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The triage of damaged proteins: degradation by the ubiquitin-proteasome pathway or repair by molecular chaperones

Carla Marques^*,,1, Weimin Guo^*,1, Paulo Pereira, Allen Taylor^*, Cam Patterson, Paul C. Evans and Fu Shang^*,2

^* Human Nutrition Research Center on Aging, Tufts University, Boston, Massachusetts, USA;
Center of Ophthalmology, IBILI, Faculty of Medicine, University of Coimbra, Coimbra, Portugal;
Carolina Cardiovascular Biology Center, University of North Carolina, Chapel Hill, Chapel Hill, North Carolina, USA; and
BHF Cardiovascular Medicine, Imperial College, London, UK

²Correspondence: Jean Mayer USDA Human Nutrition Research Center on Aging, Tufts University, Boston, MA 02111, USA. E-mail: fu.shang{at}tufts.edu

SPECIFIC AIMS

Accumulation of damaged or abnormal proteins is associated with various age-related diseases, so prompt repair and/or removal of damaged proteins are essential for the survival of cells and the health of organisms. The ubiquitin-proteasome pathway (UPP) plays a role in selective degradation of damaged or abnormal proteins, whereas molecular chaperones such as heat shock proteins are involved in refolding of denatured proteins. The specific aims of this work were to elucidate the mechanism of recognition of damaged proteins by the UPP and determine the working relationship between the UPP and molecular chaperones.

PRINCIPAL FINDINGS

1. Heat-denatured luciferase is preferentially ubiquitinated and degraded by the UPP
Thermally denatured, but not native, luciferase was ubiquitinated in a reconstituted ubiquitination system. Ubiquitination of heat-denatured luciferase depends on Ubc4, CHIP, and Hsp90. Although addition of exogenous Ubc4 alone significantly enhanced the degradation of luciferase in rabbit reticulocyte lysate (RRL), it did not show selectivity between native and denatured proteins (Fig. 1 ). In contrast, addition of exogenous CHIP dramatically promoted the degradation of heat-denatured luciferase but did not significantly destabilize the native form (Fig. 1) . A combination of Ubc4 and CHIP further increased the degradation rate of denatured proteins (Fig. 1) . Hsp90 is required for the ubiquitination of denatured luciferase. These data indicate that Ubc4 is one of the dominant E2s for denatured proteins. Hsp90, together with CHIP, is involved in recognition of denatured proteins.

View larger version (32K): [in this window] [in a new window]   Figure 1. Denatured luciferase is preferentially ubiquitinated and degraded in a CHIP and Hsp90-dependent fashion. Firefly luciferase was first labeled with 125I, then thermally denatured in the presence or absence of Hsp90. Native or thermally denatured luciferase was subjected to ubiquitination assay or degradation assay A) The ubiquitination assay was performed at 30°C for 30 min in the presence or absence of CHIP in proteasome-free reticulocyte faction II, 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 3 independent determinations, each done in duplicate.

2. Hsp70 suppresses the UPP-mediated degradation of denatured proteins in reticulocyte lysate
It has been shown that degradation of some proteins requires Hsp70. To our surprise, addition of Hsp70 to RRL inhibited the degradation of denatured luciferase by 25%, even in the presence of exogenous CHIP. Consistent with previous reports, thermally denatured luciferase was readily refolded in RRL as indicated by restoration of luciferase activity. Addition of exogenous Hsp70 increased the rate of renaturation by 30%. The inhibitory effects of Hsp70 on degradation and its stimulatory effect on renaturation indicate that molecular chaperones may compete with the UPP for denatured luciferase.

3. Enhancement of ubiquitination suppresses renaturation of denatured proteins
Addition of CHIP, the ubiquitin-ligase that promotes ubiquitination of denatured luciferase, to RRL decreased the renaturation of luciferase in a dose-dependent manner (Fig. 2 A). Addition of Ubc4, the E2 that enhances ubiquitination and degradation of denatured luciferase, further diminished the renaturation of luciferase (Fig. 2B ). To verify that the effects of CHIP and Ubc4 are due to enhanced ubiquitinating activity rather than directly inhibiting chaperone activities in RRL, we performed the experiment in a reconstituted system. The combination of Hsp70 and Hsp40 is sufficient to refold denatured luciferase (Fig. 2C ). Addition of CHIP alone or together with Ubc4 had no effect on renaturation (Fig. 2C ). However, if the ubiquitinating activity was reconstituted with E1, Ubc4, and CHIP, the Hsp70/Hsp40-mediated renaturation was almost abolished (Fig. 2C ). These data demonstrated that the effects of CHIP and Ubc4 on renaturation of luciferase are due to the enhancement of ubiquitinating activity. These data also imply that the UPP competes with chaperones for denatured luciferase.

View larger version (12K): [in this window] [in a new window]   Figure 2. CHIP and Ubc4 inhibit renaturation of denatured luciferase. Renaturation of heat-denatured luciferase was performed in RRL (A, B) or using a reconstituted system (combination of Hsp70 and Hsp40). A) 0, 2.5, 5, and 10 µg/mL of CHIP was added to the renaturation reactions. Luciferase activity was measured. 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 was 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 6 measurements.

4. Depletion of ubiquitin-activating (E1) enzyme or proteasome promote renaturation
E1, the first enzyme in the ubiquitination reaction, was depleted from RRL by immunoprecipitation with antibodies specific to E1. When 80% of E1 was removed from RRL by immunoprecipitation, the corresponding ubiquitin conjugating activity was also substantially diminished. Consistent with the hypothesis that the ubiquitin-conjugating system competes with chaperones for denatured proteins, depletion of E1 enhanced the renaturation of luciferase. To test whether proteasome-mediated proteolysis plays a role in the competition between the UPP and chaperones for denatured proteins, we examined the effect of proteasome inhibition on renaturation. We found that inhibition of proteasome activity by MG132 or clasto-lactacystin ß-lactone enhanced the renaturation of denatured luciferase. The effect of proteasome inhibitors is not due to direct enhancement of chaperone activities, as addition of MG132 had no effect on renaturation mediated by a combination of Hsp70 and Hsp40. The effect of proteasome on renaturation is not limited to the RRL, as depleting proteasome from lysate of Cos-7 cells also enhanced the renaturation activity substantially.

5. Overexpression of deubiquitinating enzymes promoted renaturation of denatured proteins
Ubiquitinated proteins that are not degraded can be refolded in cell lysates. However, ubiquitinated proteins cannot be refolded by chaperones in the reconstituted system. We hypothesized that deubiquitination of denatured proteins is required for renaturation by molecular chaperones. Consistent with this hypothesis, we found that overexpression of isopeptidases in Cos-7 cells enhanced renaturation of denatured luciferase.

CONCLUSIONS AND SIGNIFICANCE

Together, data presented here demonstrate for the first time that both the UPP and molecular chaperones recognize proteins with abnormal structures, and the two systems compete for these substrates. It appears that the cellular protein quality control systems have a triage mechanism. The first level of triage must be identification of the proteins that are damaged and require repair or removal. Once damaged proteins have been identified, a second-level decision must be made: Is the protein repairable? In principle, chaperones should have the first opportunity to fix damaged proteins. Proteins that are damaged beyond repair must be degraded by the UPP.

The ability of both the UPP and chaperones to interact with damaged or misfolded proteins in similar ways allows these pathways for repair or degradation of a given target protein to operate in a parallel or competitive manner. As illustrated in Fig. 3 , we propose that the fate of the damaged protein depends on the kinetics of interaction of the damage proteins with molecular chaperones or the chaperone component of the UPP (Hsp90). In general, proteins with exposed hydrophobic surfaces or other binding motifs interact with Hsp90. If the Hsp90-bound non-native protein is efficiently refolded with the assistance of other chaperones, such Hsp70 and Hsp40, it is removed from the triage system. If the Hsp90-bound non-native protein is not efficiently refolded by chaperones, the co-chaperone CHIP, which interacts with Hsp90 via its TPR domain and interacts with Ubc4/5 or other E2s via its U-box domain, brings the Hsp90-bound proteins to the ubiquitination machinery. Ubiquitinated substrates are often targeted to the 26S proteasome for degradation. If the ubiquitinated proteins are deubiquitinated by isopeptidases, the denatured proteins will have a second chance to be refolded by chaperones. Therefore, the relative activities of ubiquitination and deubiquitination may control the fate of denatured proteins. It appears that CHIP plays a pivotal role in the triage process by switches chaperones into components of the UPP, and deubiquitinating enzymes can reverse the action of CHIP.

View larger version (11K): [in this window] [in a new window]   Figure 3. Triage model of the protein quality control system: 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 is capable of refolding the denatured proteins in an ATP-dependent manner. If the denatured proteins cannot be refolded rapidly, CHIP, a U-box E3 that 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 will 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.

In healthy cells, the delicate balance between the UPP and chaperone controls damaged proteins to a tolerable level. However, upon aging or severe stress, functions of the UPP and chaperones may be compromised. Dysfunction of the protein quality control mechanism may be causally related to the accumulation of damaged and aggregated proteins in the cells, which is associated with various age-related diseases. A means of protecting the function of the protein quality control system may be used to prevent some age-related diseases.

FOOTNOTES

¹ These authors contributed equally to this work.

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5080fje;

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