The Small Heat Shock Protein IbpA of Escherichia coli Cooperates with IbpB in Stabilization of Thermally Aggregated Proteins in a Disaggregation Competent State

Abstract

The small heat shock proteins are ubiquitous stress proteins proposed to increase cellular tolerance to heat shock conditions. We isolated IbpA, the Escherichia coli small heat shock protein, and tested its ability to keep thermally inactivated substrate proteins in a disaggregation competent state. We found that the presence of IbpA alone during substrate thermal inactivation only weakly influences the ability of the bi-chaperone Hsp70-Hsp100 system to disaggregate aggregated substrate. Similar minor effects were observed for IbpB alone, the other E. coli small heat shock protein. However, when both IbpA and IbpB are simultaneously present during substrate inactivation they efficiently stabilize thermally aggregated proteins in a disaggregation competent state. The properties of the aggregated protein substrates are changed in the presence of IbpA and IbpB, resulting in lower hydrophobicity and the ability of aggregates to withstand sizing chromatography conditions. IbpA and IbpB form mixed complexes, and IbpA stimulates association of IbpB with substrate.The proper conformation of proteins is challenged by stress conditions. Exposure to extreme heat shock conditions results in a massive aggregation of proteins inside both prokaryotic and eukaryotic cells (1-3). Chaperones from the Hsp100 family, that is ClpB in Escherichia coli and Hsp104 in the yeast Saccharomyces cerevisiae, were implicated in the disaggregation reaction, because aggregated proteins were not eliminated in either clpB or HSP104 deletion strains (1-3). Additionally, the clpB and HSP104 gene products were identified as factors conferring thermotolerance in E. coli and S. cerevisiae (4 -7). However, in vitro studies on the reactivation of aggregated proteins showed that chaperones from the Hsp100 family alone are not sufficient for disaggregation and refolding. Other chaperone proteins are also involved in this process. E. coli Hsp70 (DnaK) and its cochaperones (DnaJ and GrpE) cooperate with ClpB and form a bi-chaperone system capable of efficient disaggregation of aggregated proteins (3,8,9). Analogous Hsp100-Hsp70 bi-chaperone systems able to disaggregate denatured protein substrates in vitro were established using chaperones from other bacterial species (10), as well as from yeast cytosol (11) and mitochondria (12, 13). However, the efficiency of refolding reaction catalyzed by these bichaperone systems depends strongly on the physical properties of protein aggregates. It was proposed that small heat shock proteins (sHsps) 1 associate with aggregated proteins and change their physical properties in such a way that chaperone-mediated disaggregation and refolding become much more efficient (14 -19). However, little is known about the molecular mechanism of these processes.Small heat shock proteins are widely distributed both in prokaryotes and eukaryotes. Members of this diverse protein family are characterized by relatively low monomeric molecular masses (15-43 kDa) and a conserved stretch of ϳ100 amino acid residues (reviewed in Refs. 20 and ...

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“…41 Likewise, in E. coli the paralogs IbpA and IbpB have been shown to be incorporated into a supramolecular structure with a mass in excess of 2 MDa. 56 The rationale…”

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“…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. IB proteins are targets of these disaggregating chaperones, which remove IB material even during IB formation in a steady IB construction/deconstruction process [26,27].…”

Escherichia coli heat-shock proteins IbpA/B are involved in resistance to oxidative stress induced by copper

“…sHsps bind denatured proteins and facilitate their refolding by the ATP-dependent molecular chaperones of the Hsp70 family (Haslbeck et al, 2005). In E. coli cells, the substrates bound to IbpA/B are refolded by DnaK and its co-chaperones DnaJ and GrpE in cooperation with the AAA + protein ClpB (Veinger et al, 1998;Mogk et al, 2003a, b;Matuszewska et al, 2005). The lack of IbpA/B proteins results in increased aggregation of heat-denatured proteins in E. coli cells.…”

“…It was also reported that removal of aggregated proteins at a recovery temperature of 30 u C was delayed in the absence of IbpA/B (Mogk et al, 2003a;Jiao et al, 2005). Both IbpA and IbpB are required during substrate inactivation in vitro, to efficiently stabilize denatured protein in a folding-competent state (Matuszewska et al, 2005). It is known that IbpA and IbpB interact in vitro; however, the nature of cooperation between IbpA and IbpB is not fully understood.…”

“…It is known that IbpA and IbpB interact in vitro; however, the nature of cooperation between IbpA and IbpB is not fully understood. Some in vivo and in vitro data suggest that IbpA may be responsible for targeting IbpB to denatured and aggregated proteins (Kuczyń ska-Wiśnik et al, 2002;Matuszewska et al, 2005).…”