Small heat shock protein activity is regulated by variable oligomeric substructure

Abstract

The alpha-crystallins are members of the small heat shock protein family of molecular chaperones that have evolved to minimize intracellular protein aggregation; however, they are also implicated in a number of protein deposition diseases. In this study, we employed novel mass spectrometry techniques to investigate the changes in quaternary structure associated with this switch from chaperone to adjuvant of aggregation. We replicated the oligomeric rearrangements observed for post-translationally modified alpha-crystallins, without altering the protein sequence, by refolding the alpha-crystallins in vitro. This refolding resulted in a loss of dimeric substructure concomitant with an augmentation of substrate affinity. We show that packaging of small heat shock proteins into dimeric units is used to control the level of chaperone function by regulating the exposure of hydrophobic surfaces. We propose that a bias toward monomeric substructure is responsible for the aberrant chaperone behavior associated with the alpha-crystallins in protein deposition diseases.

FIGURE 1.

A, binding of ANS to αB and αB_R in both PBS (dark gray bars) and ammonium acetate (light gray bars) revealed that the refolded protein has more exposed hydrophobic surfaces. The fluorescence observed was 343 and 394 fluorescence units in PBS and 1152 and 1310 fluorescence units in ammonium acetate for αB and αB_R, respectively. B, similarly, an increase in the amount of kynurenine (Kyn) binding was observed for the refolded protein. C, assaying the chaperone activity of αB toward apo-α-lactalbumin (αLac) revealed that both proteins reduced the rate of aggregation relative to the control (circles). αB_R (inverted triangles) reduced aggregation more efficiently than αB(squares), however, reflecting increased substrate affinity or capacity.

FIGURE 2.

A, shown are the MS/MS spectra of αB_R at accelerating voltages of 110 V (lower panel) and 170 V (upper panel). Dissociation of monomers from the parent oligomers resulted in singly and doubly stripped oligomers, with more of the latter at the higher voltage. B, plotting the relative abundance of the species as a function of acceleration voltage demonstrated the sequential nature of the dissociation. C, an expansion of the area boxed in A shows the very clear separation of peaks achieved. Doubly stripped species of 23–32 subunits are clearly resolved.

FIGURE 3.

A, histograms derived from MS/MS data show the relative abundances of the oligomeric species that comprise αA and αB. The lower and upper panels correspond to the proteins before and after in vitro refolding, respectively. In each case, the tendency to favor even-numbered oligomers that was evident for the native proteins (lower panels) was abolished (upper panels) by the process of in vitro refolding. B, shown is the percentage of doubly stripped oligomers (DSOs; relative to singly stripped) as a function of initial kinetic energies for αA and αB before and after in vitro refolding. •, oligomers containing an even number of subunits; ○, oligomers containing an odd number of subunits. The dissociation profiles show that oligomers of the α-crystallins with an even number of subunits show differential dissociation behavior compared with odd-numbered oligomers and that this difference is lost upon refolding.

FIGURE 4.

A, we propose that the α-crystallins can exist as oligomers with dimeric or monomeric substructure and combinations thereof. The transition from a relatively inactive, low affinity state to an active high affinity state is dictated by the ratio of the types of substructure in the assembly. Dissociation of dimers to monomers leads to the exposure of surfaces in the former contact regions, thereby increasing substrate affinity. B, we propose that, at an ideal ratio of substructures, the net protection conferred by the proteins is maximized. The observation that the α-crystallins are present in a number of age-related protein deposition diseases indicates, however, that they can also be hyperactivated, giving rise to co-precipitation phenomena in vivo. We suggest that sequence changes such as post-translational modifications or mutations disrupt their dimeric substructure sufficiently such that a critical level of substrate affinity is exceeded. At this point, the proteins lose the ability to limit the number of substrates they bind, leading to aberrant chaperone activity and co-precipitation with client proteins.