E3 ubiquitin ligase TRIM47 drives endothelial dysfunction in immune checkpoint inhibitor-associated myocarditis via K48-linked polyubiquitination and degradation of SCO1

Abstract

Immune checkpoint inhibitor-associated myocarditis (ICIAM) is a rare yet fatal clinical adverse event in cancer patients receiving immunotherapy. Previous studies have primarily focused on the pathophysiological roles of various inflammatory cells in the development of ICIAM. However, the relationship between endothelial barrier dysfunction and the onset of myocarditis remains unexplored. Here we performed a comprehensive analysis to examine the role of TRIM47 in regulating endothelial inflammation and its impact on the pathogenesis of ICIAM.

Through bioinformatics analysis, we found that TRIM47 expression was upregulated in endothelial cells in the ICIAM model, correlating with markers of endothelial inflammation (Figure 1A-B, D). We utilized WB analysis to confirm the upregulation of TRIM47 and other key inflammatory markers. The results showed that increased TRIM47 expression was associated with elevated levels of these adhesion molecules, indicating the involvement of TRIM47 in endothelial cell activation (Figure 1C). Knockdown of TRIM47 via AAV-mediated gene silencing effectively reversed the ICIAM phenotype (Figure 1E, F).

We investigated the mechanistic role of TRIM47 in regulating the inflammatory response by performing PPI analysis. Using IP and mass spectrometry, we identified SCO1 as a binding partner of TRIM47 (Figure 2A). This interaction was further confirmed by Co-IP assays, which revealed that the B30.2/SPRY subdomain of TRIM47 directly interacted SCO1 (Figure 2B-D). We observed that TRIM47 promoted K48-linked polyubiquitination of SCO1, leading to its subsequent degradation.

To further explore this mechanism, we employed molecular docking to predict the binding affinities between TRIM47 and SCO1 (Figure 2E). The results suggested that a stable and energetically favorable interaction between TRIM47 and SCO1, further supporting our hypothesis that TRIM47 regulates SCO1 degradation via direct interaction. To evaluate the functional impact of TRIM47 overexpression and SCO1 degradation on endothelial cells, cells transfected with TRIM47 overexpression plasmids exhibited decreased cell migration and wound closure, consistent with a heightened inflammatory state, which could be reversed by transfecting cells with SCO1 overexpression plasmids (Figure 2F).

We also examined the effect of TRIM47 on AMPK signaling in endothelial cells. Using bioinformatics analysis and WB, we found that TRIM47 overexpression reduced AMPK phosphorylation, whereas TRIM47 silencing restored AMPK signaling. (Figure 2G).

In summary, our study provides compelling evidence that TRIM47 serves as a regulator of endothelial inflammation in ICIAM, which exacerbates endothelial dysfunction and inflammatory responses. Findings suggest that targeting TRIM47 or restoring SCO1 expression could represent a novel strategy for managing ICIAM. Further investigations are warranted to evaluate the clinical applicability of TRIM47.Figure 1Figure 2