Mimicking phosphorylation of alphaB-crystallin affects its chaperone activity

Abstract

AlphaB-crystallin is a member of the sHsp (small heat-shock protein) family that prevents misfolded target proteins from aggregating and precipitating. Phosphorylation at three serine residues (Ser19, Ser45 and Ser59) is a major post-translational modification that occurs to alphaB-crystallin. In the present study, we produced recombinant proteins designed to mimic phosphorylation of alphaB-crystallin by incorporating a negative charge at these sites. We employed these mimics to undertake a mechanistic and structural investigation of the effect of phosphorylation on the chaperone activity of alphaB-crystallin to protect against two types of protein misfolding, i.e. amorphous aggregation and amyloid fibril assembly. We show that mimicking phosphorylation of alphaB-crystallin results in more efficient chaperone activity against both heat-induced and reduction-induced amorphous aggregation of target proteins. Mimick-ing phosphorylation increased the chaperone activity of alphaB-crystallin against one amyloid-forming target protein (kappa-casein), but decreased it against another (ccbeta-Trp peptide). We observed that both target protein identity and solution (buffer) conditions are critical factors in determining the relative chaperone ability of wild-type and phosphorylated alphaB-crystallins. The present study provides evidence for the regulation of the chaperone activity of alphaB-crystallin by phosphorylation and indicates that this may play an important role in alleviating the pathogenic effects associated with protein conformational diseases.

Figure 1. Mimicking phosphorylation of αB-crystallin affects its structure as monitored by fluorescence spectroscopy

Intrinsic tryptophan fluorescence at 37 °C (A) and 60 °C (B), ANS fluorescence at 37 °C (C) and 60 °C (D) and FRET analysis at 37 °C (E) and 60 °C (F) of αB-WT (◆), αB-1P (▲), αB-2P (◇) and αB-3P (□). The proteins (100 μg/ml) were incubated in 50 mM phosphate buffer (pH 7.2) at each temperature for 30 min before the spectra were measured.

Figure 2. Oligomeric distribution of αB-WT and αB-3P

(A) Nanospray mass spectra of αB-WT and αB-3P. The peaks at 10080 and 10200 m/z, corres-ponding to species with two charges per subunit for αB-WT and αB-3P respectively, were iso-lated and subjected to collision-induced dissociation. The doubly stripped oligomers were used to calculate the oligomeric distribution of the intact proteins as described previously [24]. (B) His-tograms quantifying the relative abundance of the various oligomeric species for αB-WT and αB-3P.

Figure 3. Phosphorylation mimics of αB-crystallin are more effective at preventing amyloid fibril formation by RCMκ-casein

(A) ThT binding curves of RCMκ-casein (500 μg/ml) incubated at 37 °C in 50 mM phosphate buffer (pH 7.2) in the absence (■) or presence of αB-WT (◆), αB-1P (▲), αB-2P (◇) or αB-3P (□). The chaperone was at 500 μg/ml and the ThT fluorescence was monitored by an in situ assay for 15 h. The change in ThT fluorescence of each sample is shown. This experiment was performed four times and the results shown are representative. (B) SEC of RCMκ-casein before and after fibril formation and after incubation in the presence of αB-WT and αB-3P. The proteins were loaded on to a Superdex 200HR 10/30 column and eluted in 50 mM phosphate buffer (pH 7.2) at a flow rate of 0.4ml/min. Calibration of the column was performed using (1) Blue Dextran, void; (2) thyroglobulin, 670 kDa; (3) γ-globulin, 158 kDa; (4) ovalbumin, 44 kDa; and (5) myoglobin, 17 kDa.

Figure 4. Fibril formation by RCMκ-casein in the presence of αB-crystallin

Electron micrographs of RCMκ-casein (500 μg/ml) after incubation at 37 °C in 50 mM phosphate buffer (pH 7.2) for 15 h in the absence (A) or presence of αB-WT (B) or αB-3P (C). The chaperone was present at 500 μg/ml. Scale bar, 500 nm.

Figure 5. Mimicking phosphorylation of αB-crystallin decreases its ability to prevent amyloid fibril formation by ccβ-Trp

(A) ThT binding curves of ccβ-Trp (150 μg/ml) incubated at 37 °C in 50 mM phosphate buffer (pH 7.8) in the absence (■) or presence of αB-WT (◆), αB-1P (▲), αB-2P (◇) or αB-3P (□). The chaperone was at 130 μg/ml and the ThT fluorescence was monitored by an in situ assay for 15 h. The change in ThT fluorescence of each sample is shown. (B) After the ThT assay, the insoluble (pellet) fractions were separated and the proteins were resolved by SDS/PAGE. This experiment was performed a minimum of three times and the results shown are representative.

Figure 6. Fibril formation by ccβ-Trp in the presence of αB-crystallin

Electron micrographs of ccβ-Trp (150 μg/ml) after incubation at 37 °C in 50 mM phosphate buffer (pH 7.8) for 15 h in the absence (A) or presence of αB-WT (B, C) or αB-3P (D, E). The chaperone was present at 130 μg/ml. Scale bar, 500 nm.

Figure 7. Phosphorylation mimics of αB-crystallin are more effective at preventing the heat-induced amorphous aggregation of target proteins

Aggregation curves of (A) β_L-crystallin (500 μg/ml) and (C) catalase (cat) incubated at 60 °C in 50 mM phosphate buffer (pH 7.2) and of (E) alcohol dehydrogenase (ADH) incubated at 42 °C in 50 mM phosphate buffer (pH 7.2) containing 100 mM NaCl and 2 mM EDTA. The target proteins were incubated in the absence (■) or presence of αB-WT (◆), αB-1P (▲), αB-2P (◇) or αB-3P (□) and the change in light scattering was measured at 340 nm. In (A, C), the chaperones were added at a final concentration of 100 μg/ml; in (E), they were added at a final concentration of 50 μg/ml. (B, D, F) After the aggregation assay, the soluble and insoluble fractions were separated and the proteins were resolved by SDS/PAGE. Each experiment was performed a minimum of three times and results shown are representative.

Figure 8. Effect of mimicking phosphorylation of αB-crystallin on its chaperone ability to prevent DTT-induced amorphous aggregation of target proteins

Aggregation curves of (A) insulin (250 μg/ml) and (C, E) α-lactalbumin (500 μg/ml) incubated in the absence (■) or presence of αB-WT (◆), αB-1P (▲), αB-2P (◇) or αB-3P (□). (A) Insulin was incubated at 37 °C in 50 mM phosphate buffer (pH 7.2) with 10 mM DTT. α-Lactalbumin was incubated at 37 °C in (C) 50 mM phosphate buffer (pH 7.2) containing 100 mM NaCl with 20 mM DTT, or (E) 100 mM ammonium acetate buffer (pH 6.8) with 20 mM DTT. In each case, the chaperone (250 μg/ml) was added to the reaction mixture and the change in light scattering was measured at 340 nm. (B, D, F) After the aggregation assay, the soluble and insoluble fractions were separated and the proteins were resolved by SDS/PAGE. Each experiment was performed a minimum of three times and the results shown are representative.

Figure 9. Effect of solution condition on the thermal stability and oligomeric distribution of αB-WT and αB-3P

Thermal denaturation curves of αB-WT (◆) and αB-3P (□) in 50 mM phosphate buffer (pH 7.2) containing 100 mM NaCl (A) and 100 mM ammonium acetate buffer (pH 6.8) (B). The proteins were incubated at 250 μg/ml and the temperature was increased at the rate of 1 °C·min⁻¹. Precipitation of the proteins was monitored by measuring the change in light scattering at 360 nm. (C) SEC coupled with multi-angle laser light scattering. αB-WT and αB-3P (1.5 mg/ml) were loaded on to a Superose 6HR 10/30 column and eluted with 100 mM ammonium acetate (pH 6.8) at a flow rate of 0.5 ml/min. The solid lines represent the absorption profile of the eluted protein, and circles represent the molecular mass obtained as a function of the elution volume.