Hydrophilic protein associated with desiccation tolerance exhibits broad protein stabilization function

Abstract

The ability of certain plants, invertebrates, and microorganisms to survive almost complete loss of water has long been recognized, but the molecular mechanisms of this phenomenon remain to be defined. One phylogenetically widespread adaptation is the presence of abundant, highly hydrophilic proteins in desiccation-tolerant organisms. The best characterized of these polypeptides are the late embryogenesis abundant (LEA) proteins, first described in plant seeds >20 years ago but recently identified in invertebrates and bacteria. The function of these largely unstructured proteins has been unclear, but we now show that a group 3 LEA protein from the desiccation-tolerant nematode Aphelenchus avenae is able to prevent aggregation of a wide range of other proteins both in vitro and in vivo. The presence of water is essential for maintenance of the structure of many proteins, and therefore desiccation stress induces unfolding and aggregation. The nematode LEA protein is able to abrogate desiccation-induced aggregation of the water-soluble proteomes from nematodes and mammalian cells and affords protection during both dehydration and rehydration. Furthermore, when coexpressed in a human cell line, the LEA protein reduces the propensity of polyglutamine and polyalanine expansion proteins associated with neurodegenerative diseases to form aggregates, demonstrating in vivo function of an LEA protein as an antiaggregant. Finally, human cells expressing LEA protein exhibit increased survival of dehydration imposed by osmotic upshift, consistent with a broad protein stabilization function of LEA proteins under conditions of water stress.

Fig. 1.

LEA protein prevents aggregation of water-soluble proteomes subjected to desiccation. (A and B) Light scattering measured as apparent absorbance (A340) of human (T-REx293) and nematode (A. avenae) water-soluble proteomes, respectively, in the hydrated state (white), after vacuum drying and rehydration (black), and after drying with AavLEA1 (gray). **, significance at P < 0.01; ns, not significant. (C) Coomassie blue-stained polyacrylamide gel of soluble and insoluble fractions of the water-soluble proteome of A. avenae. Control, not dried; desiccated, vacuum-dried and rehydrated; desiccated + LEA, dried with AavLEA1. AavLEA1 is indicated by an arrow. sup, Soluble fractions; pel, insoluble fractions. Size markers (M) range from 250 to 10 kDa.

Fig. 2.

LEA protein reduces aggregation of water-soluble proteome during both dehydration and rehydration. Light scattering measured as apparent absorbance (A340) of human water-soluble proteome, with addition of varying molar ratios of AavLEA1 (A) and with LEA protein added at different time points, as indicated (B). *, significance at P < 0.05; **, significance at P < 0.01; ***, significance at P < 0.001; ns, not significant.

Fig. 3.

Expression of LEA protein in a human cell line. (A) Confocal microscopy of T-REx293-LEA15 cells and T-REx293 cells with (Induced) or without (Uninduced) addition of tetracycline. AavLEA1 is immunostained green, and nuclei are stained blue with DAPI. (Scale bar: 5 μm.) (B) Immunoblotting using immunopurified antibody against AavLEA1. A nonspecific protein is shown as a loading control.

Fig. 4.

LEA protein reduces protein aggregation in vivo. (A) Confocal microscopy of T-REx293-LEA15 cells after transfection with EGFP-HDQ74 expression construct, with (Induced) or without (Uninduced) expression of LEA protein. AavLEA1 is immunostained red, EGFP-HDQ74 is green, and nuclei are stained blue with DAPI. Indicative EGFP-HDQ74 aggregates are shown by arrowheads. (Scale bar: 40 μm.) Percentage of T-REx293-LEA15 cells with EGFP-HDQ74 aggregates with (filled) or without (open) LEA protein induction were assessed where both AavLEA1 and EGFP-HDQ74 were expressed for 24 h (B), 48 h (C), or where EGFP-HDQ74 is expressed for 48 h and AavLEA1 for the last 24 h (D). (E) Inhibition of protein degradation pathways with 10 μM lactacystin and 10 mM 3MA does not affect LEA protein antiaggregation activity. Standard deviation of multiple replicates is shown as vertical bars. All results are highly significant as evaluated by logistic regression analysis (SI Fig. 9).

Fig. 5.

LEA protein reduces aggregate formation by EGFP-A37 in vivo. (A) Confocal microscopy of T-REx293-LEA15 cells after transfection with EGFP-A37 expression construct, with (Induced) or without (Uninduced) expression of LEA protein. AavLEA1 is immunostained red, EGFP-A37 is green, and nuclei are stained blue with DAPI. Indicative EGFP-A37 aggregates are shown by arrowheads. (Scale bar: 40 μm.) (B) Percentage of T-REx293-LEA15 cells with EGFP-A37 aggregates with (filled) or without (open) LEA protein induction were assessed where both AavLEA1 and EGFP-A37 were expressed for 48 h. ***, significant at P < 0.0001 by logistic regression analysis (SI Fig. 9E).

Fig. 6.

LEA protein expression improves osmotolerance of human cells. Shown are T-REx293-LEA15 cells subjected to 100 mM NaCl over 48 h (A), and various osmolytes at 400 mOsm for 24 h (B), either with (filled) or without (open) induction of AavLEA1 expression. Viability is assessed by measuring metabolic rate compared with untreated controls. *, significant differences between paired values (induced vs. uninduced) at P < 0.05 using the Student t test.