The Molecular Chaperone, α-Crystallin, Inhibits Amyloid Formation by Apolipoprotein C-II

Abstract

Under lipid-free conditions, human apolipoprotein C-II (apoC-II) exists in an unfolded conformation that over several days forms amyloid ribbons. We examined the influence of the molecular chaperone, ␣-crystallin, on amyloid formation by apoC-II. Time-dependent changes in apoC-II turbidity (at 0.3 mg/ml) were suppressed potently by substoichiometric subunit concentrations of ␣-crystallin (1-10 g/ml). ␣-Crystallin also inhibits time-dependent changes in the CD spectra, thioflavin T binding, and sedimentation coefficient of apoC-II. This contrasts with stoichiometric concentrations of ␣-crystallin required to suppress the amorphous aggregation of stressed proteins such as reduced ␣-lactalbumin. Two pieces of evidence suggest that ␣-crystallin directly interacts with amyloidogenic intermediates. First, sedimentation equilibrium and velocity experiments exclude high affinity interactions between ␣-crystallin and unstructured monomeric apoC-II. Second, the addition of ␣-crystallin does not lead to the accumulation of intermediate sized apoC-II species between monomer and large aggregates as indicated by gel filtration and sedimentation velocity experiments, suggesting that ␣-crystallin does not inhibit the relatively rapid fibril elongation upon nucleation. We propose that ␣-crystallin interacts stoichiometrically with partly structured amyloidogenic precursors, inhibiting amyloid formation at nucleation rather than the elongation phase. In doing so, ␣-crystallin forms transient complexes with apoC-II, in contrast to its chaperone behavior with stressed proteins.

Small Heat‐shock Proteins and Clusterin: Intra‐ and Extracellular Molecular Chaperones with a Common Mechanism of Action and Function?

“…In cell culture experiments in the presence of clusterin, conflicting results were obtained with clusterin enhancing oxidative stress caused by A in [27] but being protective under the conditions used in [28]. Our in vitro studies have shown that fibril formation by apolipoprotein C-II (apoC-II) is potently inhibited by clusterin and -crystallin [16,31]. When exerting this effect, neither chaperone forms a complex with apoC-II.…”

“…The diagram represents the interaction of sHsps and clusterin with amorphously aggregating target proteins. When interacting with amyloid fibril-forming target proteins (under slowly aggregating conditions), complexation may not occur; instead, the partially folded protofibril species is stabilized and large-scale aggregation is prevented [16,[26][27][28]31].…”

Crystallin proteins and amyloid fibrils

“…This may, at least in part, explain why comparatively high molar ratios of αB-crystallin are required to inhibit fibril formation by κ-casein compared to other systems which have been studied (i.e. the molar ratio (target protein: αB-crystallin) to completely inhibit fibril formation for α-synuclein is 1.0: 0.25 [93], apoC-II is 1.0: 0.02 [97] and ccβ-Trp 1.0: 0.1 [95] compared to > 1.0: 1.0 for RCMκ-casein [95]). These differences cannot be accounted for by simple comparison of the masses of the target proteins, as discussed above (mass of α-synuclein is 14.5 kDa, RCMκ-casein is 19.0 kDa, apoC-II is 8.8 kDa and ccβ-Trp is 2.2 kDa).…”

“…Also, the chaperone activity of αB-crystallin can be easily compared with well-established data on its chaperone activity against amorphously aggregating target proteins. Thus, studies have shown that αB-crystallin inhibits the fibrillation of α-synuclein [48, 83,93], β2-microglobulin [94] (Esposito, Carver, et al, unpublished results), κ-casein [95,96], the ccβ-Trp peptide [95], apoC-II [97], and the prion protein (Ecroyd, unpublished results). In doing so, αB-crystallin acts as a chaperone under a range of solvent conditions, including at pH values as low as 2.5 against β2-microglobulin fibrillation [94].…”

“…Studies such as those with apolipoprotein C-II (apoC-II) have shown that, in preventing amyloid fibril formation, sHsps can act at very low substoichiometric ratios and do not form stable complexes with partially folded intermediates of the aggregating target protein [97]. Instead the sHsp and target protein are proposed to form a transient complex by which the sHsp stabilizes the aggregating protein before releasing it so that it may refold back to its native state via the reversible on-folding pathway [97]. This is in contrast to its mechanism of action in preventing fibril formation by α-synuclein and κ-casein, whereby αB-crystallin forms a stable, soluble chaperone-target protein complex with the partially folded intermediate of these target proteins (i.e.…”