Active Solubilization and Refolding of Stable Protein Aggregates By Cooperative Unfolding Action of Individual Hsp70 Chaperones

Abstract

Hsp70 is a central molecular chaperone that passively prevents protein aggregation and uses the energy of ATP hydrolysis to solubilize, translocate, and mediate the proper refolding of proteins in the cell. Yet, the molecular mechanism by which the active Hsp70 chaperone functions are achieved remains unclear. Here, we show that the bacterial Hsp70 (DnaK) can actively unfold misfolded structures in aggregated polypeptides, leading to gradual disaggregation. We found that the specific unfolding and disaggregation activities of individual DnaK molecules were optimal for large aggregates but dramatically decreased for small aggregates. The active unfolding of the smallest aggregates, leading to proper global refolding, required the cooperative action of several DnaK molecules per misfolded polypeptide. This finding suggests that the unique ATP-fueled locking/unlocking mechanism of the Hsp70 chaperones can recruit random chaperone motions to locally unfold misfolded structures and gradually disentangle stable aggregates into refoldable proteins.

DnaJ Recruits DnaK to Protein Aggregates

“…The sample was then diluted (final protein concentration 1 M) in the same buffer containing 6.4 M DnaK, 0.25 M GrpE, 0.05-10 M DnaJ. Reactivation was measured at 25°C in the presence of the ATP-regenerating system, as previously described (34).…”

Bacterial Inclusion Bodies: Discovering Their Better Half

“…For example, HtpG can interact with DnaK/DnaJ/GrpE to further promote refolding of aggregated proteins in an ATP-dependent manner [21]. Alternatively, ClpB can also assist in cooperation with DnaK/DnaJ/GrpE in ATP-driven refolding of aggregated proteins [20,22,23]. The small heat shock proteins (sHSPs) IbpA and IbpB stabilize and decrease the size of protein aggregates [24] thereby promoting DnaK/DnaJ/GrpE and ClpB mediated refolding [25] and further possible proteolysis.…”

Firefly luciferase mutants as sensors of proteome stress

“…Hsp70 members are highly multifunctional proteins that have been shown to play a key role in proteome maintenance, such as in de novo protein folding (co-or post-translational), protein translocation across membranes (Lyman and Schekman, 1997;Matlack et al, 1999;Young et al, 2003), refolding of stress damaged proteins (Ben-Zvi et al, 2004;Schroder et al, 1993;Sharma et al, 2010), in preventing protein aggregation (Auluck et al, 2002;Broadley and Hartl, 2009;Klucken et al, 2004;Sakahira et al, 2002;Warrick et al, 1999), disaggregation (Ben-Zvi and Goloubinoff, 2001;Diamant et al, 2000;Liberek et al, 2008;Shorter, 2011) and degradation of irreparable misfolded proteins (Bercovich et al, 1997;Fisher et al, 1997;Urushitani et al, 2004). These essential and diverse cellular functions of Hsp70 are attributed to its physical interaction with various co-chaperones such as Hsp40, NEFs and with proteins such as HIP, HOP and CHIP.…”

“…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.…”