Abstract
Amyloid fibril formation occurs from a wide range of peptides and proteins and is typically associated with a loss of protein function and/or a gain of toxic function, as the native structure of the protein undergoes major alteration to form a cross β-sheet array. It is now well recognised that some amyloid fibrils have a biological function, which has led to increased interest in the potential that these so-called functional amyloids may either retain the function of the native protein, or gain function upon adopting a fibrillar structure. Herein, we investigate the molecular chaperone ability of α-crystallin, the predominant eye lens protein which is composed of two related subunits αA- and αB-crystallin, and its capacity to retain and even enhance its chaperone activity after forming aggregate structures under conditions of thermal and chemical stress. We demonstrate that both eye lens α-crystallin and αB-crystallin (which is also found extensively outside the lens) retain, to a significant degree, their molecular chaperone activity under conditions of structural change, including after formation into amyloid fibrils and amorphous aggregates. The results can be related directly to the effects of aging on the structure and chaperone function of α-crystallin in the eye lens, particularly its ability to prevent crystallin protein aggregation and hence lens opacification associated with cataract formation.
Highlights
The crystallin proteins are primarily found within the mammalian eye lens where they form part of the protein array that focuses light onto the retina via a supramolecular, liquid-like order [1,2,3].Crystallins are highly stable proteins, as there is very limited protein turnover in the lens, in its centre [4,5]. α-crystallin, the predominant protein of the human lens, is a heterogeneous oligomer comprised of two closely related subunits, αA- and αB-crystallin, in a ratio of approximately 3:1, respectively [6]. α-crystallin oligomers are composed of between 15 and 50 of these subunits and have an average mass of approximately 700 kDa [7,8]
2.1. α-Crystallin Amyloid Fibril Formation α-crystallin was extracted from bovine lenses and purified as described by Chiou et al (1979) [45]. α-crystallin amyloid fibrils were formed by partial unfolding in denaturant at elevated temperature [4,46] and the transformation from native to fibrillar state was confirmed by transmission electron microcopy (TEM) (Figure 1A,B)
The fibrils ranged in length from 20 nm to 1 μM and showed the characteristic morphologies previously described for α-crystallin amyloid fibrils [46]
Summary
The crystallin proteins are primarily found within the mammalian eye lens where they form part of the protein array that focuses light onto the retina via a supramolecular, liquid-like order [1,2,3].Crystallins are highly stable proteins, as there is very limited protein turnover in the lens, in its centre [4,5]. α-crystallin, the predominant protein of the human lens, is a heterogeneous oligomer comprised of two closely related subunits, αA- and αB-crystallin, in a ratio of approximately 3:1, respectively [6]. α-crystallin oligomers are composed of between 15 and 50 of these subunits and have an average mass of approximately 700 kDa [7,8]. Α-crystallin, the predominant protein of the human lens, is a heterogeneous oligomer comprised of two closely related subunits, αA- and αB-crystallin, in a ratio of approximately 3:1, respectively [6]. Α-crystallin oligomers are composed of between 15 and 50 of these subunits and have an average mass of approximately 700 kDa [7,8]. The two α-crystallin subunits are members of the small heat-shock protein (sHsp) family of molecular chaperone proteins. Their chaperone action is important in maintaining the stability and solubility of other crystallin proteins. Significant research activity has been undertaken into the mechanism by which αA- and αB-crystallin act as chaperones, both as homo- and hetero-oligomers. Delineation of the means by which sHsps act as chaperones could have significant potential therapeutically
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