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Published on: March 24, 2019
Post-Translational Modifications of the DUX4 Protein Impact Toxic Function in FSHD Cell Models
Renatta N Knox1, Jocelyn O Eidahl2, Lindsay M Wallace2
1Department of Neurology, Washington University School of Medicine, St. Louis, MO.
Objective:
Facioscapulohumeral muscular dystrophy (FSHD) is caused by abnormal de-repression of the myotoxic transcription factor DUX4. Although the transcriptional targets of DUX4 are known, the regulation of DUX4 protein and the molecular consequences of this regulation are unclear. Here, we used in vitro models of FSHD to identify and characterize DUX4 post-translational modifications (PTMs) and their impact on the toxic function of DUX4.
Methods:
We immunoprecipitated DUX4 protein and performed mass spectrometry to identify PTMs. We then characterized DUX4 PTMs and potential enzyme modifiers using mutagenesis, proteomics, and biochemical assays in HEK293 and human myoblast cell lines.
Results:
We identified 17 DUX4 amino acids with PTMs, and generated 55 DUX4 mutants designed to prevent or mimic PTMs. Five mutants protected cells against DUX4-mediated toxicity and reduced the ability of DUX4 to transactivate FSHD biomarkers. These mutagenesis results suggested that DUX4 toxicity could be counteracted by serine/threonine phosphorylation and/or inhibition of arginine methylation. We therefore sought to identify modifying enzymes that could play a role in regulating DUX4 PTMs. We found several enzymes capable of modifying DUX4 protein in vitro, and confirmed that protein kinase A (PKA) and protein arginine methyltransferase (PRMT1) interact with DUX4.
Interpretation:
These results support that DUX4 is regulated by PTMs and set a foundation for developing FSHD drug screens based mechanistically on DUX4 PTMs and modifying enzymes. ANN NEUROL 2023;94:398-413.
Insights
Facioscapulohumeral muscular dystrophy (FSHD) is linked to the DUX4 protein. Researchers identified key DUX4 post-translational modifications (PTMs) that influence its toxicity, offering new therapeutic targets for FSHD.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Facioscapulohumeral muscular dystrophy (FSHD) is a genetic disorder characterized by progressive muscle weakness.
- The disease is primarily caused by the aberrant expression of the double-homeobox 4 (DUX4) gene.
- While DUX4's transcriptional targets are known, its protein regulation and downstream effects remain incompletely understood.
Purpose of the Study:
- To investigate the post-translational modifications (PTMs) of the DUX4 protein.
- To characterize how these PTMs impact DUX4's toxic function in FSHD.
- To identify potential enzyme modifiers regulating DUX4 PTMs.
Main Methods:
- Utilized in vitro FSHD models, including HEK293 and human myoblast cell lines.
- Employed immunoprecipitation and mass spectrometry to identify DUX4 PTMs.
- Performed mutagenesis, proteomics, and biochemical assays to characterize PTMs and enzyme interactions.
Main Results:
- Identified 17 DUX4 amino acids with PTMs and created 55 DUX4 mutants.
- Demonstrated that specific DUX4 mutants reduced DUX4-mediated toxicity and biomarker transactivation.
- Found that serine/threonine phosphorylation and arginine methylation inhibition counteract DUX4 toxicity.
- Confirmed interactions between DUX4 and key enzymes: protein kinase A (PKA) and protein arginine methyltransferase 1 (PRMT1).
Conclusions:
- DUX4 protein is subject to significant post-translational modifications.
- These PTMs play a crucial role in regulating DUX4's toxic activity in FSHD.
- DUX4 PTMs and their modifying enzymes represent promising targets for novel FSHD therapeutic strategies.
