Fu Shang

Regulation of Ubiquitin-conjugating Enzymes by Glutathione Following Oxidative Stress

Upon oxidative stress cells show an increase in the oxidized glutathione (GSSG) to reduced glutathione (GSH) ratio with a concomitant decrease in activity of the ubiquitinylation pathway. Because most of the enzymes involved in the attachment of ubiquitin to substrate proteins contain active site sulfhydryls that might be covalently modified (thiolated) upon enhancement of GSSG levels (glutathiolation), it appeared plausible that glutathiolation might alter ubiquitinylation rates upon cellular oxidative stress. This hypothesis was explored using intact retina and retinal pigment epithelial (RPE) cell models. Exposure of intact bovine retina and RPE cells to H2O2 (0.1–1.7 μmol/mg) resulted in a dose-dependent increase in the GSSG:GSH ratio and coincident dose-dependent reductions in the levels of endogenous ubiquitin-activating enzyme (E1)-ubiquitin thiol esters and endogenous protein-ubiquitin conjugates and in the ability to formde novo retinal protein-125I-labeled ubiquitin conjugates. Oxidant-induced decrements in ubiquitin conjugates were associated with 60–80% reductions in E1 and ubiquitin-conjugating enzyme (E2) activities as measured by formation of ubiquitin thiol esters. When GSH levels in RPE cells recovered to preoxidation levels following H2O2 removal, endogenous E1 activity and protein-ubiquitin conjugates were restored. Evidence that S thiolation of E1 and E2 enzymes is the biochemical link between cellular redox state and E1/E2 activities includes: (i) 5-fold increases in levels of immunoprecipitable, dithiothreitol-labile35S-E1 adducts in metabolically labeled, H2O2-treated, RPE cells; (ii) diminished formation of E1- and E2-125I-labeled ubiquitin thiol esters, oligomerization of E225K, and coincident reductions in protein-125I-labeled ubiquitin conjugates in supernatants from nonstressed retinas upon addition of levels of GSSG equivalent to levels measured in oxidatively stressed retinas; and (iii) partial restoration of E1 and E2 activities and levels of protein-125I-labeled ubiquitin conjugates in supernatants from H2O2-treated retinas when GSSG:GSH ratios were restored to preoxidation levels by the addition of physiological levels of GSH. These data suggest that the cellular redox status modulates protein ubiquitinylation via reversible S thiolation of E1 and E2 enzymes, presumably by glutathione.

Ubiquitin-proteasome pathway and cellular responses to oxidative stress

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.

Activity of Ubiquitin-dependent Pathway in Response to Oxidative Stress UBIQUITIN-ACTIVATING ENZYME IS TRANSIENTLY UP-REGULATED

Relations between the ubiquitin pathway and cellular stress have been noted, but data regarding responses of the ubiquitin pathway to oxidative stress are scanty. This paper documents the response of this pathway to oxidative stress in lens cells. A brief exposure of lens epithelial cells to physiologically relevant levels of H2O2 induces a transient increase in activity of the ubiquitin-dependent pathway. Ubiquitin conjugation activity was maximal and increased 3.5–9.2-fold over the activity noted in untreated cells by 4 h after removal of H2O2. By 24 h after removal of H2O2, ubiquitin conjugation activity returned to the level noted in untreated cells. In parallel to the changes in ubiquitin conjugation activity, the activity of ubiquitin-activating enzyme (E1), as determined by thiol ester formation, increased 2–6.7-fold during recovery from oxidation. Addition of exogenous E1 resulted in an increase in ubiquitin conjugation activity and in the levels of ubiquitin carrier protein (E2)-ubiquitin thiol esters in both the untreated cells and the H2O2-treated cells. These data suggest that E1 is the rate-limiting enzyme in the ubiquitin conjugation process and that the increases in ubiquitin conjugation activity which are induced upon recovery from oxidation are primarily due to increased E1 activity. The oxidation- and recovery-induced up-regulation of E1 activity is primarily due to post-synthetic events. Substrate availability and up-regulation of E2 activities also appear to be related to the enhancement in ubiquitinylation upon recovery from oxidative stress. The oxidation-induced increases in ubiquitin conjugation activity were associated with an increase in intracellular proteolysis, suggesting that the transient increase in ubiquitinylation noted upon recovery from oxidative stress may play a role in removal of damaged proteins from the cells.

REDOX REGULATION OF UBIQUITIN-CONJUGATING ENZYMES : MECHANISTIC INSIGHTS USING THE THIOL-SPECIFIC OXIDANT DIAMIDE

The ubiquitin–proteasome pathway (UPP) regulates critical cell processes, including the cell cycle, cytokine‐induced gene expression, differentiation, and cell death. Recently we demonstrated that this pathway responds to oxidative stress in mammalian cells and proposed that activities of ubiquitin‐activating enzyme (E1) and ubiquitin‐conjugating enzymes (E2s) are regulated by cellular redox status (i.e., GSSG:GSH ratio). To test this hypothesis, we altered the GSSG:GSH ratio in retinal pigment epithelial cells with the thiol‐specific oxidant, diamide, and assessed activities of the UPP. Treatment of cells with diamide resulted in a dose‐dependent increase in the GSSG:GSH ratio resulting from loss of GSH and a coincident increase in GSSG. Increases in the GSSG:GSH ratio from 0.02 in untreated cells to ≥0.5 in diamide‐treated cells were accompanied by dose‐dependent reductions in the levels of endogenous Ub‐protein conjugates, endogenous E1∼ubiquitin thiol esters, and de novo ubiquitin‐conjugating activity. As determined by the ability to form E1‐ubiquitin and E2s‐ubiquitin thiol esters, E1 and E2s were both inhibited by elevated GSSG:GSH ratios. Inhibition of E1 was associated with the formation of E1‐protein mixed disulfides. Activities of E1 and E2s gradually recovered to preoxidation levels, coincident with gradual recovery of the GSSG:GSH ratio. These data support S‐thiolation/dethiolation as a mechanism regulating E1 and E2 activities in response to oxidant insult. Ubiquitin‐dependent proteolytic capacity was regulated by the GSSG:GSH ratio in a manner consistent with altered ubiquitin‐conjugating activity. However, ubiquitin‐independent proteolysis was unaffected by changes in the GSSG:GSH ratio. Potential adaptive and pathological consequences of redox regulation of UPP activities are discussed.

Glycation-altered proteolysis as a pathobiologic mechanism that links dietary glycemic index, aging, and age-related disease (in nondiabetics)

Epidemiologic studies indicate that the risks for major age‐related debilities including coronary heart disease, diabetes, and age‐related macular degeneration (AMD) are diminished in people who consume lower glycemic index (GI) diets, but lack of a unifying physiobiochemical mechanism that explains the salutary effect is a barrier to implementing dietary practices that capture the benefits of consuming lower GI diets. We established a simple murine model of age‐related retinal lesions that precede AMD (hereafter called AMD‐like lesions). We found that consuming a higher GI diet promotes these AMD‐like lesions. However, mice that consumed the lower vs. higher GI diet had significantly reduced frequency (P < 0.02) and severity (P < 0.05) of hallmark age‐related retinal lesions such as basal deposits. Consuming higher GI diets was associated with > 3 fold higher accumulation of advanced glycation end products (AGEs) in retina, lens, liver, and brain in the age‐matched mice, suggesting that higher GI diets induce systemic glycative stress that is etiologic for lesions. Data from live cell and cell‐free systems show that the ubiquitin–proteasome system (UPS) and lysosome/autophagy pathway [lysosomal proteolytic system (LPS)] are involved in the degradation of AGEs. Glycatively modified substrates were degraded significantly slower than unmodified substrates by the UPS. Compounding the detriments of glycative stress, AGE modification of ubiquitin and ubiquitin‐conjugating enzymes impaired UPS activities. Furthermore, ubiquitin conjugates and AGEs accumulate and are found in lysosomes when cells are glycatively stressed or the UPS or LPS/autophagy are inhibited, indicating that the UPS and LPS interact with one another to degrade AGEs. Together, these data explain why AGEs accumulate as glycative stress increases.

Ubiquitin-dependent lysosomal degradation of the HNE-modified proteins in lens epithelial cells

4‐Hydroxynonenal (HNE), a highly reactive lipid peroxidation product, may adversely modify proteins. Accumulation of HNE‐modified proteins may be responsible for pathological lesions associated with oxidative stress. The objective of this work was to determine how HNE‐modified proteins are removed from cells. The data showed that αB‐crystallin modified by HNE was ubiquitinated at a faster rate than that of native αB‐crystallin in a cell‐free system. However, its susceptibility to proteasome‐dependent degradation in the cell‐free system did not increase. When delivered into cultured lens epithelial cells, HNE‐modified αB‐crystallin was degraded at a faster rate than that of unmodified αB‐crystallin. Inhibition of the lysosomal activity stabilized HNE‐modified αB‐crystallin, but inhibition of the proteasome activity alone had little effect. To determine if other HNE‐modified proteins are also degraded in a ubiquitin‐dependent lysosomal pathway, lens epithelial cells were treated with HNE and the removal of HNE‐modified proteins in the cells was monitored. The levels of HNE‐modified proteins in the cell decreased rapidly upon removal of HNE from the medium. Depletion of ATP or the presence of MG132, a proteasome/lysosome inhibitor, resulted in stabilization of HNE‐modified proteins. However, proteasome‐specific inhibitors, lactacystin‐β‐lactone and epoxomicin, could not stabilize HNE‐modified proteins in the cells. In contrast, chloroquine, a lysosome inhibitor, stabilized HNE‐modified proteins. The enrichment of HNE‐modified proteins in the fraction of ubiquitin conjugates suggests that HNE‐modified proteins are preferentially ubiquitinated. Taken together, these findings show that HNE‐modified proteins are degraded via a novel ubiquitin and lysosomal‐dependent but proteasome‐independent pathway.

Lutein and zeaxanthin supplementation reduces photooxidative damage and modulates the expression of inflammation-related genes in retinal pigment epithelial cells

Oxidative damage and inflammation are related to the pathogenesis of age-related macular degeneration (AMD). Epidemiologic studies suggest that insufficient dietary lutein and zeaxanthin intake or lower serum zeaxanthin levels are associated with increased risk for AMD. The objective of this work is to test the protective effects of lutein and zeaxanthin against photooxidative damage to retinal pigment epithelial cells (RPE) and oxidation-induced changes in expression of inflammation-related genes. To mimic lipofuscin-mediated photooxidation in vivo, we used ARPE-19 cells that accumulated A2E, a lipofuscin fluorophore and photosensitizer, as a model system to investigate the effects of lutein and zeaxanthin supplementation. The data show that supplementation with lutein or zeaxanthin in the medium resulted in accumulation of lutein or zeaxanthin in the RPE cells. The concentrations of lutein and zeaxanthin in the cells were 2- to 14-fold of that detected in the medium, indicating that ARPE-19 cells actively take up lutein or zeaxanthin. As compared with untreated cells, exposure of A2E-containing RPE to blue light resulted in a 40-60% decrease in proteasome activity, a 50-80% decrease in expression of CFH and MCP-1, and an~20-fold increase in expression of IL-8. The photooxidation-induced changes in expression of MCP-1, IL-8, and CFH were similar to those caused by chemical inhibition of the proteasome, suggesting that inactivation of the proteasome is involved in the photooxidation-induced alteration in expression of these inflammation-related genes. Incubation of the A2E-containing RPE with lutein or zeaxanthin prior to blue light exposure significantly attenuated the photooxidation-induced inactivation of the proteasome and photooxidation-induced changes in expression of MCP-1, IL-8, and CFH. Together, these data indicate that lutein or zeaxanthin modulates inflammatory responses in cultured RPE in response to photooxidation. Protecting the proteasome from oxidative inactivation appears to be one of the mechanisms by which lutein and zeaxanthin modulate the inflammatory response. Similar mechanisms may explain salutary effects of lutein and zeaxanthin in reducing the risk for AMD.

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

Accumulation of damaged proteins is causally related to many age‐related diseases. The ubiquitin‐proteasome pathway (UPP) plays a role in selective degradation of damaged proteins, whereas molecular chaperones, such as heat shock proteins, are involved in refolding denatured proteins. This work demonstrates for the first time that the UPP and molecular chaperones work in a competitive manner and that the fates of denatured proteins are determined by the relative activities of the UPP and molecular chaperones. Enhanced UPP activity suppresses the refolding of denatured proteins whereas elevated chaperone activity inhibits the degradation of denatured proteins. CHIP, a co‐chaperone with E3 activity, plays a pivotal role in determining the fates of the damaged proteins. The delicate balance between UPP‐mediated degradation and refolding of denatured proteins is governed by relative levels of CHIP and other molecular chaperones. Isopeptidases, the enzymes that reverse the actions of CHIP, also play an important role in determining the fate of denatured proteins.

Vitamin C and vitamin E restore the resistance of GSH-depleted lens cells to H2O2

A decline in reduced glutathione (GSH) levels is associated with aging and many age-related diseases. The objective of this study was to determine whether other antioxidants can compensate for GSH depletion in protection against oxidative insults. Rabbit lens epithelial cells were depleted of > 75% of intracellular GSH by 25-200 microM buthionine sulfoximine (BSO). Depletion of GSH by BSO alone had little direct effect on cell viability, but resulted in an approximately 30-fold increase in susceptibility to H(2)O(2)-induced cell death. Experimentally enhanced levels of nonprotein sulfhydryls other than GSH (i.e., N-acetylcysteine) did not protect GSH-depleted cells from H(2)O(2)-induced cell death. In contrast, pretreatment of cells with vitamin C (25-50 microM) or vitamin E (5-40 microM), restored the resistance of GSH-depleted cells to H(2)O(2). However, concentrations of vitamin C > 400 microM and vitamin E > 80 microM enhanced the toxic effect of H(2)O(2). Although levels of GSH actually decreased by 10-20% in cells supplemented with vitamin C or vitamin E, the protective effects of vitamin C and vitamin E on BSO-treated cells were associated with significant ( approximately 70%) decreases in oxidized glutathione (GSSG) and concomitant restoration of the cellular redox status (as indicated by GSH:GSSG ratio) to levels detected in cells not treated with BSO. These results demonstrate a role for vitamin C and vitamin E in maintaining glutathione in its reduced form. The ability of vitamin C and vitamin E in compensations for GSH depletion to protect against H(2)O(2)-induced cell death suggests that GSH, vitamin C, and vitamin E have common targets in their actions against oxidative damage, and supports the preventive or therapeutic use of vitamin C and E to combat age- and pathology-associated declines in GSH. Moreover, levels of these nutrients must be optimized to achieve the maximal benefit.

H2O2-mediated oxidative stress activates NF-κB in lens epithelial cells

In the mammalian lens, intracellular oxidants produced by photo-oxidative processes and exposure to toxic chemicals constitute stresses that produce cellular oxidative damage, result in changes in gene expression, and are causally related to cataract formation. Currently, it is believed that H(2)O(2) is the major oxidant to which the lens is exposed. In this report, we examine the activation and regulation of the oxidant-sensitive transcription factor, NF-kappa B, by H(2)O(2)-mediated oxidative stress in lens epithelial cells. Lens epithelial cells treated with H(2)O(2) demonstrated at 1 h a strong activation of NF-kappa B which returned to basal levels by 2 h. Under proteasome inhibition using both MG132 and lactacystin, H(2)O(2)-mediated activation of NF-kappa B was prevented, implicating the involvement of proteasome degradation of I kappa B proteins as being necessary for this activation. However, Western blot analysis demonstrated no degradation of I kappa B-alpha, -beta, or -epsilon associated with H(2)O(2)-mediated NF-kappa B activation. In comparison, when cells were treated with the cytokine TNF-alpha, NF-kappa B was strongly activated and degradation of both I kappa B-alpha and -beta was observed. These results clearly demonstrate that H(2)O(2)-mediated oxidative stress activates NF-kappa B in lens epithelial cells, which may subsequently lead to changes in gene expression. The results also reveal that different signaling pathways in the activation of NF-kappa B in lens epithelial cells are utilized by H(2)O(2) and TNF-alpha. These different pathways of NF-kappa B activation may be required to effect specific NF-kappa B-dependent gene expression in response to these different stimuli.