Molecular Chaperones and DNA Replication

Division Of Basic Sciences - Nci

Investigators

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Abstract

The first aim of our research is to elucidate the mechanism of action of Hsp90 and the role of the Hsp70 chaperone in protein remodeling with Hsp90. Hsp90 is a highly conserved and highly expressed molecular chaperone. Eukaryotic Hsp90 is known to control the stability and the activity of several hundred substrate proteins. Moreover, it is important for the growth and survival of cancer cells, and drugs targeting Hsp90 are currently being developed. Studies have shown Hsp90 inhibitors exhibit synergistic antitumor activity when used in combination therapies. To gain insight into the mechanism of action of Hsp90 chaperones, we are studying both bacterial and eukaryotic Hsp90. Unlike protein remodeling by eukaryotic Hsp90, which requires many Hsp90 cochaperones, E. coli Hsp90, Hsp90Ec, is independent of Hsp90 cochaperones and thus provides a simpler system to explore the mechanism of Hsp90. We previously discovered that E. coli Hsp90 functions synergistically with E. coli Hsp70 (DnaK) in protein reactivation in vitro. Hsp70 is also an ATP-dependent highly conserved and highly expressed molecular chaperone. Additionally, we showed that E. coli Hsp90 and Hsp70 directly interact and that yeast Hsp90 and Hsp70 also directly interact. Residues in the middle domain of Hsp90 were shown to interact with residues in the nucleotide-binding domain of Hsp70 that are known to interact with J-domain proteins. In a recent study to explore the role of J-domain cochaperones in protein refolding by Hsp90 and Hsp70, we discovered that J-domain proteins facilitate and stabilize the interaction between E. coli Hsp90 and DnaK and between yeast Hsp82 and Ssa1. We tested the hypothesis that J-domain proteins participate in the collaboration between Hsp90 and Hsp70 by simultaneously interacting with Hsp90 and Hsp70. Using E. coli Hsp90, Hsp70 (DnaK), and an E. coli J-domain protein, (CbpA), we detected a ternary complex containing all three proteins. The interaction involved the J-domain of CbpA, the DnaK binding region of E. coli Hsp90, and the J-domain protein binding region of DnaK, where Hsp90 also binds. Our results also showed that E. coli Hsp90 forms binary complexes with E. coli J-domain proteins, DnaJ and CbpA, and that yeast Hsp90, Hsp82, binds a yeast J-domain protein, Ydj1. Altogether the results suggest that binary and ternary complexes of chaperones and cochaperones are transient intermediates in the pathway of protein remodeling and are likely important in the transfer of substrates from Hsp70 to Hsp90. The second aim of our research is to elucidate the mechanism of action of Clp/Hsp100 chaperones in protein disaggregation and proteolysis. Some Clp chaperones, including E. coli ClpB and yeast Hsp104, have the remarkable ability to disaggregate and reactivate insoluble protein aggregates in collaboration with Hsp70 and Hsp70 cochaperones. ClpB is of interest because it is a promising target for the development of antimicrobial drugs since its activity is linked to virulence in many pathogenic bacteria. Our group has been studying the mechanism of action of E. coli ClpB and its collaboration with DnaK and DnaK cochaperones, DnaJ and GrpE. Previous work demonstrated that DnaK and its cochaperones act before ClpB by binding aggregates and recruiting ClpB to the aggregate. Our previous work showed that the collaboration between ClpB and Dnak involves a direct interaction of the middle domain of ClpB with the nucleotide-binding domain of DnaK. Recently, we explored the possibility that E. coli homologs of Hsp70 (DnaK), Hsp90, and ClpB, in combination with two DnaK cochaperones, DnaJ and GrpE, could promote protein disaggregation and reactivation under conditions where the Hsp70-Hsp90 and Hsp70-ClpB bichaperone systems are ineffective. Our results demonstrated that all three chaperones and two chaperones function together to overcome the inhibition of protein disaggregation and reactivation observed when the concentration of DnaK approaches physiological concentrations. ATP hydrolysis and substrate binding by all three chaperones are essential for the collaborative function. The work further showed that ClpB acts early in protein reactivation with DnaK and its cochaperones and that E. coli Hsp90 acts at a later stage, after ClpB. The results highlight the collaboration among chaperones to regulate and maintain proteostasis. Some Clp chaperones associate with a proteolytic component forming ATP-dependent proteases. Adaptor and anti-adaptor proteins regulate Clp proteases. In ongoing collaborations with Susan Gottesman (LMB, NCI) and Alexandra Deaconescu (Rutgers University), we are studying the regulation of the ClpXP protease by an adaptor protein, RssB, and anti-adaptor proteins, IraP, IraD, and IraM. The RssB adaptor protein specifically targets RpoS, the stationary phase sigma factor of E. coli, for degradation by ClpXP during exponential growth. IraP, IraD, and IraM are induced under various stress conditions and inhibit RssB activity, thereby preventing RpoS degradation. A crystal structure of full length RssB bound to beryllium fluoride phosphomimic has been obtained. The results from in vivo, in vitro and structural studies suggest that phosphorylation of RssB modulates the conformation of RssB. Altogether the studies are revealing the mechanism of regulation of proteolysis by RssB and anti-adaptor proteins.