Ubiquitin-proteasome pathway and cellular responses to oxidative stress

Abstract

The ubiquitin-proteasome pathway (UPP) is the primary cytosolic proteolytic machinery for the selective degradation of various forms of damaged proteins. Thus, the UPP is an important protein quality control mechanism. In the canonical UPP, both ubiquitin and the 26S proteasome are involved. Substrate proteins of the canonical UPP are first tagged by multiple ubiquitin molecules and then degraded by the 26S proteasome. However, in noncanonical UPP, proteins can be degraded by the 26S or the 20S proteasome without being ubiquitinated. It is clear that a proteasome is responsible for selective degradation of oxidized proteins, but the extent to which ubiquitination is involved in this process remains a subject of debate. Whereas many publications suggest that the 20S proteasome degrades oxidized proteins independent of ubiquitin, there is also solid evidence indicating that ubiquitin and ubiquitination are involved in degradation of some forms of oxidized proteins. A fully functional UPP is required for cells to cope with oxidative stress and the activity of the UPP is also modulated by cellular redox status. Mild or transient oxidative stress up-regulates the ubiquitination system and proteasome activity in cells and tissues and transiently enhances intracellular proteolysis. Severe or sustained oxidative stress impairs the function of the UPP and decreases intracellular proteolysis. Both the ubiquitin-conjugating enzymes and the proteasome can be inactivated by sustained oxidative stress, especially the 26S proteasome. Differential susceptibilities of the ubiquitin-conjugating enzymes and the 26S proteasome to oxidative damage lead to an accumulation of ubiquitin conjugates in cells in response to mild oxidative stress. Thus, increased levels of ubiquitin conjugates in cells seem to be an indicator of mild oxidative stress.

Fig.1. Recognition and degradation of oxidatively modified proteins by 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 oxidation, proteins could be unfolded with exposure of the recognition signals, such as hydrophobic patches. Some of the oxidized (unfolded) proteins can be recognized and degraded by the 20S proteasome directly whereas others are recognized by molecular chaperones. With the help of other chaperones or co-factors, molecular chaperones are capable of refolding the denatured proteins in an ATP-dependent manner. If the denatured proteins cannot be refolded rapidly, the chaperone bound substrates are ubiquitinated (yellow triangles) by chaperone-interacting ubiquitin-ligases, such as CHIP. The ubiquitinated substrates are 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.

Figure 2. The response of UPP to oxidative stress

The UPP is an important protein quality control mechanism for selective degradation of various damaged proteins, including oxidized proteins. However, the components of the UPP are also targets of oxidative insults. The differential susceptibility of different components of the UPP dictates the response of the UPP to different levels of oxidative stress. Mild oxidative stress increases substrate availability and up-regulates the ubiquitin conjugation systems, therefore, enhancing intracellular protein degradation. The timely degradation of oxidatively modified proteins prevents their accumulation and aggregation. Sustained oxidative stress inactivates the proteasome without inhibiting the ubiquitination system and results in the accumulation of ubiquitin conjugates in the cells. Age- and stress-related inactivation of the proteasome may be related to the accumulation of ubiquitin-containing inclusion bodies in cells of various age-related diseases. Extensive oxidative stress not only inactivates the proteasome, but also inhibits the ubiquitination system and results in a decrease in levels of newly formed ubiquitin conjugates and intracellular degradation. Oxidative inactivation of the UPP will accelerate the accumulation of oxidative damaged proteins and reduce cell viability.

Figure 3. Ubiquitination responses to various types of oxidative stress

Panel A. Lymphoblastoid cells (L-40) from a normal human at a concentration of 0.5 ×10⁶ cells/ml were treated with 80 ng/ml of neocarzinostatin (NCS) for 0 – 4h. Equal amounts of soluble protein (50 μg) from each exposure group were separated by SDS-PAGE and transferred to nitrocellulose. Endogenous ubiquitin conjugates were detected by Western blotting analysis of cellular extracts using an anti-ubiquitin antiserum. Panel B. 23 month old Emory mice were injected i.p.with 20 mg/kg paraquat dissolved in 0.9% saline, 24 h prior to sacrifice. Endogenous ubiquitin conjugates in the soluble fraction were detected in 50 μg protein from each exposure group as in panel A. Panel C. Rabbit lens epithelial (RLE) cells were treated with a single bolus of 500 μM H₂O₂ for 1h and then were allowed to recover in normal medium for the times indicated, in the absence (upper panel) or presence (lower panel) of proteasome inhibitor (lactacystin). The cells were lysed and endogenous ubiquitin conjugates were determined as in panel A. Panel D. RLE cells were treated with a constant level of H₂O₂ for 6h. De novo ubiquitin conjugation assays were performed using endogenous conjugating enzymes and substrates along with exogenous¹²⁵I-labeled ubiquitin. Panel E. Lenses from 3 or 29 month old rats were treated with a single bolus of 500 μM H₂O₂ for 30 min. Proteins were extracted from the cortex of these lenses and levels of endogenous ubiquitin conjugates were determined as described in panel A. Panel F. Human retinal pigment epithelial (RPE) cells were treated with the indicated levels of H₂O₂ for 5 min with (REC) or without additional 10 min recovery in PBS. Levels of endogenous ubiquitin conjugates were detected as described in panel A. Panel G. Bovine retinas were treated with the indicated concentrations of H₂O₂ for 30 min. Levels of endogenous ubiquitin conjugates were detected as described in panel A. Panel H. Human RPE cells were preloaded with lipofuscin and then exposed to blue light for the time indicated. Levels of endogenous ubiquitin conjugates were detected. Panel I. Human RPE cells were treated with 250 μM diamide for the time indicated with or without recovery. De novo ubiquitin conjugates were formed using endogenous conjugating enzymes and endogenous substrates with exogenous¹²⁵I-labeled ubiquitin.