Mechanistic Insight of Tardigrade Anhydrobiosis via Protein Structural Analysis

Tardigrades are microscopic organisms with extraordinary tolerance to environmental stresses such as desiccation and thus offer unique solutions to bio-preservation, anti-aging, and interstellar travel. Recent studies revealed a collection of cytoplasmic and secretory abundant soluble proteins (CAHSs and SAHSs respectively) contributing to tardigrades extreme resilience. Using computational tools, I examined 39 CAHSs and 28 SAHSs from three representative tardigrade species. Both protein families possess a conserved central region and two highly variable terminal regions. Phylogenetic analysis suggests that CAHSs and SAHSs have distinct sequences despite functional similarity. AlphaFold predicts that CAHSs central region forms a long and amphiphilic -helix whereas SAHSs folds into {beta}-barrel. Since dehydration leads to the increase of intracellular protein concentration, I used AlphaFold to simulate CAHS oligomerization and find that they preferably dimerize via their central helix motifs. Examination of CAHS dimers reveals a strong inter-helix interaction. The dimerized anti-parallel helix bundle has hydrophobic and hydrophilic sides, resembling lipid-interacting proteins like ApoE. Empirical tests using mammalian fibroblast cells expressing the representative RvCAHS3 show that CAHSs concentrated on intracellular membranes instead of in proteinaceous condensates during dehydration. Moreover, RvCAHS3 significantly improves cell survival as measured by the stimulation-evoked release of Ca2+ from membrane-enclosed internal stores like endoplasmic reticulum. Taken together, these results suggest that CAHS inclines to dimerize and further forms a mesh on intracellular membranes to reinforce them during environmental stresses. By so doing, CAHSs can protect the integrity and the functionality of membrane-enclosed organelles. This finding implicates new strategies to preserve biomolecules, cells, and tissues under challenging conditions or for easy transportation.