Intrinsically disordered proteins as molecular shields

Abstract

The broad family of LEA proteins are intrinsically disordered proteins (IDPs) with several potential roles in desiccation tolerance, or anhydrobiosis, one of which is to limit desiccation-induced aggregation of cellular proteins. We show here that this activity, termed molecular shield function, is distinct from that of a classical molecular chaperone, such as HSP70 - while HSP70 reduces aggregation of citrate synthase (CS) on heating, two LEA proteins, a nematode group 3 protein, AavLEA1, and a plant group 1 protein, Em, do not; conversely, the LEA proteins reduce CS aggregation on desiccation, while HSP70 lacks this ability. There are also differences in interaction with client proteins - HSP70 can be co-immunoprecipitated with a polyglutamine-containing client, consistent with tight complex formation, whereas the LEA proteins can not, although a loose interaction is observed by Förster resonance energy transfer. In a further exploration of molecular shield function, we demonstrate that synthetic polysaccharides, like LEA proteins, are able to reduce desiccation-induced aggregation of a water-soluble proteome, consistent with a steric interference model of anti-aggregation activity. If molecular shields operate by reducing intermolecular cohesion rates, they should not protect against intramolecular protein damage. This was tested using the monomeric red fluorescent protein, mCherry, which does not undergo aggregation on drying, but the absorbance and emission spectra of its intrinsic fluorophore are dramatically reduced, indicative of intramolecular conformational changes. As expected, these changes are not prevented by AavLEA1, except for a slight protection at high molar ratios, and an AavLEA1-mCherry fusion protein is damaged to the same extent as mCherry alone. A recent hypothesis proposed that proteomes from desiccation-tolerant species contain a higher degree of disorder than intolerant examples, and that this might provide greater intrinsic stability, but a bioinformatics survey does not support this, since there are no significant differences in the degree of disorder between desiccation tolerant and intolerant species. It seems clear therefore that molecular shield function is largely an intermolecular activity implemented by specialist IDPs, distinct from molecular chaperones, but with a role in proteostasis.

Fig. 1

Light scattering measured as apparent absorbance (A₃₄₀) of CS (black bars) after (A) heat stress, or (B) desiccation stress, in the presence of five-fold molar excess HSP70 (dark grey), AavLEA1, or Em (both light grey). Non-stressed CS is taken as control (white). *** denotes significance at p < 0.001 and ** denotes p < 0.01 using one-way ANOVA, plus Tukey post test; ns, not significant.

Fig. 2

(A) Immunoprecipitation (IP) after expression of empty vector pFLAG-CMV5a or FlagHDQ138 and AavLEA1-HA (left panels), Em-HA (middle panels), or HSP70-HA (right panels). IP was performed with anti-Flag-M2 affinity gel followed by immunoblotting with anti-HA antibody (top row of panels). The inputs from the total cell lysates were probed with antibodies against HA (middle row) or Flag (bottom row) to detect the molecular shield or chaperone, and the polyQ protein, respectively. The asterisks in the top left and top middle panels show the expected position of any HA signal. (B) Example FRET analysis of EGFP-HDQ74 (donor) and AavLEA1-mCherry (acceptor) interactions in a live cell, showing signal in the donor channel upon excitation at donor wavelength (dx/dm), signal in the acceptor channel upon excitation at acceptor wavelength (ax/am), and donor normalized and unmixed FRET transfer efficiency dFRET.

Fig. 3

Light scattering measured as apparent absorbance (A₃₄₀) of T-REx293 water-soluble proteome after in vitro desiccation stress (black bars), (A) in the presence of variable molar ratios of ficoll 70 (dark grey), and (B) in the presence of 1 : 1 or 1 : 2 molar ratio of AavLEA1 (light grey) or ficoll 70/Dextran 42 (dark grey), respectively, or with 1 : 2 : 1 ratio of proteome: ficoll 70/Dextran 42: AavLEA1 (mid grey). The non-dried water-soluble proteome is taken as control (white). *, ** and *** denote significance at p < 0.05, p < 0.01 and p < 0.001, respectively, using one-way ANOVA, plus Tukey post test; ns, not significant.

Fig. 4

AavLEA1 provides limited protection of mCherry during desiccation. (A) Absorbance and (B) fluorescence emission spectra of mCherry, before and after four cycles of drying and rehydration. (C) Effect of drying on mCherry in the absence or presence of AvLEA1 or BSA (control) at molar ratios of 1 : 1 and 1 : 5. (D) Effect of desiccation on mCherry and an AavLEA1-mCherry fusion protein. Absorbance at 587 nm (the absorbance maximum of the intrinsic fluorophore) was measured before and after two and four cycles of drying and rehydration. Data were normalised, with the absorbance of the untreated sample represented as 1, for ease of comparison. All experiments were carried out in triplicate; error bars indicate ± 1 SD. *** denotes significance at p < 0.001 and * denotes p < 0.05 using one-way ANOVA, plus Tukey post test; ns, not significant.

Fig. 5

For each protein in D. radiodurans, the score reported by 0j.py, reflecting low complexity, is plotted against the number of amino acids from that protein predicted by Foldindex to be in an intrinsically disordered domain. A linear model is shown, but is a poor fit given the lack of a correlation between low complexity (as measured by 0j.py) and intrinsic disorder. Use of SEG produces very similar results (data not shown).

Fig. 6

Models for molecular shield function. (A) In the absence of molecular shields, partially denatured proteins (shaded circles) will interact and adhere at some rate, indicated by the double-headed arrow. (B) In the basic molecular shield model, shield proteins (represented by lines) are entropic chains that do not interact with other proteins but occupy space in solution and reduce the collision rate of aggregating species (indicated by a smaller double-headed arrow). (C) Evidence suggests a loose association of shield proteins with other polypeptides thereby forming a dynamic, three-dimensional protective barrier around aggregating species. Such interactions might also involve partial folding of the molecular shield on the surface of the misfolded client protein, potentially allowing a degree of entropy transfer that might facilitate refolding of the client, as proposed by Tompa and Csermely.