Abstract
Purpose: :
Ferritin, a member of family of iron storage proteins, is found in a wide variety of different human tissues including cornea, retina and RPE. Ferritin has been shown to increase in response to stress and inflammation and to sequester iron-II in the cell. In the eye rising levels of ferritin were detected in hyper-ferritinemia cataract and age-related macular degeneration. Ferritin expression under oxidative or temperature stress conditions and its protective effect on cell viability suggest that ferritin lacking iron (apoferritin) may have a role in the formation, stabilization or maintenance of the native conformation of proteins.
Methods: :
To test this hypothesis we studied the influence of apoferritin on the unfolding and refolding of citrate synthase (CS) in vitro using aggregation assay, size exclusion chromatography, SDS-PAGE and Western blot analysis, and fluorescent spectroscopy. The native state of CS was determined by measuring catalytic activity.
Results: :
Here we show that at stoichiometric amounts apoferritin protects CS catalytic activity, stabilize the aggregation of CS under heat stress,and acts as a chaperone-like molecule in these folding reactions in vitro. Furthermore, apoferritin promotes the functional refolding of CS after guanidinium hydrochloride denaturation. Experiments on biotin label transfer demonstrated that iron-free apoferritin participates in transient protein-protein interactions with protein substrates, whereas ferritin does not show this interaction.
Conclusions: :
These results confirm that apoferritin has chaperone-like activity in vitro and suggests that in addition to its iron binding activity apoferritin might have a role in protecting and maintaining the native conformation of proteins in the eye.
Keywords: protein structure/function • chaperones • stress response