ABSTRACT
External stresses or mutations may cause labile proteins to lose their distinct native conformations and seek alternatively stable aggregated forms. Molecular chaperones that specifically act on protein aggregates were used here as a tool to address the biochemical nature of stable homo- and hetero-aggregates from non-pathogenic proteins formed by heat-stress. Confirmed by sedimentation and activity measurements, chaperones demonstrated that a single polypeptide chain can form different species of aggregates, depending on the denaturing conditions. Indicative of a cascade reaction, sub-stoichiometric amounts of one fast-aggregating protein strongly accelerated the conversion of another soluble, slow-aggregating protein into insoluble, chaperone-resistant aggregates. Chaperones strongly inhibited seed-induced protein aggregation, suggesting that they can prevent and cure proteinaceous infectious behavior in homo- and hetero-aggregates from common and disease-associated proteins in the cell.
Subject(s)
Glucosephosphate Dehydrogenase/chemistry , Malate Dehydrogenase/chemistry , Peptides/chemistry , Protein Conformation , Serum Albumin, Bovine/chemistry , Animals , Catalysis , Cattle , Centrifugation , Dimerization , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Endopeptidase Clp , Escherichia coli Proteins/metabolism , Glucosephosphate Dehydrogenase/metabolism , Heat-Shock Proteins/metabolism , Malate Dehydrogenase/metabolism , Molecular Chaperones/metabolism , Protein Binding , Rabbits , Serum Albumin, Bovine/metabolism , Spectrometry, Fluorescence , Subcellular Fractions , Swine , Temperature , Time FactorsABSTRACT
Active protein-disaggregation by a chaperone network composed of ClpB and DnaK + DnaJ + GrpE is essential for the recovery of stress-induced protein aggregates in vitro and in Escherichia coli cells. K-glutamate and glycine-betaine (betaine) naturally accumulate in salt-stressed cells. In addition to providing thermo-protection to native proteins, we found that these osmolytes can strongly and specifically activate ClpB, resulting in an increased efficiency of chaperone-mediated protein disaggregation. Moreover, factors that inhibited the chaperone network by impairing the stability of the ClpB oligomer, such as natural polyamines, dilution, or high salt, were efficiently counteracted by K-glutamate or betaine. The combined protective, counter-negative and net activatory effects of K-glutamate and betaine, allowed protein disaggregation and refolding under heat-shock temperatures that otherwise cause protein aggregation in vitro and in the cell. Mesophilic organisms may thus benefit from a thermotolerant osmolyte-activated chaperone mechanism that can actively rescue protein aggregates, correctly refold and maintain them in a native state under heat-shock conditions.