␣B-Crystallin is one of the six known mammalian small heat-shock proteins (sHsps). These are characterized by the presence of a conserved sequence of 80 -100 residues, which constitutes the putative C-terminal domain. Like other sHsps, ␣B-crystallin forms multimeric globular complexes, often in combination with related sHsps. Here we show that in a yeast two-hybrid system, ␣B-crystallin can specifically interact with itself as well as with ␣A-crystallin and Hsp27. Analyses of the separate domains show that the conserved C-terminal domain (C␣B) is essential for this interaction between subunits. To try and detect residues that are important in subunit interaction, the C␣B domain was used in a twohybrid screen as bait to select randomly mutated C␣B mutants. In this way we obtained nine mutants that were still able to interact with wild-type C␣B despite the presence of up to 15 replacements. Similarly, we obtained 16 mutants that were unable to bind, because of the presence of just three to nine replacements. In binding C␣B mutants, lysine residues were most often replaced by glutamic acid residues, and in non-binding C␣B mutants, acidic residues were often found to be replaced by non-charged residues. This indicates that negative charges are important for subunit interaction and we propose a model to explain this role of acidic residues. Furthermore, we observed that two homologs of ␣B-crystallin, ␣A-crystallin and Hsp27, generally interact similarly with the binding and non-binding C␣B mutants as does ␣B-crystallin. This suggests that interactions involved in the complex formation of these three sHsps are largely comparable.␣B-Crystallin is a protein with dual function. It is an abundant structural protein in the vertebrate eye lens (1). Outside the lens it is especially expressed in tissues with high oxidative activity such as heart, striated muscle, and kidney, but also in degenerative disorders of the nervous system (2-4). ␣B-Crystallin belongs to the family of small heat shock proteins (sHsps) 1 (5, 6). The sHsps vary in molecular mass between 12 and 43 kDa and are characterized by the presence of an 80 -100-amino acid long conserved C-terminal region. Most members of this family form large globular particles, up to 700 kDa in size, which can consist of a variable number of subunits.They are capable of preventing the aggregation of unfolding proteins (7-9). This chaperone-like activity may contribute to the most important in situ property of the sHsps, namely their ability to increase the stress tolerance of cells (10). In many cell types ␣B-crystallin is constitutively expressed, while in others the expression can be induced by different kinds of stress (11,12). In normal cells, ␣B-crystallin is mainly present as a soluble cytoplasmic protein. It can form mixed complexes with ␣A-crystallin, as well as with Hsp27 and Hsp20 (9, 13-16). Under certain stress conditions, ␣B-crystallin interacts with intermediate filaments, and in this way it is probably able to modulate the intermediate network (17)(18)(19).The t...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.