AI-derived-intrinsically disordered proteins from antarctic nematode stabilize human cells under freezing stress

Cryopreservation and low-temperature stress pose a significant threat to cellular integrity due to ice crystal formation, dehydration, and apoptosis. Extremophiles such as the Antarctic nematode, Plectus murrayi, are a unique biological animal model of novel protective molecules to address this challenge. Here, we investigated the cryoprotective potential of Intrinsically Disordered Proteins (IDPs) derived from P. murray predicted by the "in-house" bioinformatic tool. Transcriptomic analysis of P. murray identified dur-1, a gene responsible for its freezing tolerance and homologous to the gene plin4 in humans. Functional validation studies in human 293T cells confirmed that overexpression of plin4 reduced apoptosis and enhanced proliferation significantly under low temperature stress at both 4 °C and -20 °C, indicating a cross-species conservation of its protective role. Furthermore, the molecular docking predicted strong binding affinities between Plin4 and P. murray-derived IDPs (1, 2, and 3), with IDP2 showing particular promise. Further validation in human skin cell lines (HaCaT, BJ) confirmed that IDP1 and IDP2 significantly enhanced cell viability under both 4 °C and freeze-dehydration (-20 °C) stresses, while IDP3 was effective specifically at 4 °C. However, the protective effect of IDP individually performed not stably along with the duration of low-temperature stress exposure of cells, while the mixture of three IDPs showed more efficient cellular uptake and perinuclear localization. Our findings identify a novel type of biomimetic cryoprotectants with significant potential for applications in cryobiology, biobanking, and the development of therapies for cold-induced cellular damage.