PMC

Expression of CHIP inhibits renaturation of denatured luciferase in Cos-7 cells. Cos-7 cells were transfected with control plasmid or with plasmids encoding wt CHIP or mutant CHIP. Thermally denatured luciferase was delivered into the cells by a liposome-based protein delivery reagent (BioPorter). Renaturation of the delivered luciferase was monitored by the recovery of luciferase activity. Each point represents the mean of six measurements.

Inhibition or removal of proteasome enhances renaturation of denatured luciferase. A) Renaturation was performed in RRL in the presence or absence of proteasome inhibitors MG132 or clasto-lactacystin β-lactone (LAC). B) Renaturation was performed in a reconstituted renaturation reaction (Hsp70 and Hsp40) in the presence or absence of MG132. C) Renaturation was performed in Cos-7 cell lysate with or without depletion of the proteasome by centrifugation. Luciferase activity was measured at the indicated time points. Each point represents the mean of 12 measurements.

Depleting E1 enhances the renaturation of luciferase in reticulocyte lysate. E1 was depleted from RRL by immunoprecipitation with antibodies specific to E1. As a control, mock immunoprecipitation was performed using preimmune IgG. A) Shows the effectiveness of the immunoprecipitation; the upper panel shows the depletion of E1 and the lower panel shows the loss of ubiquitin conjugating activity. B) Shows the effects of depleting E1 on renaturation. Luciferase activity was measured at 0, 30, and 60 min of incubation. For each point a total of six replicates were measured.

Expression of deubiquitinating enzyme promotes renaturation of denatured luciferase. Cos-7 cells were transfected with control plasmid or plasmid encoding the HA-tagged cezanne, a deubiquitinating enzyme, and chaperone activity was compared in the cell lysate. A) Shows the expression of cezanne, which were detected by Western blotting using antibody to HA. B) Shows the activity of cezanne as indicated by the decrease in levels of. endogenous ubiquitin conjugates. C) Shows the effect of cezanne on renaturation of denatured luciferase. D) Shows the activity of UBPY as indicated by the decrease in levels of endogenous ubiquitin conjugates. E) Shows the effect of UBPY on renaturation of denatured luciferase. Each point represents the mean of 12 measurements.

CHIP and Ubc4 inhibit renaturation of denatured luciferase. Renaturation of heat-denatured luciferase was performed in RRL (A and B) or using a reconstituted system (combination of Hsp70 and Hsp40) (C). A) 0, 2.5, 5 and 10 µg/ml of CHIP was added to the renaturation reactions. Luciferase activity was measured at the indicated time points. B) 5 µg/ml CHIP with or without 5 µg/ml Ubc4 was added to the renaturation reaction. C) The renaturation was mediated by the combination of Hsp70 and Hsp40. Effects of ubiquitination on renaturation were determined by addition of ubiquitin (Ub), E1, Ubc4 and CHIP as indicated in the figure. Luciferase activity was measured at time points indicated. Each point represents the mean of six measurements.

Hsp70 reduces UPP-mediated degradation of denatured luciferase and promotes renaturation. ¹²⁵I-labeled firefly luciferase was denatured in the presence of Hsp90 as described in legend to . A) the degradation assay was performed in Ubc4 supplemented RRL. As indicated in the figure CHIP and Hsp70 were added to test their effects on degradation of denatured luciferase. The percentage of degradation was calculated from acid-soluble radioactivity recovered in supernatants after 90 min of incubation at 37°C. B) The renaturation assay was performed in RRL with or without addition of Hsp70. The renaturation was monitored by restoration of luciferase activity, which was expressed as a relative activity and the units were arbitrary. Values are means ± sd of three independent determinations.

Denatured luciferase is preferentially ubiquitinated and degraded in a CHIP and Hsp90-dependent fashion. Firefly luciferase was first labelled with ¹²⁵I and then thermally denatured in the presence or absence of Hsp90. Native or thermally denatured luciferase was subjected to ubiquitination assay or degradation assay as described in experimental procedures. A) the ubiquitination assay was performed at 30°C for 30 min in the presence or absence of CHIP in proteasome-free reticolucyte faction II, which was supplemented with ubiquitin and Ubc4. The ubiquitinated luciferase was indicated by the species with higher molecular weight than that of free luciferase. B) The degradation assay was performed in reticulocyte with or without supplementation of Ubc4 or CHIP. Percentage of degradation was calculated from acid-soluble radioactivity recovered in supernatants after 90 min of incubation at 37°C. Values are means ± SD of three independent determinations; each was done in duplicate.

Triage model of the protein quality control system: the interaction between molecular chaperones and the UPP. This model predicts that most, if not all, proteins have intrinsic signals for interaction with molecular chaperones or the ubiquitination system. These signals (red) are hidden in properly folded native proteins and they are not recognized by the protein quality control systems. Upon environmental stress, such as heat or oxidation, proteins could be unfolded with exposure of the recognition signals, such as hydrophobic patches. The unfolded proteins are recognized by Hsp90 or Hsp70. With the help of other chaperones or co-factors, Hsp70 can refold the denatured proteins in an ATP-dependent manner. If the denatured proteins cannot be refolded rapidly, CHIP, a U-box E3, which interacts with Hsp90 and Hsp70 with its TPR domain, triggers the ubiquitination of Hsp90/hsp70-bound substrates. The ubiquitinated substrates will be recognized and degraded by the 26S proteasome. If the ubiquitinated proteins were deubiquitinated by isopeptidases, the denatured proteins would have a second chance to be refolded by molecular chaperones. The parallel/competitive functional relationship between the UPP and molecular chaperones assures the efficiency of the protein quality control systems to get rid of abnormal proteins.