Sequential mechanism of solubilization and refolding of stable protein aggregates by a bichaperone network

Abstract

A major activity of molecular chaperones is to prevent aggregation and refold misfolded proteins. However, when allowed to form, protein aggregates are refolded poorly by most chaperones. We show here that the sequential action of two Escherichia coli chaperone systems, ClpB and DnaK-DnaJ-GrpE, can efficiently solubilize excess amounts of protein aggregates and refold them into active proteins. Measurements of aggregate turbidity, Congo red, and 4,4-dianilino-1,1-binaphthyl-5,5-disulfonic acid binding, and of the disaggregation͞refolding kinetics by using a specific ClpB inhibitor, suggest a mechanism where (i) ClpB directly binds protein aggregates, ATP induces structural changes in ClpB, which (ii) increase hydrophobic exposure of the aggregates and (iii) allow DnaK-DnaJ-GrpE to bind and mediate dissociation and refolding of solubilized polypeptides into native proteins. This efficient mechanism, whereby chaperones can catalytically solubilize and refold a wide variety of large and stable protein aggregates, is a major addition to the molecular arsenal of the cell to cope with protein damage induced by stress or pathological states.Hsp104 ͉ ClpB ͉ DnaK ͉ Congo red ͉ protein disaggregation

Walker‐A threonine couples nucleotide occupancy with the chaperone activity of the AAA+ ATPase ClpB

“…We produced large aggregates of two substrates: glucose-6-phosphate dehydrogenase (G6PDH) and malate dehydrogenase (MDH) that can be reactivated by ClpB with the DnaK system of cochaperones. 8,12 We incubated the aggregates with ClpB in the absence and presence of different nucleotides and then determined the amount of ClpB bound to the aggregated particles (see Fig. 4).…”

“…5 ClpB reactivates aggregated proteins in cooperation with the DnaK chaperone system. [6][7][8] The ClpB activity is linked to extraction of single polypeptides from aggregated particles and their forced unfolding by translocation through a narrow channel located at the center of the hexameric ring. 9 The substrate translocation by ClpB is driven by ATP hydrolysis and is analogous to the degradationpreceding unfolding/translocation of substrates by the proteasome and other ATP-dependent proteases.…”

Firefly luciferase mutants as sensors of proteome stress

“…Using model proteins like β-galactosidase and firefly luciferase, many studies have addressed the role of the Hsp70 (DnaK)-Hsp40 (DnaJ)-Hsp110 (GrpE) system (also called KJE system) in re-activation of stress denatured proteins both in vitro and in vivo (Ben-Zvi et al, 2004;Goloubinoff et al, 1999;Pinto et al, 1991;Schroder et al, 1993). The refolding reaction is generally slow and is strictly dependent on ATP hydrolysis.…”

“…Soon after the discovery of Hsp104 disaggregase in yeast, an orthologous protein called ClpB was shown to possess disaggregation activity in E. coli and in chloroplasts and mitochondria of higher eukaryotes . Several in vitro studies have shown that the Hsp104 or ClpB system alone has little or no disaggregation activity and requires collaboration with the Hsp70 system for effective disaggregation (Glover and Lindquist, 1998;Goloubinoff et al, 1999).…”

Reactivation of protein aggregates by mortalin and Tid1—the human mitochondrial Hsp70 chaperone system

“…In organisms and cellular compartments that also express Hsp100/ClpB homologues, the Hsp70-Hsp40 unfoldase chaperone systems benefit from a powerful synergic mechanism of forceful disaggregation that may act both upstream and downstream to the polypeptide unfoldase activity of Hsp70 chaperones (Glover and Lindquist 1998;Goloubinoff et al 1999;Weibezahn et al 2005). Thus, in the yeast cytoplasm, a large number of Hsp70-Hsp40 can collaborate with the ClpB-like (Hsp104) disaggregating co-chaperone to prevent the formation of misfolded species and even solubilizeresistant prions.…”