The chaperone system formed by DnaK, DnaJ and GrpE mediates stress‐dependent negative modulation of the Escherichia coli heat shock response, probably through association with the heat shock promoter‐specific sigma32 subunit of RNA polymerase. Interactions of the DnaK system with sigma32 were analysed. DnaJ and DnaK bind free, but not RNA polymerase‐bound, sigma32 with dissociation constants of 20 nM and 5 muM respectively. Association and dissociation rates of DnaJ‐sigma32 complexes are 5900‐ and 20‐fold higher respectively than those of DnaK‐sigma32 complexes in the absence of ATP. ATP destabilizes DnaK‐sigma32 interactions. DnaJ, through rapid association with sigma32 and stimulation of hydrolysis of DnaK‐bound ATP, mediates efficient binding of DnaK to sigma32 in the presence of ATP, resulting in DnaK‐DnaJ‐sigma32 complexes containing ADP. GrpE binding to these complexes stimulates nucleotide release and subsequent complex dissociation by ATP. We propose that the principles of this cycle also operate in other chaperone activities of the DnaK system. DnaK and DnaJ cooperatively inhibit sigma32 activity in heat shock gene transcription and GrpE partially reverses this inhibition. These data indicate that reversible inhibition of sigma32 activity through transient association of DnaK and DnaJ is a central regulatory element of the heat shock response.
The DnaK (Hsp70), DnaJ, and GrpE heat shock proteins of Escherichia coli constitute a cellular chaperone system for protein folding. Substrate interactions are controlled by the ATPase activity of DnaK which itself is regulated by the nucleotide exchange factor GrpE. To understand the structure-function relationship of this chaperone system, the quaternary structures of DnaK, GrpE, and DnaK-GrpE complexes were analyzed by gel filtration chromatography, dynamic light scattering, analytical ultracentrifugation, and native gel electrophoresis. GrpE formed dimers in solution. DnaK formed monomers, dimers, and higher mole mass oligomers, the equilibrium between these forms being dependent on the DnaK concentration. The behavior of DnaK and GrpE in gel filtration and dynamic light scattering suggested elongated shapes of both molecules. In the absence of added nucleotides, DnaK and GrpE formed stable complexes containing one molecule of DnaK and two molecules of GrpE. A 44-kDa N-terminal ATPase fragment of DnaK also formed complexes with GrpE with the same 1:2 stoichiometry. DnaK-GrpE complex formation was unaffected by elimination of DnaK-bound nucleotides or addition of saturating concentrations of a DnaK peptide substrate. These findings allow the correlation of DnaK-GrpE interactions with a role for GrpE in the functional cycle of the DnaK chaperone system.
Small heat shock proteins (sHsps) are ubiquitous molecular chaperones that bind denatured proteins in vitro, thereby facilitating their subsequent refolding by ATP-dependent chaperones. The mechanistic basis of this refolding process is poorly defined. We demonstrate that substrates complexed to sHsps from various sources are not released spontaneously. Dissociation and refolding of sHsp bound substrates relies on a disaggregation reaction mediated by the DnaK system, or, more efficiently, by ClpB/DnaK. While the DnaK system alone works for small, soluble sHsp/substrate complexes, ClpB/DnaK-mediated protein refolding is fastest for large, insoluble protein aggregates with incorporated sHsps. Such conditions reflect the situation in vivo, where sHsps are usually associated with insoluble proteins during heat stress. We therefore propose that sHsp function in cellular protein quality control is to promote rapid resolubilization of aggregated proteins, formed upon severe heat stress, by DnaK or ClpB/DnaK.
Paracrine-acting proteins are emerging as a central mechanism by which bone marrow cell-based therapies improve tissue repair and heart function after myocardial infarction (MI). We carried out a bioinformatic secretome analysis in bone marrow cells from patients with acute MI to identify novel secreted proteins with therapeutic potential. Functional screens revealed a secreted protein encoded by an open reading frame on chromosome 19 (C19orf10) that promotes cardiac myocyte survival and angiogenesis. We show that bone marrow-derived monocytes and macrophages produce this protein endogenously to protect and repair the heart after MI, and we named it myeloid-derived growth factor (MYDGF). Whereas Mydgf-deficient mice develop larger infarct scars and more severe contractile dysfunction compared to wild-type mice, treatment with recombinant Mydgf reduces scar size and contractile dysfunction after MI. This study is the first to assign a biological function to MYDGF, and it may serve as a prototypical example for the development of protein-based therapies for ischemic tissue repair.
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