Pharmacologic shifting of a balance between protein refolding and degradation mediated by Hsp90

Abstract

The role of the abundant stress protein Hsp90 in protecting cells against stress-induced damage is not well understood. The recent discovery that a class of ansamycin antibiotics bind specifically to Hsp90 allowed us to address this problem from a new angle. We find that mammalian Hsp90, in cooperation with Hsp70, p60, and other factors, mediates the ATP-dependent refolding of heat-denatured proteins, such as firefly luciferase. Failure to refold results in proteolysis. The ansamycins inhibit refolding, both in vivo and in a cell extract, by preventing normal dissociation of Hsp90 from luciferase, causing its enhanced degradation. This mechanism also explains the ansamycin-induced proteolysis of several protooncogenic protein kinases, such as Raf-1, which interact with Hsp90. We propose that Hsp90 is part of a quality control system that facilitates protein refolding or degradation during recovery from stress. This function is used by a limited set of signal transduction molecules for their folding and regulation under nonstress conditions. The ansamycins shift the mode of Hsp90 from refolding to degradation, and this effect is probably amplified for specific Hsp90 substrates.

Figure 1

Effect of heat shock on firefly luciferase expressed in vivo in the presence of HA. Luciferase activities (A) and levels of ³⁵S-labeled luciferase protein (B) in SW620 colon carcinoma cells subjected to heat shock at 42°C and recovery at 37°C in the presence (□) and absence (•) of HA. Ten minutes before heat shock, cultures received 40 μg/ml cycloheximide. Values measured in DMSO-treated control cells maintained at 37°C throughout are set to 100%. Note that, although normally localized in peroxisomes, more than 90% of luciferase expressed in SW620 cells was localized in the soluble supernatant fraction of a high-speed centrifugation (18), as uptake into peroxisomes is inefficient. Luciferase did not form insoluble aggregates during incubation at 42°C.

Figure 2

Specific binding of Hsp90 in cell extracts to GA affinity beads. Extracts of ³⁵S-labeled SW620 cells (see Fig. 1) were incubated with GA coupled to Affi-Gel 10 (13) or Affi-Gel 10 alone, as indicated, followed by 10 washes in lysis buffer (14) and by elution with SDS buffer. Eluates were analyzed by SDS/PAGE and autoradiography (³⁵S) or immunoblotting with anti-Hsp90 antibody (anti-Hsp90). Total protein (25 μg) was analyzed in lane T. In lanes 1–4, 875 μg of total protein was incubated with 10-μl beads for 60 min at 25°C.

Figure 3

Refolding of firefly luciferase in RL. (A) Schematic representation of the luciferase-myc-His fusion construct used as a substrate (FaXa, factor Xa cleavage site; c-myc, c-myc epitope; 6His, 6Histidine-tag). The factor Xa cleavage site is separated from the c-myc epitope by a 14-amino acid spacer. (B) Refolding of thermally denatured (T) and chemically denatured (C) luciferase in control lysate (RL) in the presence and absence of ATP and in HA-treated lysate as indicated. The activity of an equivalent amount of native luciferase is set to 100%. (C) Effect of increasing concentrations of HA and GA on the refolding of chemically denatured luciferase (○, ♦) and on the de novo folding of newly translated luciferase (□). Enzyme activities were measured after 40 min of refolding or after 60 min of translation. Specific luciferase activities were calculated as the ratio of enzyme activity:full-length luciferase with the specific activity in untreated RL set to 1.0. Note that refolding was not inhibited when purified luciferase was incubated with HA or GA before or during chemical denaturation, and free drug was removed by gel filtration before refolding; inhibition persisted, however, when drug-treated RL was gel-filtered before the addition of unfolded luciferase (not shown). In contrast to Hsp90, luciferase itself did not bind to GA-agarose beads.

Figure 4

Isolation and characterization of chaperone-bound luciferase from RL. Immunoisolation of chaperone complexes containing unfolded luciferase-myc-His (U) from control lysate and from lysate treated with HA or GA using chemically denatured (A) and thermally denatured (B) luciferase. In A, lanes 1 and 2, native luciferase (N) was added. In B, lanes 1 and 2, thermal denaturation of luciferase was carried out in ATP-depleted lysate. Complexes bound to protein G-Sepharose were eluted with ATP (A) or SDS (S). Eluted fractions were analyzed by SDS/PAGE followed by Coomassie blue staining (Ai and B) or immunoblotting with anti-Hsp90 and anti-Hsc70 (Aii). (C) Luciferase complexes were released from Sepharose beads with factor Xa and eluates immunoblotted with antibodies against Hip, Hsp40, and p23. (D) Time course of ATP-dependent elution of chaperones from complexes with luciferase isolated as in A from control lysate (−HA) and HA-treated lysate (+HA). Sepharose beads were incubated for 1 min at 25°C in 200 μl of buffer B with 1 mM ATP/5 mM Mg²⁺, and the supernatant was removed. This procedure was repeated five times, followed by a final elution with SDS. ATP eluates were analyzed as in A. Proteins were quantified by densitometry and plotted as the amount of total chaperone protein released up to a given time.

Figure 5

Protein degradation from HA-trapped Hsp90 complexes. (A) Time course of luciferase degradation in control RL (−HA) and in HA-treated (+HA) RL with and without ATP. Luciferase immunoblots are shown. Amounts of luciferase protein degraded were determined by densitometry. (B) Inhibition of luciferase degradation by hemin and methylated ubiquitin (Met-Ubi) and partial reversal of inhibition by ubiquitin (Ubi) in HA-treated RL. (C) Degradation of luciferase immunoisolated as a chaperone complex from control (−HA) and HA-treated (+HA) RL upon transfer into untreated, degradation-competent RL. Time-dependent degradation is shown in percent of the total luciferase protein in the reaction.

Figure 6

HA-induced degradation of Raf-1 in human breast carcinoma cells. (A) Raf-1 immunoprecipitates from MCF-7 cells, cultured at 37°C in the presence of HA, were analyzed by SDS/PAGE and immunoblotting with anti-Raf-1 and anti-Hsp90 antibodies. Raf-1 (74 kDa) migrates as a double-band due to phosphorylation. Direct immunoblotting of cells confirmed the degradation of essentially all Raf-1. (B) Time-dependent formation of Hsp90:Raf-1 complex and Raf-1 degradation. Depending on the efficiency of Hsp90 coimmunoprecipitation with anti-Raf-1 antibody (probably less than 100%), the results are consistent with a 1:1 or 1:2 stoichiometry of Raf-1:Hsp90 in the complex. Note that HA caused an approximately 2-fold increase in total Hsp90 (32) (not shown).