstrated that PDK4 mediates dexamethasone-induced skeletal muscle atrophy. Mechanistically, PDK4 phosphorylates and degrades myogenin via recruitment of E3 ubiquitin ligase, muscle atrophy F-box. Rescue of muscle regeneration by genetic ablation of PDK4 or overexpression of non-phosphorylatable myogenin mutant indicates PDK4 as an amenable therapeutic target in muscle atrophy.

Dataset Information

Caspase-dependent regulation of the ubiquitin-proteasome system through direct substrate targeting.

Publications

Caspase-dependent regulation of the ubiquitin-proteasome system through direct substrate targeting.

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