Inhibition of protein aggregation in vitro and in vivo by a natural osmoprotectant

Abstract

Small organic molecules termed osmolytes are harnessed by a variety of cell types in a wide range of organisms to counter unfavorable physiological conditions that challenge protein stability and function. Using a well characterized reporter system that we developed to allow in vivo observations, we have explored how the osmolyte proline influences the stability and aggregation of a model aggregation-prone protein, P39A cellular retinoic acid-binding protein. Strikingly, we find that the natural osmolyte proline abrogates aggregation both in vitro and in vivo (in an Escherichia coli expression system). Importantly, proline also prevented aggregation of constructs containing exon 1 of huntingtin with extended polyglutamine tracts. Although compatible osmolytes are known to stabilize the native state, our results point to a destabilizing effect of proline on partially folded states and early aggregates and a solubilizing effect on the native state. Because proline is believed to act through a combination of solvophobic backbone interactions and favorable side-chain interactions that are not specific to a particular sequence or structure, the observed effect is likely to be general. Thus, the osmolyte proline may be protective against biomedically important protein aggregates that are hallmarks of several late-onset neurodegenerative diseases including Huntington's, Alzheimer's, and Parkinson's. In addition, these results should be of practical importance because they may enable protein expression at higher efficiency under conditions where aggregation competes with proper folding.

Fig. 1.

Osmotic upshift suppresses protein aggregation. (A) (Upper) Time course of the bulk FlAsH fluorescence signal at 530 nm (excitation 500 nm) of E. coli BL21(DE3) cells expressing P39A tetra-Cys CRABP and subjected at the time of isopropyl-β-d-thiogalactoside induction to medium at differing osmolalities (from 100 to 300 mM NaCl). Note the behavior of the completely soluble tetra-Cys CRABP. (Lower) Examples of fluorescence microscopy images of the P39A tetra-Cys CRABP-expressing cells in low-salt medium (Left) and high-osmolality (+300 mM NaCl) medium (Right) taken 180 min after induction. (Magnification: ×1,000.) (B) Effects of the osmolality of the nutrient medium on the partitioning of P39A tetra-Cys CRABP between soluble and insoluble fractions. Note that tetra-Cys CRABP remains soluble at all sodium chloride concentrations (data not shown).

Fig. 2.

Proline internalized at early times in vivo inhibits aggregation. (A) Time evolution of the aggregation of P39A tetra-Cys CRABP monitored by the bulk FlAsH fluorescence signal of labeled cells, under depleted (−ProP) and up-regulated (+ProP) ProP transporter conditions in the E. coli strain WG710. Expression of the soluble tetra-Cys CRABP is not influenced by the amount of the ProP transporter; it remains the same in the absence of the ProP transporter (−ProP) and upon its overexpression (+ProP). (B) Proline influx induced at early times leads to soluble expression of P39A tetra-Cys CRABP. Proline uptake by the ProP transporter was initiated at different times after induction by addition of arabinose and 300 mM NaCl. The expression curves in the absence of ProP (−ProP) and in the presence of fully activated ProP (+ProP) E. coli WG710 are included for comparison.

Fig. 3.

Proline inhibits aggregation at early stages in vitro. The aggregation of 15 μM P39A tetra-Cys CRABP in vitro in the absence or presence of 500 mM proline added at different times is shown. The time evolution of the aggregation was monitored either by FlAsH fluorescence (Upper) or by determination of the quantity of soluble protein remaining after high-speed centrifugation (Lower).

Fig. 4.

Proline inhibits aggregation of a polyQ-containing chimera in vitro and in vivo. (A) Time evolution of the aggregation of tetra-Cys CRABP Htt53 and tetra-Cys CRABP Htt20 in E. coli BL21(DE3) cells cultured in medium of low salt or high osmolality (+300 mM NaCl). Note that the expression curves for tetra-Cys CRABP Htt20 in a low-salinity medium and with 300 mM NaCl-containing medium overlap and that the protein remains soluble over the entire expression cycle. (B) Proline (500 mM) was added to 5 μM aggregating tetra-Cys CRABP Htt53 in vitro at various time points after initiation of the aggregation reaction [by bringing the solution to 1 M urea (28)].

Fig. 5.

A schematic model of proline action on various species in the aggregation pathway. The disfavored species are shown with shadowing, and favored species are indicated by boxes. The solvophobic properties of proline disfavor species with greater solvent-exposed surface area, e.g., partially folded (nucleus) and unfolded ensembles. At early stages, intramolecular interactions are favored by the addition of proline, leading to a relative stabilization of the native state. In the later phases, intermolecular interactions are favored, promoting the formation of aggregated species that effectively sequester solvent-accessible surface area.