Introduction

Expression of late embryogenesis abundant (LEA) proteins is highly correlated with desiccation tolerance in anhydrobiotic animals, selected land plants, and bacteria. Genes encoding two LEA proteins, one localized to the cytoplasm/nucleus (AfrLEA2) and one targeted to mitochondria (AfrLEA3m), were stably transfected into human HepG2 cells. A trehalose transporter was used for intracellular loading of this disaccharide. Cells were rapidly and uniformly desiccated to low water content (<0.12 g H 2 O/g dry weight) with a recently developed spin-drying technique. Immediately on rehydration, control cells without LEA proteins or trehalose exhibited 0% membrane integrity, compared with 98% in cells loaded with trehalose and expressing AfrLEA2 or AfrLEA3m; surprisingly, AfrLEA3m without trehalose conferred 94% protection. Cell proliferation across 7 d showed an 18-fold increase for cells dried with AfrLEA3m and trehalose, compared with 27-fold for nondried controls. LEA proteins dramatically enhance desiccation tolerance in mammalian cells and offer the opportunity for engineering biostability in the dried state.water stress | biopreservation | intrinsically disordered proteins | osmolyte | Artemia franciscana

Anhydrobiotic animals survive virtually complete loss of cellular water. The mechanisms that explain this phenomenon are not fully understood but often include the accumulation of low molecular weight solutes such as trehalose and macromolecules like Late Embryogenesis Abundant (LEA) proteins. Here we report for the first time the occurrence of a mitochondria-targeted LEA gene (Afrlea3m) product in an animal species. The deduced molecular mass of the 307-amino acid polypeptide from the brine shrimp Artemia franciscana is 34 kDa. Bioinformatic analyses reveal features typical of a Group 3 LEA protein, and subcellular localization programs predict targeting of the mature peptide to the mitochondrial matrix, based on an N-terminal, amphipathic presequence. Real-time quantitative PCR shows that Afralea3m mRNA is expressed manyfold higher in desiccation-tolerant embryonic stages when compared with intolerant nauplius larvae. Mitochondrial localization of the protein was confirmed by transfection of human hepatoma cells (HepG2/C3A) with a nucleotide construct encoding the first 70 N-terminal amino acids of AfrLEA3m in-frame with the nucleotide sequence for green fluorescence protein. The chimeric protein was readily incorporated into mitochondria of these cells. Successful targeting of a protein to human mitochondria by use of an arthropod signaling sequence clearly reveals the highly conserved nature of such presequences, as well as of the import machinery. Finally, mitochondria isolated from A. franciscana embryos, which naturally contain AfrLEA3m and trehalose, exhibit resistance to water stress (freezing) as evidenced by an unchanged capacity for oxidative phosphorylation on succinate ؉ rotenone, a resistance that is absent in mammalian mitochondria lacking AfrLEA3m.Fluctuation in cellular water content is a universal problem confronting a variety of organisms, and any environmental stress that impacts cellular water poses a risk to life (1). Nevertheless, some animals are able to cope with virtually complete loss of cellular water for prolonged times (2, 3), a phenomenon termed anhydrobiosis. Tolerance to water stress (i.e. evaporative water loss, freezing, and osmotic removal of water) is most likely achieved by several different mechanisms designed to protect and repair the structures and functions of cells and tissues. Many desiccation-tolerant organisms respond to water stress by intracellular accumulation of selected sugars, amino acids and derivatives, and methylamines (often occurring with urea) termed "compatible" osmolytes (4 -7). Organic solutes such as the non-reducing sugar trehalose can actually stabilize biological structures during severe drying (8 -12). Evidence indicates that the presence of small stress proteins and Late Embryogenesis Abundant (LEA) 2 proteins is also important for cellular protection during drying in eukaryotic cells (13)(14)(15)(16). However, the issue of how subcellular compartments in nucleated cells are protected during water stress has received far less attention (17)(18)(...

SUMMARY Cellular conditions experienced during energy-limited states –elevated calcium, shifts in cellular adenylate status, compromised mitochondrial membrane potential – are precisely those that trigger, at least in mammals, the mitochondrion to initiate opening of the permeability transition pore, to assemble additional protein release channels, and to release pro-apoptotic factors. These pro-apototic factors in turn activate initiator and executer caspases. How is activation of mitochondria-based pathways for the signaling of apoptotic and necrotic cell death avoided under conditions of hypoxia, anoxia, diapause, estivation and anhydrobiosis?Functional trade-offs in environmental tolerance may have occurred in parallel with the evolution of diversified pathways for the signaling of cell death in eukaryotic organisms. Embryos of the brine shrimp, Artemia franciscana, survive extended periods of anoxia and diapause, and evidence indicates that opening of the mitochondrial permeability transition pore and release of cytochrome c (cyt-c) do not occur. Further, caspase activation in this crustacean is not dependent on cyt-c. Its caspases display regulation by nucleotides that is consistent with `applying the brakes' to cell death during energy limitation. Unraveling the mechanisms by which organisms in extreme environments avoid cell death may suggest possible interventions during disease states and biostabilization of mammalian cells.

Anhydrobiotic animals protect cellular architecture and metabolic machinery in the dry state, yet the molecular repertoire supporting this profound dehydration tolerance is not fully understood. For the desiccation-tolerant crustacean, Artemia franciscana, we report differential expression of two distinct mRNAs encoding for proteins that share sequence similarities and structural features with late-embryogenesis abundant (LEA) proteins originally discovered in plants. Bioinformatic analyses support assignment of the LEA proteins from A. franciscana to group 3. This eucoelomate species is the most highly evolved animal for which LEA gene expression has been reported. It is becoming clear that an ensemble of micromolecules and macromolecules is important for establishing the physical conditions required for cellular stabilization during drying in nature.