The chaperone action of α-crystallin

Author

Arezou Ghahghaei, University of Wollongong

Year

2006

Degree Name

Doctor of Philosophy

Department

Department of Chemistry - Faculty of Science

Recommended Citation

Ghahghaei, Arezou, The chaperone action of α-crystallin, PhD thesis, Department of Chemistry, University of Wollongong, 2006. http://ro.uow.edu.au/theses/525

Abstract

α-Crystallin is the principal lens protein. It is a member of the small heat shock protein family (sHsp) and acts as a molecular chaperone by stabilizing proteins under stress conditions through the formation of a soluble sHsp target protein complex to prevent their aggregation.

Macromolecular crowding is ubiquitous in all types of cells and describes the normal conditions inside a cell. The concentration of macromolecules inside a cell is very high (up to 300 mg/mL) arising from species such as polysaccharides, proteins and nucleic acids and therefore it greatly promotes the self-assembly of proteins. Thus, there is a major difference between in vivo and in vitro conditions such as those used in most studies of protein behaviour and properties.

Appropriate destabilizing conditions (i.e. heating or reduction) cause damage and misfolding of proteins, which as a result expose previously buried hydrophobic regions. Hydrophobic interactions of nearby molecules cause selfassociation and aggregate formation. Aggregation of intermediately folded peptide or protein molecules also leads to the formation of amyloid fibrils, highly-ordered β-sheet structures associated with a number of neurodegenerative disorders such as Alzheimer’s, Parkinson’s, and Creutzfeldt-Jakob diseases.

In vitro, much work has been published on the interactions of α-crystallin with target proteins in dilute solutions. In order to better understand the chaperone activity of α-crystallin under conditions more closely resembling the intracellular environment, its interaction with a range of destabilized proteins (ovotransferrin, βL-crystallin, insulin, α-lactalbumin, αs- and κ-casein) in the presence of dextran (68 kDa) have been examined using visible absorption spectroscopy, tryptophan fluorescence spectroscopy, ANS binding, TEM, HPLC and NMR spectroscopy studies.

In the presence of dextran, the rate and extent of aggregation of reduced ovotransferrin, insulin, α-lactalbumin and βL-crystallin was accelerated. Under these conditions, α-crystallin was less effective in preventing aggregation and precipitation of target proteins. It is proposed that a kinetic competition exists between aggregation of target proteins and the chaperone action of α-crystallin which supports the hypothesis that α-crystallin interacts more effectively with slowly aggregating rather than rapidly aggregating target proteins.

Amyloid fibril formation by α-lactalbumin, αs- and κ-casein was verified by a sigmoidal increase in Thioflavin T fluorescence over time. α-Crystallin prevented amyloid formation in αs- and κ-casein. In the presence of dextran, the rate of amyloid formation by α-lactalbumin, αs- and κ-casein was enhanced. Under these conditions, α-crystallin was less effective in preventing amyloid formation of κ-casein and this was supported by TEM, CD, NMR spectroscopy and HPLC studies.

Subunit exchange is an important feature of sHsp chaperone action. This study found that subunit exchange of αA-crystallin increased with increasing temperature and decreased as a result of interaction with reduced ovotransferrin. It was further demonstrated that the presence of the dextran markedly reduced the rate of subunit exchange of αA-crystallin and with increasing temperature, this effect was exacerbated. Moreover, in the presence of reduced ovotransferrin, dextran further slowed the subunit exchange of αAcrystallin.

Aggregation of β-lactoglobulin occurs mainly via intermolecular disulphide bond exchange. Upon heating, β-lactoglobulin aggregated which increased with increasing pH. The presence of dextran or DTT led to more rapid aggregation and precipitation of β-lactoglobulin. α-Crystallin prevented the aggregation of heat-stressed β-lactoglobulin and was a more efficient chaperone at higher pH values. In the presence of DTT, however, α-crystallin was a less efficient chaperone due to faster aggregation of heated and reduced β-lactoglobulin.

In order to obtain further structural and functional information on the C-terminal extension, α-crystallin from dogfish (Squalus acanthias) was studied to allow comparisons with bovine α-crystallin to be made. Chaperone assays under heat and reduction stresses as well as NMR spectroscopy showed that the Cterminal extension of dogfish α-crystallin was very flexible and had a similar structure and function to that of bovine α-crystallin. Its chaperone action under heat stress was found to be comparable to bovine α-crystallin but it was a better chaperone under reduction stress.