Molecular Characterization of Tripartite Motif-containing (TRIM) E3 Ubiquitin Ligases in the Heart

• Applicant: Privatdozent Ashraf Rangrez, Ph.D.
• Subject Area: Cardiology, Angiology
• Term: since 2016
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 299281625

We previously identified Dysbindin, a protein involved in schizophrenia pathogenesis, as a novel activator of Serum Response Factor-signaling promoting cardiomyocyte hypertrophy. Furthermore, we postulate that Dysbindin likely play a role in cardiac autophagy as we observed strong downregulation of Dysbindin upon activation of autophagy in mice and substantial activation of autophagy when Dysbindin is upregulated. Based on these interesting preliminary results, one of the objectives of our current DFG application is to study and understand the role of Dysbindin in autophagy and/or associated protein trafficking utilizing Dysbindin-KO mice and in vitro models of Dysbindin overexpression and knockdown.A yeast-two hybrid screen identified several potential Dysbindin interactors, including TRIM24 (Tripartite Motif-containing 24), an E3 ubiquitin ligase. Further characterization of this interaction in vitro revealed that TRIM24 and TRIM32 (another known E3 ligase of Dysbindin) oppositely regulate Dysbindin in cardiomyocytes (NRVCMs): TRIM32 was found to degrade dysbindin, whereas TRIM24 protected Dysbindin from its degradation. TRIM24, also called transcription intermediary factor 1 alpha (TIF1alpha), belongs to a subclass of TRIM E3 ligases including TRIM28 (TIF1ß) and TRIM33 (TIF1gamma), and is a cofactor that is known to interact with distinct transcription factors in other cell types. However, the potential role of TRIM24 in cardiac epigenetics and its direct/indirect targets in cardiomyocytes are not known yet. As a first step towards this, we recently performed proteome profiling of cardiomyocytes where TRIM24 was knocked-down. Interestingly, Tropomyosins (Tpm) 1, 2 and 3 were significantly upregulated upon TRIM24 knockdown in neonatal rat cardiomyocytes. We thus now aim to decode how TRIM24 regulates these Tropomyosins, if they are direct substrates or regulated by TRIM24 via its epigenetic function to get further insights into the role of TRIM24 in cardiomyocyte pathophysiology using loss- and gain-of-function approaches in vitro and by generating a cardiac-specific TRIM24-conditional knockout mouse model.Finally, using human Affymetrix data, we found significant expression of several of the yet uncharacterized TRIMs in the heart, many of which were also found to be differentially regulated in myocardium under cardiac disease settings like heart failure, cardiomyopathies, myocardial infarction, and atrial fibrillation. Further validations of these results by qPCR revealed significant upregulation of TRIM22 in HCM, TRIM61 in both HCM and DCM, whereas, though not significant, there was a trend of dysregulation of TRIM67 in DCM, HCM and ICM. Thus, our last objective is to elucidate the role of TRIM22, TRIM61 and TRIM67 in cardiomyocyte physiology using loss- and gain-of-function approaches in vitro.

• DFG Programme: Research Grants