Review: Mechanisms of Disaggregation and Refolding of Stable Protein Aggregates by Molecular Chaperones Anat Peres Ben-Zvi and Pierre Goloubinoff Department of Plant Sciences, A. Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel Received January 31, 2001, and in revised form March 28, 2001; published online July 25, 2001 Molecular chaperones are essential for the cor- rect folding of proteins in the cell under physiolog- ical and stress conditions. Two activities have been traditionally attributed to molecular chaperones: (1) preventing aggregation of unfolded polypep- tides and (2) assisting in the correct refolding of chaperone-bound denatured polypeptides. We dis- cuss here a novel function of molecular chaperones: catalytic solubilization and refolding of stable pro- tein aggregates. In Escherichia coli, disaggregation is carried out by a network of ATPase chaperones consisting of a DnaK core, assisted by the cochap- erones DnaJ, GrpE, ClpB, and GroEL-GroES. We suggest a sequential mechanism in which (a) ClpB exposes new DnaK-binding sites on the surface of the stable protein aggregates; (b) DnaK binds the aggregate surfaces and, by doing so, melts the in- correct hydrophobic associations between aggre- gated polypeptides; (c) ATP hydrolysis and DnaK release allow local intramolecular refolding of na- tive domains, leading to a gradual weakening of improper intermolecular links; (d) DnaK and GroEL complete refolding of solubilized polypeptide chains into native proteins. Thus, active disaggre- gation by the chaperone network can serve as a central cellular tool for the recovery of native pro- teins from stress-induced aggregates and actively remove disease-causing toxic aggregates, such as polyglutamine-rich proteins, amyloid plaques, and prions. © 2001 Academic Press Key Words: aggregate solubilization; DnaK; GroEL; ClpB; malate dehydrogenase; heat shock; stress. Protein misfolding and protein aggregation. An- finsen demonstrated that all the information neces- sary for a polypeptide chain to fold correctly into a three-dimensional structure is encoded in its pri- mary sequence (Anfinsen, 1973). However, under physiological conditions of high protein concentra- tions and cellular crowding (Ellis, 1997; Minton, 2000; van den Berg et al., 1999), especially at high temperatures, many proteins tend to form stable insoluble aggregates devoid of biological activity (Jaenicke, 1995). Various stresses, such as heat and cold shock, dehydration, oxidation, salt and osmotic shock, as well as mutations, can increase the pro- pensity of native proteins to partially unfold and seek alternatively stable, but misfolded states (Jaenicke, 1995; Radford, 2000). Aggregation occurs when folding/unfolding intermediates become trapped in partially misfolded states that succes- sively associate, mainly through hydrophobic sur- faces, into an oligomeric continuum of increasingly larger, more stable, and less soluble complexes (De Bernardez Clark et al., 1999; Jaenicke, 1995). In the aggregated state, inactive proteins are enriched in anti-parallel -strands (Fink, 1998). They appear as amorphous structures, such as inclusion bodies and heat shock granules, or as ordered fibers, such as amyloid plaques and prion particles (McLaurin et al., 2000; Prusiner, 1998; Serio and Lindquist, 2000). Experimental approaches to observe protein aggregates in vitro include low-speed sedimenta- tion, light scattering, and spectroscopic analysis of thioflavin-T or Congo red binding (Jaenicke, 1995; Klunk et al., 1999; LeVine, 1999; Lomakin et al., 1999). In addition to the loss of function and cellular encumbrance, aggregates are generally more hydro- phobic, which can impair membrane function, as in the case of neurodegenerative amyloid plaques. Ag- gregates may even become infectious by promoting aggregation of native conformers, as with prions (Prusiner, 1998; Serio and Lindquist, 2000). Most molecular chaperones prevent aggregation of unfolded polypeptides. Molecular chaperones are composed of several distinct classes of sequence- conserved proteins, most of which are stress induc- ible (Hsps). Major classes of chaperones are Hsp100 (ClpA/B/X, HslU), Hsp90 (HtpG), Hsp70 (DnaK), Hsp60 (GroEL), and the small Hsps (IbpA/B) (Esch- erichia coli chaperones in parentheses), which have housekeeping functions under physiological condi- Journal of Structural Biology 135, 84 –93 (2001) doi:10.1006/jsbi.2001.4352, available online at http://www.idealibrary.com on 84 1047-8477/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.