RiAFP enhances cold tolerance via an antioxidant-centered, ice-independent mechanism

Low temperature challenges the structural stability and functional integrity of biological macromolecules and disrupts cellular homeostasis even under non-freezing conditions. Although antifreeze proteins (AFPs) are classically recognized for inhibiting ice crystal growth at subzero temperatures, their bioactivities under non-freezing cold stress remain poorly characterized. Here, we explore the cytoprotective potential of RiAFP, a hyperactive antifreeze protein from the beetle Rhagium inquisitor, as a biological macromolecule using mammalian cell systems and vertebrate models. RiAFP expression in HEK-293T cells significantly enhances cell viability under cold stress by preserving membrane integrity. In vivo, transgenic zebrafish expressing RiAFP exhibit increased resistance to cold-induced developmental defects, improved locomotor performance, and enhanced survival, accompanied by attenuated tissue damage, particularly in the gills. Integrated transcriptomic and biochemical analyses reveal that RiAFP selectively promotes oxidative phosphorylation and stabilizes mitochondrial electron transport chain function. This coordinated response maintains redox homeostasis, thereby effectively mitigating cold-induced oxidative stress. These findings demonstrate that RiAFP functions as a multifunctional biological macromolecule that confers cold tolerance through an antioxidant-centered, ice-independent mechanism. This work expands the functional repertoire of antifreeze proteins beyond classical cryoprotection and highlights their potential as bioactive macromolecules for low-temperature biotechnology and stress-resilient biological systems.