ATP-enhanced molecular chaperone functions of the small heat shock protein human alphaB crystallin

Abstract

We report direct experimental evidence that human alphaB-crystallin, a member of the small heat shock protein family, actively participates in the refolding of citrate synthase (CS) in vitro. In the presence of 3.5 mM ATP, CS reactivation by alphaB-crystallin was enhanced approximately twofold. Similarly, 3.5 mM ATP enhanced the chaperone activity of alphaB-crystallin on the unfolding and aggregation of CS at 45 degrees C. Consistent with these findings, cell viability at 50 degrees C was improved nearly five orders of magnitude in Escherichia coli expressing alphaB-crystallin. SDS/PAGE analysis of cell lysates suggested that alphaB-crystallin protects cells against physiological stress in vivo by maintaining cytosolic proteins in their native and functional conformations. This report confirms the action of alphaB-crystallin as a molecular chaperone both in vitro and in vivo and describes the enhancement of alphaB-crystallin chaperone functions by ATP.

Figure 1

ATP enhances the effects of human αB-crystallin on the refolding and reactivation of chemically denatured CS. Effects of human αB-crystallin on the reactivation of 150 nM chemically denatured CS in the absence (A) and presence (B) of 3.5 mM ATP. ▿, 0 nM αB; ▴, 50 nM αB (1:3 αB-to-CS molar ratio); □, 100 nM αB (1:1.5); and •, 150 nM αB (1:1). (Inset) Effects of 3.5 mM ATP on the reactivation of chemically denatured CS by human αB-crystallin shown as percent reactivation at t = 60 min for selected molar ratios of αB-to-CS.

Figure 2

ATP enhances the suppression of CS unfolding and aggregation by human αB-crystallin. Effects of human αB-crystallin on the aggregation at 45°C of 500 nM CS in the absence (A) and presence (B) of 3.5 mM ATP. •, 0 nM αB; □, 12.5 nM αB (1:40 αB-to-CS molar ratio); ▴, 25 nM αB (1:20); and ▿, 50 nM αB (1:10). (Inset) Effects of 3.5 mM ATP on the suppression of CS unfolding and aggregation by human αB-crystallin shown as relative aggregation at t = 60 min for selected molar ratios of αB-to-CS.

Figure 3

Fluorescence spectroscopy of human αB-crystallin in the presence and absence of ATP. Shown are emission spectra for human αB-crystallin in the absence of ATP (trace 1) and in the presence of 350 μM and 3.5 mM ATP (traces 2 and 3, respectively). A similar decrease in peak fluorescence intensity was observed for human αB-crystallin in the presence of 350 nM-350 μM ATP (data not shown). Quenching was observed at concentrations of ATP between 350 nM and 350 μM whereas 3.5 mM ATP resulted in both quenching and a red shift. Spectra were corrected for background emission Raman scattering. Spectra of the buffers (including ATP) were subtracted from the spectra of the protein samples.

Figure 4

Protective effect of human αB-crystallin expression on cell viability and protein stability at 50°C in vivo. (A) The protection of human αB-crystallin expression (pET16b–αB) vs. Control Culture 1 (pET16b; un-induced control) and Control Culture 2 (pET16b–β-galactosidase; overexpressed β-galactosidase) on CFUs at 0, 2, 4, 6, and 8 h after heat shock at 50°C. (B) SDS/PAGE analysis of crude cell lysates of bacterial cultures overexpressing β-galactosidase (Control Culture 2) or (C) human αB-crystallin before induction of protein expression (UI = un-induced cells) and at 0, 2, 4, 6, and 8 h after heat shock at 50°C. Expression of human αB-crystallin was associated directly with protection of protein stability (most evident at early time points) and cell viability in these experiments.