Molecular Chaperones and DNA Replication

Division Of Basic Sciences - Nci

Investigators

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Abstract

Molecular chaperones function during non-stress conditions to facilitate folding of newly synthesized proteins, to remodel protein complexes, and to target for degradation regulatory proteins and misfolded proteins. During cell stress, chaperones play an essential role in preventing the appearance of folding intermediates that lead to irreversibly damaged and aggregated proteins. They promote recovery from stress by disaggregating and reactivating proteins, a process not long ago thought to be impossible. They are also involved in delivering damaged proteins to compartmentalized proteases. Protein aggregation and misfolding are primary contributors to a large number of human diseases, including Alzheimers, Parkinsons, type II diabetes, cystic fibrosis, and prion diseases. Understanding how chaperones function and how they interact with proteases will provide the foundation for discovering cures and preventions for the many diseases caused by protein misfolding, premature degradation, and formation of toxic protein aggregates. To better understand the mechanisms involved in ATP-dependent protein remodeling and disagggregation we have been studying protein disaggregation by two members of the Clp/Hsp100 family of molecular chaperones, ClpB of prokaryotes and its yeast homolog, Hsp104. Both ClpB and Hsp104 have been shown to dissolve insoluble aggregated proteins in combination with a second molecular chaperone system composed of DnaK, DnaJ and GrpE in E. coli and Hsp70, Hsp40 and NEF in yeast. We are currently investigating the function of the DnaK/Hsp70 system in conjunction with ClpB/Hsp104 and the interaction between the two chaperone systems. Although ClpB and Hsp104 have high sequence homology, they show species specificity for their DnaK/Hsp70 partner. Both in vivo and in vitro ClpB acts with DnaK of E. coli and is inactive with yeast Hsp70. Similarly, Hsp104 acts with yeast Hsp70 and is inactive with E. coli DnaK. To gain insight into the species specificity of the two chaperones, we have exchanged one or more of the four domains of ClpB and Hsp104 by genetic engineering. We are now testing the chimeras for their ability to cooperate with the DnaK or Hsp70 system both in vivo and in vitro. Our results suggest that the middle domain of ClpB/Hsp104 is involved in the collaboration with the DnaK/Hsp70 system. To identify amino acid residues in the middle-domain of ClpB involved in the interaction between the two chaperones, we have constructed site-directed mutants. We chose regions that would be anticipated to be surface exposed based on the model of the crystal structure. We obtained some mutants that are defective in collaborating with the DnaK chaperone system in protein disaggregation, although their DnaK independent activities are similar to wild type. Thus, we have identified specific ClpB residues and regions that are important for the collaboration between ClpB and DnaK. The further characterization of these chimeras and mutants will more clearly define the interaction of the two chaperone systems and shed light on the mechanism of action of the DnaK/Hsp70 system in its collaboration with ClpB/Hsp104. We have also been exploring the mechanism ATP utilization by ClpB, which is a hexameric protein containing 12 ATP binding sites arranged in two rings of six. Each ClpB protomer contributes one nucleotide-binding site to each ring. Results from subunit mixing experiments show that when ClpB acts alone, approximately six active and six inactive nucleotide-binding sites are required for optimal protein remodeling. The location of the active and inactive sites in the hexamer is not important. Approximately one protomer with two hydrolytically active ATP binding sites per hexamer is sufficient to support remodeling activity, indicating that ClpB can act by a probabilistic mechanism in the absence of the DnaK system. However, when ClpB acts in conjunction with the DnaK system, introduction of approximately one protomer with two inactive ATP binding sites blocks protein disaggregation, supporting a sequential mechanism of ATP utilization by the two rings. Taken together the results suggest that the mechanism of ATP utilization by ClpB is adaptable and can vary from probabilistic to sequential depending on the presence of the DnaK system and the specific substrate. Altogether these studies will provide insight into the role of these important chaperones in protein homeostasis and in diseases of protein aggregation. We are also studying another Clp/Hsp100 chaperone, ClpX, which associates with a proteolytic component, ClpP, forming an ATP-dependent protease, ClpXP. In vitro, we found that ClpXP degrades FtsZ, the major cytoskeletal protein in bacteria and a tubulin homolog. FtsZ performs an essential role in bacterial cell division by polymerizing and forming the FtsZ-ring at midcell where division occurs. We found that ClpXP degrades both polymers and monomers of FtsZ and that the N-terminal domain of ClpX is important for recognition of FtsZ. By deletion analysis of FtsZ, we found that the C-terminus of FtsZ contains a ClpX recognition signal. In ongoing studies we are further characterizing the interaction of FtsZ with ClpX and with ClpXP. In vivo, we have shown that FtsZ is turned over at a slower rate in a clpX deletion mutant than in a wild-type strain. Overexpression of ClpXP results in increased FtsZ degradation. Overproduction also results in filamentation of cells. These results suggest that ClpXP may participate in cell division by modulating the equilibrium between free and polymeric FtsZ via degradation of FtsZ filaments and protomers. Additional studies are ongoing to elucidate the mechanism by which ClpXP modulates cell division.