CAND1 drives intestinal ischemia/reperfusion injury via SCFFBXO6-PKM2-mediated mitochondrial fission and apoptosis.

BACKGROUND

Intestinal ischemia/reperfusion (I/R) injury is a critical clinical condition associated with high mortality, in which the ubiquitin‒proteasome system (UPS) plays a pivotal pathogenic role. Cullin-associated and neddylation-dissociated 1 (CAND1), a critical regulator of cellular protein homeostasis, governs the ubiquitination and degradation of abnormal protein substrates by regulating the assembly of SKP1‒Cullin1‒F-box (SCF) E3 ubiquitin ligase complexes. However, the mechanisms by which CAND1 regulates SCF complex assembly and its potential therapeutic role in intestinal I/R injury remain unclear.

OBJECTIVES

This study aims to elucidate the molecular mechanisms by which CAND1 mediates intestinal I/R injury and to identify potential therapeutic strategies targeting CAND1.

METHODS

Intestinal I/R was induced by superior mesenteric artery (SMA) occlusion in mice. Four weeks before I/R challenge, AAV-CAND1 was injected into the mice via the tail vein. Evodiamine (evo) was administered daily via intraperitoneal injection for three days before I/R challenge. Caco-2 cells were subjected to hypoxia/reoxygenation (H/R) treatment in vitro to simulate intestinal I/R in mice.

RESULTS

Excessive oxidative stress during intestinal I/R injury triggers mitochondrial fission and apoptosis. We identified CAND1 as a key regulator in this process, demonstrating upregulated expression during intestinal I/R injury. CAND1 knockdown attenuated reactive oxygen species (ROS) overproduction, mitochondrial fission, and apoptosis. Mechanistically, CAND1 inhibited Cullin1-FBXO6-PKM2 complex assembly and reduced PKM2 ubiquitination and degradation, thereby increasing PKM2 stability. Phosphorylated PKM2 formed dimers and translocated to mitochondria, where it activated Drp1-dependent fission pathway, worsening oxidative stress and apoptosis. Through molecular docking, evo was identified as a potential small-molecule candidate targeting CAND1. CAND1 may be inhibited by evo, thereby alleviating intestinal I/R injury.

CONCLUSION

CAND1 suppresses Cullin1-FBXO6-PKM2 complex assembly and PKM2 ubiquitination, promoting PKM2 dimerization-mediated mitochondrial fission and apoptosis in intestinal I/R injury. CAND1 is likely inhibited by evo, thereby alleviating mitochondrial dynamic alterations and cell death during intestinal I/R injury.