Molecular Chaperones and DNA Replication

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

One of our aims is to elucidate the mechanism of action of Hsp90 and the interplay between Hsp90 and the Hsp70 chaperone system. The Hsp90 family of heat shock proteins is one of the most abundantly expressed and highly conserved families of molecular chaperones. Eukaryotic Hsp90 is known to control the stability and the activity of several hundred substrate proteins, referred to in the field as clients. Moreover, it is important for the growth and survival of cancer cells, and drugs targeting Hsp90 are currently being developed. To gain insight into the mechanism of action of this important family of chaperones, we are studying Hsp90 from E. coli (Hsp90Ec) and yeast (Hsp82). We discovered that Hsp90Ec and the E. coli Hsp70 chaperone system (DnaK, DnaJ and GrpE) act synergistically in protein reactivation in vitro and that Hsp90Ec and DnaK directly interact in the absence of cochaperones. We previously demonstrated that a region of Hsp90Ec in the middle domain of the protein is important for the interaction with DnaK. Additionally, we identified a region in the nucleotide-binding domain of DnaK that interacts with Hsp90Ec. The region of DnaK we identified as important for the interaction with Hsp90Ec overlaps with the region of DnaK that interacts with the J-domain of DnaJ. We found that yeast Hsp90, Hsp82, and Hsp70, Ssa1, directly interact in vitro in the absence of the yeast Hop homolog, Sti1, which was thought to be a bridging protein between Hsp82 and Ssa1 prior to this work. We identified a region in the middle domain of yeast Hsp90 that is required for the interaction with Ssa1 by constructing and analyzing Hsp82 mutants in residues homologous to those we had identified in Hsp90Ec as being important for interaction with DnaK. In vivo results using Hsp82 substitution mutants showed that several residues in this region were important or essential for growth at high temperature. Moreover, mutants in this region were defective in interaction with Ssa1 in cell lysates. In vitro, we found that the purified Hsp82 mutant proteins were defective in direct physical interaction with Ssa1 and in protein remodeling in collaboration with Ssa1 and cochaperones. This region of Hsp90 is also important for interactions with several Hsp90 cochaperones and client proteins, suggesting that collaboration between Hsp70 and Hsp90 in protein remodeling may be modulated through competition between Hsp70 and Hsp90 cochaperones for the interaction surface. We additionally showed that the regions of Hsp90 and Hsp70 that were important for the interaction between the two chaperones were the regions of direct interaction. We constructed single cysteine substitution mutants in Hsp90 and Hsp70 and performed crosslinking experiments. The results of the crosslinking experiments showed that the direct interaction is between a site in the middle domain of Hsp90 and a site in Hsp70 that was previously shown to bind an Hsp70 cochaperone, a J-domain protein (JDP). The same region of interaction was demonstrated in both E. coli and yeast. This work suggests that the direct interaction between Hsp90 and Hsp70 chaperones of E. coli and yeast is an intermediate in the pathway of protein remodeling and likely important in the transfer of the clients from Hsp70 to Hsp90. A second aim is to understand how J-domain proteins (JDPs) target substrates for protein remodeling by Hsp70. Hsp70 is a highly conserved molecular chaperone that plays an important role in protein folding by coupling ATP hydrolysis to the binding and release of substrates. Its function is facilitated by two cochaperones, a J-domain protein that binds substrates and stimulates Hsp70 ATPase and a nucleotide exchange factor. We have explored the ability of J-domain proteins, including E. coli DnaJ and yeast Ydj1, to bind and target substrates for protein remodeling by Hsp70. We constructed substitution mutants in the two C-terminal domains (CTD I and CTD II) of DnaJ and Ydj1 in regions of the proteins not previously shown to bind substrates and tested the ability of the mutants to bind peptides and substrates and to reactivate denatured proteins in collaboration with Hsp70 of E. coli (DnaK) or yeast (Ssa1). Our results show that some of the DnaJ and Ydj1 variants with substitutions in CTD I and CTD II are defective in binding substrates and peptides, indicating that residues in multiple, and previously unidentified, regions of CTD I and CTD II are involved in substrate binding. Moreover, the ability of the mutant proteins to bind different substrates varied, suggesting DnaJ and Ydj1 possess substrate specificity. Additionally, mutants showed defects in reactivation of denatured substrates with DnaK/Ssa1, showing that the residues involved in substrate binding are important for J-domain protein function in collaboration with an Hsp70 protein. Together, these results indicate that J-domain proteins have multiple sites that are important for binding substrates and that the sites exhibit substrate specificity. A third aim is to clarify the mechanism of action of Clp chaperones in protein remodeling and proteolysis and the regulation of Clp proteases by adaptors and anti-adaptors. Bacterial Clp proteases are comprised of an ATP-dependent chaperone component and a protease component. They have analogous structures and functions to the eukaryotic proteasome and the importance of the proteasome in cancer is well documented. E. coli ClpXP is a two-component ATP-dependent protease that unfolds and degrades proteins bearing specific recognition signals. One important substrate of ClpXP is RpoS, the stationary phase RNA polymerase sigma factor of E. coli. ClpXP, like many Clp proteases are regulated by adaptor proteins and anti-adaptor proteins. One ClpXP adaptor protein, RssB, is essential to target RpoS for degradation during exponential cell growth. In response to various stress conditions, one of the several anti-adaptor proteins, IraP, IraM or IraD, interacts with RssB to block RpoS degradation. In collaboration with Susan Gottesman's group (NCI), current work is ongoing to gain an understanding of how one of the anti-adaptor proteins, IraP, physically and functionally interacts with RssB to block RpoS degradation by ClpXP. In our ongoing collaboration, we are also exploring the mechanism of ClpXP regulation by RssB and the inhibition of RssB by other anti-adaptor proteins. Altogether the work is revealing the mechanisms of regulation of proteases by adaptors and anti-adaptors.