E3 ligase TRIM65 alleviates intestinal ischemia/reperfusion injury through inhibition of TOX4-mediated apoptosis

Abstract Intestinal ischemia-reperfusion (II/R) injury is an urgent clinical disease with high incidence and mortality, and impaired intestinal barrier function caused by excessive apoptosis of intestinal cells is an important cause of its serious consequences. Tripartite motif-containing protein 65 (TRIM65) is an E3 ubiquitin ligase that is recently reported to suppress the inflammatory response and apoptosis. However, the biological function and regulation of TRIM65 in II/R injury are totally unknown. We found that TRIM65 was significantly decreased in hypoxia-reoxygenation (H/R) induced intestinal epithelial cells and II/R-induced intestine tissue. TRIM65 knockout mice markedly aggravated intestinal apoptosis and II/R injury. To explore the molecular mechanism of TRIM65 in exacerbating II/R-induced intestinal apoptosis and damage, thymocyte selection-associated high mobility group box factor 4 (TOX4) was screened out as a novel substrate of TRIM65 using the yeast two-hybrid system. TRIM65 binds directly to the N-terminal of TOX4 through its coiled-coil and SPRY structural domains. Immunofluorescence confocal microscopy showed that they can co-localize both in the cytoplasm and nucleus. Furthermore, TRIM65 mediated the K48 ubiquitination and degradation of TOX4 depending on its E3 ubiquitin ligase activity. In addition, TRIM65 inhibits H/R-induced intestinal epithelial apoptosis via TOX4. In summary, our results indicated that TRIM65 promotes ubiquitination and degradation of TOX4 to inhibit apoptosis in II/R. These findings provide a promising target for the clinical treatment of II/R injury.