Dicarboxylic amino acids and glycine‐betaine regulate chaperone‐mediated protein‐disaggregation under stress

Abstract

SummaryActive 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. Kglutamate 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.

Native folding of aggregation-prone recombinant proteins in Escherichia coli by osmolytes, plasmid- or benzyl alcohol–overexpressed molecular chaperones

“…In comparison, a short heat shock (42ЊC for 30 minutes) before IPTG induction resulted in a 4-fold increase of the DnaK concentration but only a 2.5-fold increase of the IMAC yields. This confirms that, independent of the aggregation-prevention and curing activity of this molecular chaperone, chemical chaperones can efficiently favor the native-folding pathway of protein in vivo as well as in vitro (Diamant et al 2001(Diamant et al , 2003.…”

“…The presence of high concentrations of osmolytes, such as betaine or K-glutamate, can strongly stabilize heat-labile proteins during heat stress in vivo and favor native refolding of urea-denatured proteins in vitro (Diamant et al 2001(Diamant et al , 2003. The results shown in Figure 1 confirm that the cellular accumulation of a viscous osmolyte such as betaine, during massive E8R synthesis, may strongly favor the funneling of early folding intermediates into the native-folding pathway.…”

“…When expressed fused in frame with bacterial NusA , 25-40% of the E8R was found in the soluble fraction after 30 minutes centrifugation at 100 000 ϫ g, depending on the growth conditions (Fig 1). The bacteria were grown with inducer either at 20ЊC for 20 hours or at 37ЊC for 4 hours, with and without addition of 0.5 M NaCl and 5 mM glycine betaine (betaine) to cause accumulation of betaine in the bacteria (Diamant et al 2003). Western blot analysis showed that NaCl treatment did not induce significant variations in the expression of the total E8R protein or of the molecular chaperone DnaK (Fig 1).…”

“…Moreover, physiological (molar) amounts of osmolytes can efficiently promote the in vitro spontaneous native refolding of urea-denatured proteins (Diamant et al 2001(Diamant et al , 2003. This may be a direct consequence of osmolyte viscosity, which by slowing down rates of structure acquisition would reduce the thermodynamically less favorable, accidental misfolding events to a greater extent than the thermodynamically more favorable native-folding events (Anfinsen 1972).…”

“…Thus, under extreme unnatural conditions, such as massive overexpression of a single recombinant protein in the cell, the posttranslational folding machinery made of molecular chaperones that can normally prevent and correct protein misfolding, may become rapidly overwhelmed. To overcome this problem, protocols were applied to reduce the rate of protein synthesis and folding by lowering the growth temperature and the concentration of the inducer, by increasing the concentration of viscous osmolytes (Diamant et al 2003) or by increasing the levels of molecular chaperones by plasmid coexpression (Goloubinoff et al 1989b;.…”