| Summary: | Human Hsp27 is a member of the small heat shock protein family that is over-expressed during cellular stress and that is involved in biological functions ranging from inhibition of apoptosis to regulation of cellular glutathione levels. In addition, Hsp27 is an ATP-independent molecular chaperon that binds to unfolding peptides and inhibits their precipitation. Roles for Hsp27 in several human diseases have also been proposed. For example, the expression of Hsp27 by several human tumors has been noted as a potential diagnostic feature or a therapeutic target. Increasing evidence indicates that the biological functions of Hsp27 are linked to the reversible self-association of the protein to form large oligomers in a process that is at least in part regulated by reversible phosphorylation of three Ser residues. The three-dimensional structure of Hsp27 is not available, and relatively few rigorous physical studies of the protein have been reported. In the present study, analytical ultracentrifugation has been used to define self-association of Hsp27 and selected variants as a function of protein concentration, pH, temperature, and ionic strength to evaluate the role of structural domains believed to be functionally significant. These results are correlated with the chaperon activity, as determined by monitoring the inhibition of insulin unfolding, and with the kinetics of subunit exchange, monitored by fluorescence resonance energy transfer. The results establish that wild-type Hsp27 forms a distribution of oligomers that ranges from dimers to at least 32-mers and that oligomerization is highly regulated by temperature but not ionic strength or pH. Moreover, the oligomeric size of Hsp27 increases with increased temperature in a manner that correlates well with increased chaperon activity and rate of subunit exchange. Comparison of results from all three types of experiments obtained for the wild-type protein to those obtained with Hsp27 variants has led to the development of a model for Hsp27 self-association and chaperon activity. === Medicine, Faculty of === Biochemistry and Molecular Biology, Department of === Graduate
|
|---|
