Late embryogenesis abundant (LEA) proteins are intrinsically disordered proteins (IDPs) commonly found in anhydrobiotic organisms and are frequently correlated with desiccation tolerance. Herein we report new findings on AfrLEA6, a novel group 6 LEA protein from embryos of Artemia franciscana. Assessment of secondary structure in aqueous and dried states with circular dichroism (CD) reveals 89% random coil in the aqueous state, thus supporting classification of AfrLEA6 as an IDP. Removal of water from the protein by drying or exposure to trifluoroethanol (a chemical de-solvating agent) promotes a large gain in secondary structure of AfrLEA6, predominated by α-helix and exhibiting minimal β-sheet structure. We evaluated the impact of physiological concentrations (up to 400 mM) of the disaccharide trehalose on the folding of LEA proteins in solution. CD spectra for AfrLEA2, AfrLEA3m, and AfrLEA6 are unaffected by this organic solute noted for its ability to drive protein folding. AfrLEA6 exhibits its highest concentration in vivo during embryonic diapause, drops acutely at diapause termination, and then declines during development to undetectable values at the larval stage. Maximum cellular titer of AfrLEA6 was 10-fold lower than for AfrLEA2 or AfrLEA3, both group 3 LEA proteins. Acute termination of diapause with H₂O₂ (a far more effective terminator than desiccation in this Great Salt Lake, UT, population) fostered a rapid 38% decrease in AfrLEA6 content of embryos. While the ultimate mechanism of diapause termination is unknown, disruption of key macromolecules could initiate physiological signaling events necessary for resumption of development and metabolism.

Group 1 (Dur-19, PF00477, LEA_5) Late Embryogenesis Abundant (LEA) proteins are present in organisms from all three domains of life, Archaea, Bacteria, and Eukarya. Surprisingly, Artemia is the only genus known to include animals that express group 1 LEA proteins in their desiccation-tolerant life-history stages. Bioinformatics analysis of circular dichroism data indicates that the group 1 LEA protein AfLEA1 is surprisingly ordered in the hydrated state and undergoes during desiccation one of the most pronounced disorder-to-order transitions described for LEA proteins from A. franciscana. The secondary structure in the hydrated state is dominated by random coils (42%) and β-sheets (35%) but converts to predominately α-helices (85%) when desiccated. Interestingly, AfLEA1 interacts with other proteins and nucleic acids, and RNA promotes liquid–liquid phase separation (LLPS) of the protein from the solvent during dehydration in vitro. Furthermore, AfLEA1 protects the enzyme lactate dehydrogenase (LDH) during desiccation but does not aid in restoring LDH activity after desiccation-induced inactivation. Ectopically expressed in D. melanogaster Kc167 cells, AfLEA1 localizes predominantly to the cytosol and increases the cytosolic viscosity during desiccation compared to untransfected control cells. Furthermore, the protein formed small biomolecular condensates in the cytoplasm of about 38% of Kc167 cells. These findings provide additional evidence for the hypothesis that the formation of biomolecular condensates to promote water stress tolerance during anhydrobiosis may be a shared feature across several groups of LEA proteins that display LLPS behaviors.

Anhydrobiosis is the remarkable phenomenon of “life without water”. It is a common technique found in plant seeds, and a rare technique utilized by some animals to temporarily stop the clock of life and enter a stasis for up to several millennia by removing all of their cellular water. If this phenomenon can be replicated, then biological and medical materials could be stored at ambient temperatures for centuries, which would address research challenges as well as enhance the availability of medicine in areas of the world where refrigeration, freezing, and cold-chain infrastructure are not developed or infeasible. Furthermore, modifying crop tissues could make them resistant to droughts, addressing one of the greatest threats to food stability around the world. This work utilizes a combination of computational techniques and novel approaches to performing biochemistry without water to elucidate the mechanisms of function of specialized proteins that are responsible for anhydrobiosis in animals, particularly the anhydrobiotic cysts of the brine shrimp Artemia franciscana. A detailed evaluation of the chemical properties of anhydrobiosis-related, intrinsically disordered proteins indicates that there are multiple protein-based strategies to achieve anhydrobiosis, but that late embryogenesis abundant (LEA) proteins are the most well understood. However, the mechanisms of LEA protein function have never been demonstrated, resulting in a wide variety of hypotheses regarding their ability to confer desiccation tolerance. This work demonstrates that a group 1 LEA protein, AfLEA1.1, and a group 6 LEA protein, AfrLEA6, undergo liquid-liquid phase separations during desiccation and thereby transiently form novel protective membraneless organelles which partition specific proteins and nucleic acids. These desiccation-induced cellular compartments are a novel mechanism to explain how LEA proteins confer desiccation tolerance, and the drivers of this behavior have been linked to the consensus sequences that define these LEA proteins. Therefore, the separation of aqueous proteins into a specialize compartment during drying is unlikely to only be a function of AfLEA1.1 and AfrLEA6, but actually the mechanism by which group 1 and group 3 LEA proteins function in plant seeds and anhydrobiotic animals. These results indicate that when water is unavailable, anhydrobiotic organisms substitute it with their own solvents.

Blood transfusions are one of the most common medical procedures in hospitals, but shortages of erythrocytes often occur due to their limited shelf life (6 weeks) when refrigerated. Preservation of erythrocytes in a dried state offers a potential solution to challenges faced with blood storage, and preservative compounds such as trehalose have been identified in organisms that survive desiccation in nature. However, these compounds do not readily cross mammalian cell membranes. Therefore, we are exploring the use of sonoporation to facilitate their delivery into erythrocytes ex vivo. In this study, we assessed the effect of cavitation activity on delivery of a fluorescent compound similar in size to trehalose (fluorescein) to human erythrocytes. Microbubbles were added to erythrocyte solutions and sonicated (2.5 MHz, 4 cycles) at various pressures and durations. Fluorescence was quantified with flow cytometry. The amplitude of broadband emissions in the first 8 seconds of sonication did not correlate with delivery (r² = 0.23), whereas after 8 seconds the broadband emissions amplitude was associated with increased delivery to erythrocytes (r² = 0.97). These results suggest that the timing of cavitation activity, rather than the amplitude alone, may be an important factor in ultrasound-mediated delivery of compounds into erythrocytes.