Protein interactions ofhsp27.

Abstract

Heat shock protein (hsp) 27 is a member of the small heat shock protein family. The amount of hsp27 in cells correlates with the ability of cells to survive toxic or stressful conditions. The protective effect of hsp27 is believed to occur as a result of hsp27 stabilizing microfilaments, assisting in re-folding of denatured proteins, or by modulating the redox status of cells. Hsp27 exists in cells as oligomers and its activities have been shown to depend on the size of oligomers. Observations have also suggested that hsp27 may interact with other proteins. Thus, how hsp27 forms oligomers is a subject of interest to biologists. This thesis project has focused on the protein-protein interactions of hsp27, both with itself and with other proteins. Using the yeast two-hybrid system, I have shown that hsp27 binds to itself via a 30-amino acid region within the conserved domain shared by the small hsp family. Interaction of hsp27 with itself or with another small hsp, alphaB-crystallin, does not require the N-terminal half or the C-terminal tail of hsp27. These results indicate that hsp27 forms dimers from which larger oligomers then assemble. To identify others proteins with which hsp27 interacts, the yeast two-hybrid system was also used to screen a cDNA library prepared from rat Sertoli cells. A novel hsp27 binding protein was identified from the Sertoli cell library and has been named PASS1 (<italic>p</italic>rotein <italic> a</italic>ssociated with <italic>s</italic>mall <italic> s</italic>tress proteins 1). PASS1 was confirmed to bind to hsp27 by GST pull-down assay and by co-immunoprecipitation, primarily via a 100-amino acid region near its N-terminal terminus. PASS1 was demonstrated by Northern hybridization and Western blot to be expressed most abundantly in Sertoli cells and in kidney. Expression of PASS1 in cultured cells was shown to inhibit the ability of hsp27 to protect the cells from heat shock. This result indicates that PASS1 is unlikely a component of the cell protective mechanism of hsp27 and suggests that PASS1 may affect the oligomer structure of hsp27 by its binding to hsp27 and thus may have regulatory effects on hsp27 <italic>in vivo</italic>.