Partially Folded Aggregation Intermediates of Human yD-, yC-, and yS-Crystallin Are Recognized and Bound by Human αB-Crystallin Chaperone

JOURNAL OF MOLECULAR BIOLOGY

卷:401

Article

Acosta-Sampson, Ligia¹  King, Jonathan¹ 

[1] MIT, Dept Biol, Cambridge, MA 02139 USA

关键词: alpha-crystallin;    cataracts;    small heat shock protein;    chaperone;    aggregation;   

DOI  :  10.1016/j.jmb.2010.05.067

来源: Elsevier

【 摘 要 】

Human gamma-crystallins are long-lived, unusually stable proteins of the eye lens exhibiting duplicated, double Greek key domains. The lens also contains high concentrations of the small heat shock chaperone alpha-crystallin, which suppresses aggregation of model substrates in vitro. Mature-onset cataract is believed to represent an aggregated state of partially unfolded and covalently damaged crystallins. Nonetheless, the lack of cell or tissue culture for anucleate lens fibers and the insoluble state of cataract proteins have made it difficult to identify the conformation of the human gamma-crystallin substrate species recognized by human alpha-crystallin. The three major human lens monomeric gamma-crystallins, gamma D, gamma C, and gamma S, all refold in vitro in the absence of chaperones, on dilution from denaturant into buffer. However, off-pathway aggregation of the partially folded intermediates competes with productive refolding. Incubation with human alpha B-crystallin chaperone during refolding suppressed the aggregation pathways of the three human gamma-crystallin proteins. The chaperone did not dissociate or refold the aggregated chains under these conditions. The alpha B-crystallin oligomers formed long-lived stable complexes with their gamma D-crystallin substrates. Using alpha-crystallin chaperone variants lacking tryptophans, we obtained fluorescence spectra of the chaperone-substrate complex. Binding of substrate gamma-crystallins with two or three of the four buried tryptophans replaced by phenylalanines showed that the bound substrate remained in a partially folded state with neither domain native-like. These in vitro results provide support for protein unfolding/protein aggregation models for cataract, with a-crystallin suppressing aggregation of damaged or unfolded proteins through early adulthood but becoming saturated with advancing age. (C) 2010 Elsevier Ltd. All rights reserved.

【 授权许可】

Free