The selective inhibition of serpin aggregation by the molecular chaperone, alpha-crystallin, indicates a nucleation-dependent specificity.
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. alpha1-Antichymotrypsin (ACT) and alpha1-antitrypsin (alpha1-AT) are two closely related members of the serpin superfamily that aggregate through nucleation-dependent and nucleation-independent pathways, respectively. The sHsp alpha-crystallin was unable to prevent the nucleation-independent aggregation of alpha1-AT, whereas alpha-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 alpha-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 alpha-crystallin interacts specifically with a non-native conformation of ACT. The finding that alpha-crystallin does not interact with alpha1-AT under these conditions suggests that alpha-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 alpha-crystallin and other sHsps.
D002850:Chromatography, Gel; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D011994:Recombinant Proteins / Q000378:metabolism; D015843:Serpins / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D038201:alpha-Crystallins / Q000378:metabolism*
D011994:Recombinant Proteins; D015843:Serpins; D038201:alpha-Crystallins