Failure of protein folding resulting in protein aggregation is a serious problem in molecular biology, biotechnology and medicine. Alzheimer's disease, prion diseases and inclusion-body myopathies are examples of pathological conditions arising from accumulation of misfolded and aggregated proteins in cells. Molecular chaperones, which belong to several heat-shock-protein (Hsp) families, inhibit protein aggregation and facilitate protein folding and assembly. Recently, novel multi-chaperone systems consisting of Hsp100, Hsp70, and Hsp40 proteins have been discovered in yeast and in Escherichia coli. Unlike previously studied chaperones, these multi-chaperone systems are capable of efficient reactivation of strongly aggregated proteins. In the E. coli system, ClpB, an Hsp100 protein with previously unknown function, cooperates with DnaK, DnaJ, and GrpE in reactivating aggregated luciferase. Our long-term goal is to understand the molecular mechanism of protein reactivation and disaggregation reactions involving Hsp100 proteins. In this proposal, we focus on ClpB as a member of the multi-chaperone system. ClpB is expressed in vivo as two gene products: 95-kDa ClpB95 and 80-kDa ClpB80. While both ClpB95 and ClpB80 exhibit ATPase activity, only the ATPase of ClpB95 is stimulated by other proteins. Our previous results show an ATP-induced self-association of ClpB, which is inhibited by ADP. We hypothesize that the mechanism of the ClpB chaperone function arises from allosteric couplings between ClpB-substrate of ClpB-co-chaperone interactions, ClpB self-association, and ATP binding and hydrolysis. We will test whether the observed differences in stimulation of the ATPase activities of ClpB95 and ClpB80 will be reflected in different protein-binding properties and chaperone activities of ClpB95 and ClkpB80. The proposed research will achieve the following aims: 1. Characterize nucleotide-dependent oligomerization of ClpB95 ClpB80. 2. Characterize interactions between ClpB95 or ClpB80 and the co-chaperones (DnaK, DnaJ, GrpE). 3. Characterize the role of ClpB95 or ClpB80 and the three co-chaperones in the disaggregation and reactivation of luciferase.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Molecular and Cellular Biophysics Study Section (BBCA)
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Wehrle, Janna P
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Kansas State University
Schools of Arts and Sciences
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Zolkiewski, Michal; Chesnokova, Liudmila S; Witt, Stephan N (2016) Reactivation of Aggregated Proteins by the ClpB/DnaK Bi-Chaperone System. Curr Protoc Protein Sci 83:28.10.1-28.10.18
Zolkiewski, Michal; Zhang, Ting; Nagy, Maria (2012) Aggregate reactivation mediated by the Hsp100 chaperones. Arch Biochem Biophys 520:1-6
Nagy, Maria; Guenther, Izabela; Akoyev, Vladimir et al. (2010) Synergistic cooperation between two ClpB isoforms in aggregate reactivation. J Mol Biol 396:697-707
Doyle, Shannon M; Shorter, James; Zolkiewski, Michal et al. (2007) Asymmetric deceleration of ClpB or Hsp104 ATPase activity unleashes protein-remodeling activity. Nat Struct Mol Biol 14:114-22
Nagy, Maria; Akoev, Vladimir; Zolkiewski, Michal (2006) Domain stability in the AAA+ ATPase ClpB from Escherichia coli. Arch Biochem Biophys 453:63-9
Zolkiewski, Michal (2006) A camel passes through the eye of a needle: protein unfolding activity of Clp ATPases. Mol Microbiol 61:1094-100
Akoev, Vladimir; Gogol, Edward P; Barnett, Micheal E et al. (2004) Nucleotide-induced switch in oligomerization of the AAA+ ATPase ClpB. Protein Sci 13:567-74
Kedzierska, Sabina; Akoev, Vladimir; Barnett, Micheal E et al. (2003) Structure and function of the middle domain of ClpB from Escherichia coli. Biochemistry 42:14242-8
Tek, Vekalet; Zolkiewski, Michal (2002) Stability and interactions of the amino-terminal domain of ClpB from Escherichia coli. Protein Sci 11:1192-8
Barnett, Micheal E; Zolkiewski, Michal (2002) Site-directed mutagenesis of conserved charged amino acid residues in ClpB from Escherichia coli. Biochemistry 41:11277-83

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