Figure 1.
Location of IF interactive domains in β3-strand (red), β8-strand (yellow), and C-terminal 155–165 residues (blue) of human wild-type αB-crystallin.
The primary sequences for human wild type αB-crystallin, human wild type αA-crystallin, and C. elegans wild type HSP12.2 were aligned using the residue numbers for human αB-crystallin in ClustalX. The boxes and colors in the αB-crystallin sequence correspond with the interactive sequences labeled on the surface of the 3D model. The amino acid substitutions in the αB-crystallin protein constructs are indicated by bold-italics. The β3-strand of αB crystallin, 73DRFSVNLDVKHFS85, was replaced with the corresponding sequences from αA-crystallin, DKFVIFLDVKHFS (αAβ3), or HSP12.2, EKFEVGLDVQFFT (CEβ3). The β8-strand of αB-crystallin, 131LTITSSLS138, was replaced with the corresponding sequences in αA-crystallin, SALSCSLS (αAβ8), or HSP12.2, STVKSHLA (CEβ8). The 155–165 residues were deleted in αB-crystallin to create the Δ155–165 protein construct. The CEβ3, CEβ8, and Δ155–165 αB-crystallin protein constructs were designed to target the desmin interaction sequences.
Figure 2.
(LEFT) Schematic of the 80,000 g (high-speed) sedimentation assay [8], [9], [30]. Desmin was assembled at 22, 37, or 44°C and centrifuged at 80,000 g. The pellet (P) will contain desmin filaments and any aggregates formed as a result of filament-filament interactions. Any αB-crystallin that associates with these filaments or their aggregates will also be cosedimented. The supernatant (S) will contain soluble αB-crystallin and also any assembly intermediates or unassembled desmin. Therefore this assay measures filament assembly and αB-crystallin binding to assembled filaments. (RIGHT) Schematic of the 2,500 g (low speed) centrifugation assay. Individual desmin filaments will not be sedimented by these sedimentation conditions, neither will αB-crystallin. Only when the assembled desmin filaments self-associate into filament aggregates, will these sediment. Therefore this assay measures filament-filament interaction. If αB-crystallin binds to these aggregates, then it too will be cosedimented. Unlike the high-speed assay, it is the aggregate-associated αB-crystallin which will sediment into the pellet fraction (P) rather than the individual filaments and their associated αB-crystallin. The supernatant (S) will contain the free desmin filaments, their associated αB crystallin, desmin assembly intermediates and the unassociated αB-crystallin particles.
Figure 3.
Analysis of negatively stained samples desmin and wild type αB-crystallin protein by electron microscopy.
The morphology of the assembled desmin filaments and the coassembled wild type (WT aB) and various αB-crystallin protein constructs was analysed by electron microscopy. Samples were negatively stained with uranyl acetate and viewed at an 100 kV accelerating voltage. Wild-type αB-crystallin formed monodisperse particles at all three temperatures (WT aB). This was also true for all the αB-crystallin protein constructs (Fig. S1). Desmin, when assembled alone, formed long smooth 10 nm filaments at all three temperatures (Des). When desmin was assembled with wild type αB-crystallin (Des + WT aB), the filaments were not aggregated and some αB-crystallin particles were seen to associate with the filaments (arrowheads). Unassociated particles are indicated (arrows). Coassembly of desmin with either αAβ3, or CEβ3 or αAβ8 αB-crystallin gave similar results to wild type αB-crystallin at all three temperatures. In contrast, both the CEβ8 and Δ155–165 αB-crystallin protein constructs increased desmin filament-filament associations at higher temperatures leading to filament aggregation along with increased αB-crystallin particle association (arrowheads). Bar = 100 nm.
Figure 4.
Analysis of αB-crystallin interaction with desmin filaments using a likelihood ratio test (LRT).
Samples from the shown combinations of desmin and αB-crystallin were analysed by the described LRT. Examples of some of the selected images are shown. Strong statistical evidence (G2 log-likelihood scores 16.8–64.2, P values = <0.001%) was found that the αB-crystallin particles of the samples tested were positively associated with the desmin filaments (see summary table) in these samples. Bar = 100 nm.
Figure 5.
Gel electrophoretic analysis of the low- and high-speed sedimentation properties of desmin and wild type and mutant αB-crystallins.
(A) The low- and high-speed sedimentation properties of each individual protein was determined at 3 different temperatures. The pellet (P) and supernatant (S) fractions were analysed by SDS-PAGE and the proportion of each protein in each fraction determined. By high-speed sedimentation assay, which measures the efficiency of desmin assembly, virtually all the desmin had pelleted at 22°, 37° and 44°C. By low-speed sedimentation assay, there was a temperature dependent increase in the proportion of desmin sedimented. The αB-crystallin remained largely in the supernatant fractions of both sedimentation assays. (B) Analysis of desmin pelleted by high- and low-speed sedimentation assay in the presence of either wild-type or the various αB-crystallin protein constructs at three different temperatures. αAβ3 αB-crystallin (aAb3 aB) reduced the proportion of desmin filaments sedimenting at low-speed at 44°C. Conversely, the CEβ8 and the Δ155–165 αB-crystallin protein constructs induced the complete low-speed sedimentation of desmin at 44°C. For each sedimentation assay, the band intensities were quantified and then combined with two other data sets to determine statistical significance and summarized in Figs. 5 and 6.
Figure 6.
Desmin sedimentation characteristics in the presence of wild type and various αB-crystallin protein constructs.
Bar chart of the low-speed (light and dark blue; lower portion of each bar) and high-speed (red) sedimentation assay data for desmin coassembled with either wild type (WT aB) or the various αB-crystallin protein constructs. The percentage of desmin in each pellet fraction at 22, 37 and 44°C was determined after both low- and high-speed sedimentation assay. The mean % from three independent experiments with its corresponding standard error was calculated for each and then plotted as a composite bar chart. The assembled desmin is pelleted by high-speed sedimentation assay. At low-speed, only the assembled filaments that have formed filament-filament interactions are pelleted. Neither temperature nor the presence of the various αB-crystallin protein constructs changed significantly the proportion of desmin pelleted in the high-speed assay. The significant differences are seen in the low-speed sedimentation assay. At 44°C, αAβ3 αB-crystallin (Des + aAb3 aB) produced a significant reduction in desmin pellet fraction (dark blue bar). Conversely, both the CEβ8 (Des + CEb8 aB) and Δ155–165 (Des + d155 aB) αB-crystallin protein constructs caused significant increases in the proportion of desmin pelleted at 44°C (dark blue bars). This was also true at 37°C for the Δ155–165 (Des + d155 aB) αB-crystallin protein construct.
Figure 7.
Cosedimentation of αB-crystallin with and without desmin filaments.
A. Cosedimentation of αB-crystallin with desmin filaments. Summary of the low-speed (light blue and dark blue) and high-speed (light red and dark red) sedimentation data for various αB-crystallin protein constructs coassembled with desmin, as quantified by gel densitometry. The percentage of αB-crystallin in the pellet fractions at 22°, 37° and 44°C was determined after both low- and high-speed sedimentation to quantify the association of αB-crystallin with the sedimented desmin filaments. The most striking observation is that both the CEβ8 (CEb8 aB + Des) and Δ155–165 (d155 aB + Des) αB-crystallin protein constructs showed significant increases in desmin binding at 44°C as shown by the high speed assay (44°C, dark red bars). Conversely, αAβ3 αB-crystallin (aAb3 aB + Des) showed significantly decreased association at 44°C at high speed. The CEβ3 (CEb3 aB) and αAβ8 (aAb8 aB) protein constructs showed similar sedimentation properties to wild type αB-crystallin. B. Aggregation of wild type and mutant αB-crystallins as measured by low- and high-speed sedimentation. Summary of the low-speed (blue) and high-speed (red) sedimentation data for the various αB-crystallin protein constructs as quantified by gel densitometry. The percentage of αB-crystallin in the pellet fractions at 22°, 37° and 44°C was determined after both low- and high-speed sedimentation assay to quantify the aggregation of the αB-crystallins. All the other protein constructs showed similar sedimentation properties to the wild type (WT aB) αB-crystallin, except Δ155–165 (d155 aB) αB-crystallin at 44°C, which showed increased aggregation by both low- (darker blue) and high-speed (darker red) sedimentation assay.
Figure 8.
Summary of the influences of αB-crystallin on desmin filaments.
The β3- and β8-strands and the Δ155–165 sequences (C-terminal domain) in αB-crystallin were identified from peptide array studies as being desmin interaction sequences. In wild type αB-crystallin these sequences contribute to the interaction of the αB-crystallin oligomers with desmin filaments to prevent their self-association and the formation of filament-filament aggregates. This activity can be increased by substituting the β3-strand from other small heat shock proteins (αA-crystallin and C. elegans HSP12.2). Substituting the β8-strand in αB-crystallin or removing the 155–165 residues appears to lead to the loss of this activity, but increases the binding of αB-crystallin to desmin filaments. This in turn will encourage increased filament-filament interactions, which in the case of the many point mutations in αB-crystallin linked to inherited myopathies, then leads to protein inclusion formation and the appearance of the histopathological feature of desmin-related myopathies – protein inclusions containing both desmin and αB-crystallin.
Table 1.
Comparison of the chaperone activities of WT and the various αB-crystallin protein constructs used in this study with different client proteins.