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We have investigated whether HspBP1, a Hsp70 binding protein, could have effect on the assembly of the bovine progesterone receptor (bPR) with a chaperone complex consisting of bovine Hsp90 (bHsp90), bovine Hsp70 (bHsp70), Hop, Ydj-1, and p23. The bPR, isolated in its native conformation, loses its function to interact with progesterone hormone in the absence of this protein complex. However, in the presence of bHsp90, bHsp70, Hop, p23 and Ydj-1, its function could be restored in vitro. Our findings here indicate that the inclusion of HspBP1 to five-protein system prevented the proper assembly of progesterone receptor-chaperone complex and induce the loss of bPR ability to interact with hormone. Immunoprecipitation assays of bPR with HspBP1 show that the presence of HspBP1 did not have any effect on the assembly of Ydj-1 and bHsp70 with the progesterone receptor. However, further assembly of Hsp90, Hop and p23 was completely prevented and the function of the bPR was lost. In vitro competition and protein folding assays indicated that the binding of HspBP1 to bHsp70 prevented the ternary complex formation of bHsp70, bHsp90, and Hop. These results indicate that HspBP1 is a negative regulator of the assembly of Hsp90, Hop and Hsp70, and thus, prevent the proper maturation of unliganded bPR with chaperones assembly system. |
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