The Selective Inhibition of Serpin Aggregation by the Molecular Chaperone, α-Crystallin, Indicates a Nucleation-dependent Specificity

Abstract

Small heat shock proteins (sHsps) are a ubiquitous family of molecular chaperones that prevent the misfolding and aggregation of proteins. However, specific details about their substrate specificity and mechanism of chaperone action are lacking. ␣ 1 -Antichymotrypsin (ACT) and ␣ 1 -antitrypsin (␣ 1 -AT) are two closely related members of the serpin superfamily that aggregate through nucleation-dependent and nucleation-independent pathways, respectively. The sHsp ␣-crystallin was unable to prevent the nucleation-independent aggregation of ␣ 1 -AT, whereas ␣-crystallin inhibited ACT aggregation in a dose-dependent manner. This selective inhibition of ACT aggregation coincided with the formation of a stable high molecular weight ␣-crystallin-ACT complex with a stoichiometry of 1 on a molar subunit basis. The kinetics of this interaction occur at the same rate as the loss of ACT monomer, suggesting that the monomeric species is bound by the chaperone. 4,4 -Dianilino-1,1 -binaphthyl-5,5 -disulfonic acid (Bis-ANS) binding and far-UV circular dichroism data suggest that ␣-crystallin interacts specifically with a non-native conformation of ACT. The finding that ␣-crystallin does not interact with ␣ 1 -AT under these conditions suggests that ␣-crystallin displays a specificity for proteins that aggregate through a nucleation-dependent pathway, implying that the dynamic nature of both the chaperone and its substrate protein is a crucial factor in the chaperone action of ␣-crystallin and other sHsps.

Small Heat-shock Proteins Prevent α-Synuclein Aggregation via Transient Interactions and Their Efficacy Is Affected by the Rate of Aggregation

“…monomeric forms that are in a disordered, intermediate state (68,69). Dynamic, transient interactions between the target protein and the sHsp are also crucial factors in determining chaperone efficacy with target proteins (70).…”

The small heat shock protein Hsp27 binds α-synuclein fibrils, preventing elongation and cytotoxicity

“…Instead, the interactions with aggregation-prone proteins are often weak and transient, and do not result in stable complexes. Thus, rather than solely acting as holdases, the molecular mechanisms underlying the interactions between sHsps and aggregating target proteins is multi-faceted and is likely to depend on the stability of the target protein (10,11,19).…”

The Effect of Molecular Chaperone, Alpha-Crystallin, on the Heat-Induced Aggregation of Beta-lactoglobulin

“…The effect of the molecular chaperone, alpha scasein, on preventing the aggregation of one of its natural targets, heat-stressed beta-lactoglobulin, has been previously demonstrated [29] , whereby Alpha scasein effectively suppressed thermally induced aggregation of beta-lactoglobulin at 70°C and pH 7.0 and this chaperone ability of alpha s -casein decreased with increasing pH which is the opposite in fact seen for alpha-crystallin. Devlin et al [21] observed that alpha-crystallin was not able to prevent the aggregation of serpin (AT), which aggregates via a polymerization mechanism rather than a nucleation-dependent mechanism. However, alpha-crystallin inhibited the aggregation of a serpin (ACT), which aggregated via a nucleation-dependent mechanism.…”

“…the aggregation is not (in the main) due to nucleationdependent processes [7,9] but arises from polymerization. In a previous study [21] indicated that alpha-crystallin did not inhibit the aggregation of a serpin (AT (antitrypsin)) which aggregated via a polymerization reaction but did inhibit a serpin aggregation (ACT (antichymotrypsin)) when occurring via a nucleation-dependent mechanism.…”