|Budget Amount *help
¥2,200,000 (Direct Cost : ¥2,200,000)
Fiscal Year 1996 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1995 : ¥1,400,000 (Direct Cost : ¥1,400,000)
We recently identified a 40-kDa heat shock protein Hsp40 in mammalian cells. Hsp40 is one of the homologues of bacterial DnaJ heat shock protein. We have already reported that Hsp40 forms complex with Hsp70 in the cytosol and translocates into the nuclei and nucleoli upon heat shock, and that both Hsp40 and Hsc70 bind to the nascent polypetide chains emerging from translating ribosomes and function as molecular chaperons, mediating their correct folding.
We here investigated heat shock-induced nuclear translocation of Hsp40 and Hsp70 in several mamalian cells including human (HeLa), rat (NRK) and Chinese hamster (HA-1). Heat-induced translocation kinetics of Hsp40 is very similar to that of Hsp70 that indicates cotraslocation. Also, both Hsps colocalize in the nucleoli in the same cells, suggesting they work together in repairing heat-denatured proteins.
We next examined biochemical characterization of purified recombinant Hsp40 and Hsc70. The Hsc70 has weak intrinsic ATPase activity. Th
e ATPase activity is stimulated 2-3 fold by Hsp40. This stimulation effect of Hsp40 is maximum at 45ﾟC,suggesting that Hsc70 and Hsp40 function together as molecular chaperones more efficiently under severe stress conditons. Also, we showed that both Hsc70 and Hsp40 act cooperatively to suppress the aggregation and restore the enzyme activity of denatured protein in an ATP-dependent manner using rhodanese as model protein. However, Hsp4- itself does not have such kind effect.
We tried to establish semi-intact cell system to investigate molecular mechanism of nuclear translocation of Hsps, but the semi-intact cell system did not work well under our experimental conditions. Hsp40 (340 amino acids) has several basic amino acid clusters which might be involved in the nuclear translocation (59-61,157-159,181-182,294-296). Therefore, these basic amino acids were changed to neutral amino acids by site-directed mutagenesis. We are now examining whether these mutant proteins translocate into the nuclei by heat shock Less