Molecular Chaperones and DNA Replication

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

Our research focuses on elucidating the mechanism of action of Hsp90 and the interplay between Hsp90 and the Hsp70 chaperone system. 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, 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. Our group has been studying Hsp90 and Hsp70 from E. coli (Hsp90Ec and DnaK, respectively) and yeast (Hsp82 and Ssa1). These model systems are excellent systems for our studies due to the high conservation of the chaperones across species. We discovered that Hsp90Ec and the DnaK 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 one of its cochaperones, DnaJ. We also showed 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. Additionally, we 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 showed that the direct interaction is between a site in the middle domain of Hsp90 and a site in Hsp70, which had been previously shown to bind J-domain proteins, which are Hsp70 cochaperones. DnaJ and CbpA are J-domain proteins in E. coli and Ydj1 is a J-domain protein in yeast. The same region of interaction between Hsp90 and Hsp70 was demonstrated in both E. coli and yeast. These results suggest 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. Recently we found that J-domain proteins facilitate and stabilize the interaction between Hsp90Ec and DnaK and between Hsp82 and Ssa1 and are exploring the role of J-domain proteins in the Hsp90-Hsp70 interaction. 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 a 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. Additionally, results showed that E. coli Hsp90 interacts with E. coli J-domain proteins, DnaJ and CbpA, and that yeast Hsp90, Hsp82, interacts with a yeast J-domain protein, Ydj1. Together these results suggest that the complexes may be transient intermediates in the pathway of collaborative protein remodeling by Hsp90 and Hsp70. In addition, we are studying the mechanism of action of Clp/Hsp100 chaperones in proteolysis. Clp chaperones unfold proteins by threading substrates through a central pore. Some Clp chaperones associate with a proteolytic component forming ATP-dependent proteases with analogous structures and functions to the eukaryotic proteasome. Adaptors and anti-adaptors regulate Clp proteases. ClpXP is an E. coli Clp protease that unfolds and degrades proteins bearing specific recognition signals. One important ClpXP substrate is RpoS, the stationary phase RNA polymerase sigma factor of E. coli. RpoS degradation by ClpXP is regulated by the ClpX adaptor protein, RssB. RssB is essential to target RpoS for degradation during exponential cell growth. In response to various stress conditions, one of the several ClpXP anti-adaptor proteins, IraP, IraM or IraD, interacts with RssB to block RpoS degradation. In collaboration with Susan Gottesman (NCI) and Alexandra Deaconescu (Brown University), current work is ongoing to gain an understanding how phosphorylation of RssB, which is known to stimulate the interaction of RssB with RpoS and to facilitate degradation, modulates the conformation of RssB. Altogether the in vivo, in vitro and structural studies are revealing the mechanism of regulation of proteolysis by the RssB adaptor and by anti-adaptors.