USP26 Promotes Autophagosome–Lysosome Fusion to Improve Mitochondrial Function in the Repair of Gut Barrier Damage by Stabilizing STX17

Aims: Intestinal mucosal barrier injury is a major consequence of intestinal ischemia–reperfusion (II/R) and contributes to poor clinical outcomes. While mitophagy sustains mitochondrial homeostasis, how impaired autophagic flux disrupts intestinal barrier function remains unclear. We investigated the role of the deubiquitinase ubiquitin-specific protease 26 (USP26) and its downstream target syntaxin 17 (STX17) in regulating mitophagy and intestinal barrier repair. Methods: II/R mouse models and oxygen–glucose deprivation/reperfusion (OGD/R)-treated Caco-2 cells were used to evaluate intestinal barrier integrity. Functional manipulation, autophagy blockade, Co-IP, ubiquitination assays, Seahorse metabolic detection, and in vivo rescue assays were performed to dissect the USP26-STX17 regulatory axis. Results: USP26 was markedly downregulated in II/R intestinal tissues and OGD/R Caco-2 cells, coinciding with impaired autophagy, mitochondrial dysfunction, and intestinal barrier disruption. USP26 overexpression preserved intestinal architecture, reduced permeability and apoptosis, and restored expression of the tight-junction proteins. Functionally, USP26 enhanced autophagic flux and mitophagy, as evidenced by increased LC3-II/LC3-I ratios, reduced p62 accumulation, elevated PINK1/Parkin signaling, improved adenosine triphosphate production, restored mitochondrial membrane potential, and normalized cellular bioenergetics; autophagy inhibition attenuated these protective phenotypes. Mechanistically, USP26 bound STX17 and stabilized it via deubiquitination. STX17 knockdown impaired autophagosome–lysosome fusion and largely abolished USP26-mediated mitochondrial and intestinal barrier protection in vitro and in vivo . Conclusions: This study identifies the USP26-STX17 axis as a previously unrecognized regulator of autophagosome–lysosome fusion and mitophagy in II/R injury. By restoring mitochondrial quality control and epithelial barrier integrity, USP26 emerges as a promising therapeutic target for the prevention and treatment of gut barrier dysfunction associated with critical illness. Antioxid. Redox Signal. 00, 000–000.