Investigating the Cytoprotective Mechanisms of the Tardigrade Damage Suppressor (Dsup) Protein in Human Cells Under Hypoxic Stress

Ischemia/reperfusion injury (IRI) is a common damage due to the restoration of blood flow following an ischemic injury. Its pathogenesis is mainly linked to the production of reactive oxygen species (ROS), which sustain cell damage and promote cell death. The tardigrade damage suppressor protein (Dsup) is a DNA-binding protein that enables tardigrades to tolerate stress conditions, including oxidative stress. We investigated the ability of the Dsup to protect human cells from IRI, using an in vitro model of hypoxia and reoxygenation. We exposed HEK293TT cells transfected with the Dsup to hypoxic injury and analyzed cell viability, oxidative stress, expression of antioxidant proteins using functional assays, and a proteomic approach to dissect the molecular mechanisms modulated by the Dsup. Dsup expression significantly enhanced cell survival following hypoxia-reoxygenation and markedly reduced intracellular ROS levels. Proteomic and Western blot analyses revealed a significant upregulation of antioxidant enzymes in Dsup-expressing cells. Furthermore, the Dsup modulated autophagy and key stress-related pathways, including the MAPK cascade. This study demonstrates that the Dsup protects human cells from IRI by reducing oxidative stress and modulating key cytoprotective pathways. Our results establish the Dsup as a promising candidate for future therapeutic applications against IRI, meriting further exploration in in vivo models.