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Electrophoretic Mobility Shift Assay EMSA for the Study of RNA-Protein Interactions: The IRE/IRP Example
Published on: December 3, 2014
TRIM28 Is an E3 Ligase of IRP2 Suppressing Ischemia/Reperfusion-Induced Myocardial Ferroptosis
Kun Zhu1, Jing Guo1, Yangli Liu1
1Laboratory of Cardiovascular Science, Beijing Clinical Research Institute (K.Z., J.G., Y. Liu, J.L., X.W., R.D., L.G., J.B., R.C., Z.L., W.Z., Y.Z., Y.W., L.D., Y. Li, D.-S.L., W.A., C.-M.C.), Beijing Friendship Hospital, Capital Medical University, China.
Background:
Myocardial ischemia/reperfusion (I/R) injury is a common and severe clinical complication in patients with ischemic heart disease after reperfusion therapy. Effective therapeutic strategies for myocardial I/R injury remain limited. Ferroptosis is a form of regulated cell death characterized by iron-dependent lipid peroxidation. However, the mechanisms underlying ferroptosis in myocardial I/R injury are not fully understood.
Methods:
Transcriptomic data from patients with heart failure and cardiomyocytes undergoing ferroptosis were analyzed. Based on the screening results, TRIM28 (tripartite motif-containing 28) expression was evaluated in ferroptotic cardiomyocytes. Cardiac I/R injury models in mice and hypoxia/reoxygenation injury models in neonatal rat ventricular myocytes were established. To explore the function of TRIM28, we used adeno-associated virus serotype 9 to achieve cardiomyocyte-specific overexpression and generated tamoxifen-inducible cardiomyocyte-specific TRIM28 knockout mice. RNA sequencing, coimmunoprecipitation coupled with mass spectrometry, and ubiquitinome profiling were applied to elucidate the underlying mechanisms. Human heart samples from patients with ischemic heart disease were used to evaluate the expression of TRIM28 and its related signaling molecules. The Connectivity Map database was used to screen potential activators of TRIM28.
Results:
We found that TRIM28 expression was downregulated in ferroptosis inducer-treated and hypoxia/reoxygenation-injured cardiomyocytes, as well as in I/R-injured mouse hearts. Cardiomyocyte-specific overexpression of TRIM28 protected the heart against I/R-induced ferroptosis, whereas its deficiency exacerbated myocardial I/R-induced ferroptotic injury. Mechanistically, TRIM28 functioned as an E3 ubiquitin ligase that directly bound to IRP2 (iron regulatory protein 2) and promoted K48-linked ubiquitination at the K877 site, leading to the downregulation of IRP2 and TFR1 (transferrin receptor 1), suppression of intracellular iron uptake, and consequent attenuation of cardiomyocyte ferroptosis. Furthermore, p55γ interacted with and upregulated TRIM28, thereby mitigating I/R-induced myocardial ferroptosis. Consistent with these findings, protein levels of TRIM28 and p55γ were decreased in heart samples from patients with ischemic heart disease, whereas IRP2 and TFR1 were increased. Last, we demonstrated that perhexiline inhibited I/R-induced myocardial ferroptosis by upregulating p55γ and TRIM28.
Conclusions:
Our study identifies TRIM28 as an essential E3 ubiquitin ligase of IRP2 and delineates that TRIM28-mediated inhibition of myocardial ferroptosis by targeting IRP2-TFR1 signaling protects the heart against I/R injury, indicating that targeting TRIM28 represents a promising therapeutic strategy for suppressing cardiomyocyte ferroptosis and I/R injury.
Insights
Tripartite motif-containing 28 (TRIM28) protects the heart from ischemia/reperfusion (I/R) injury by inhibiting ferroptosis. Upregulating TRIM28 may be a therapeutic strategy for I/R injury.
Area of Science:
- Cardiovascular Biology
- Cell Death Mechanisms
- Molecular Cardiology
Background:
- Myocardial ischemia/reperfusion (I/R) injury is a severe clinical complication with limited therapeutic options.
- Ferroptosis, an iron-dependent cell death, plays a role in I/R injury, but its underlying mechanisms are unclear.
Purpose of the Study:
- To investigate the role of tripartite motif-containing 28 (TRIM28) in myocardial I/R injury and ferroptosis.
- To elucidate the molecular mechanisms by which TRIM28 regulates ferroptosis in cardiomyocytes.
Main Methods:
- Analysis of transcriptomic data and ferroptosis models in cardiomyocytes and mice.
- Generation of cardiomyocyte-specific TRIM28 overexpression and knockout models.
- Utilized RNA sequencing, co-immunoprecipitation, mass spectrometry, and ubiquitinome profiling.
- Examined human heart samples from patients with ischemic heart disease.
Main Results:
- TRIM28 expression was reduced in I/R-injured hearts and ferroptotic cardiomyocytes.
- TRIM28 overexpression protected against I/R injury, while TRIM28 deficiency exacerbated it.
- TRIM28 acts as an E3 ubiquitin ligase targeting IRP2, reducing iron uptake and ferroptosis.
- p55γ interacts with and upregulates TRIM28, mitigating ferroptosis.
Conclusions:
- TRIM28 is a crucial E3 ubiquitin ligase for IRP2, inhibiting ferroptosis via the IRP2-TFR1 signaling pathway.
- Targeting TRIM28 offers a potential therapeutic strategy for myocardial I/R injury by suppressing ferroptosis.
