The FASEB Journal • FJ Express
Selectivity of the ubiquitin pathway for oxidatively modified proteins: relevance to protein precipitation diseases E. J. Dudek, F. Shang, P. Valverde, Q. Liu, M. Hobbs, and A. Taylor1 Laboratory for Nutrition and Vision Research, USDA Human Nutrition Research Center on Aging at Tufts University, Boston, Massachusetts, USA To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4049fje; doi: 10.1096/fj.05-4049fje SPECIFIC AIMS There is now consensus that: 1) the accumulation of oxidatively modified proteins is cytotoxic and causally related to several age-related diseases including agerelated cataracts and other amyloid diseases; and 2) that proteolytic pathways provide a quality control mechanism to limit accumulation of modified proteins. Although many researchers assume that the ubiquitin proteasome pathway (UPP) is involved in recognition and proteolytic removal of oxidatively modified proteins that are produced upon cellular stress, there has been no direct evidence to support this hypothesis. This research tested the hypothesis that the UPP is involved in the selective targeting of oxidatively damaged proteins as well as in maintenance of cellular viability upon oxidative challenge. PRINCIPAL FINDINGS 1. Oxidatively modified proteins are preferred substrates for ubiquitination Using K6W-Ub, we directly tested the hypothesis that as compared with nonoxidatively modified proteins, oxidatively modified proteins are selective substrates for ubiquitination. This ubiquitin variant allows for efficient integration into high MW ubiquitin conjugates but not for their efficient degradation or disassembly. Therefore, K6W-Ub-containing conjugates accumulate. K6W-Ub was readily incorporated into conjugates as shown by the presence of this moiety in high MW conjugates on immunoblots (Fig. 1A, lanes 1– 4). In comparison, when identical levels of the control, progenitor viral vector was used no such conjugates were observed (Fig. 1A, lanes 5– 8). Blotting with an antibody to wild-type ubiquitin showed that ubiquitination using endogenously expressed WT-ubiquitin proceeded in both groups of cells (Fig. 1B). The majority of the material containing the ubiquitin variant was bound (B) (Fig. 1A, lanes 2 and 4). Only a small fraction of the K6W-Ub-containing conjugates were found in the flowthrough (FT) fraction (Fig. 1A, lanes 1 and 3). Upon 0892-6638/05/0019-1707 © FASEB
oxidative stress there was a 1.7-fold increase in ubiquitin conjugates in the B fraction and 1.3-fold increase in the FT fraction (Fig. 1B), suggesting that protein moieties produced in response to the oxidative stress were selectively incorporated into ubiquitin conjugates. As shown in Fig. 1C, treatment of RLE cells with H2O2 increased protein carbonyls in the FT (compare lanes 3 vs. 1 and 7 vs. 5) and B fractions (compare lanes 4 vs. 2) of the cells that were infected with the K6W-Ub virus. Trace amounts of carbonyls (Fig. 1C, lanes 8 and 6) and proteins (Fig. 1D, lanes 8 and 6) must be due to experimental procedures and nonspecific binding since no Ni ligand is expressed in these lysates. Therefore, they were considered background and are corrected for in the calculations that follow. The H2O2induced increase in total carbonyls was 2.7-fold, with the increase in the B fraction being 6.8-fold and the increase in the FT being 2.1-fold (Fig. 1C). These data indicate that ubiquitin conjugates, particularly those conjugates that are formed in response to oxidative stress, include and are enriched in carbonyl-containing proteins. This is corroborated by comparing levels of protein carbonyls in the RGS(His)6K6W-Ub conjugates. The ratio of carbonyls (Fig. 1C) to K6W-Ub (Fig. 1A) in the B fraction is 10-fold higher after oxidation (lane 4) relative to the ratio in unstressed cells (lane 2). This result is corroborated by additional results that used RLE cells, as well as by data obtained from 293 cells. When 293 cells expressing HA-Ub were challenged by H2O2 , there was a ⬎15-fold increase in carbonyls in the pool of the HA-tagged high MW ubiquitin conjugates (data not shown). As indicated in Fig. 1D, the differences in ubiquitin conjugates and protein carbonyls among the samples is not due to loading variations. The levels of all observable proteins in the various B fractions were indistinguishable. The same is true for the FT fractions. Levels of GFP in the FT fractions were also identical, indicating that there were no significant 1 Correspondence: Laboratory for Nutrition and Vision Research, USDA HNRCA, Tufts University, 711 Washington St., Boston, MA 02111, USA. E- mail:
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differences in viral gene expression between the control and K6W-Ub adenoviruses used (data not shown). In support of the data above, in a reciprocal experiment we observed a marked enrichment for ubiquitin conjugates in the fraction of proteins that was first enriched for carbonyls (Fig. 1E). In these experiments only 60 M H2O2 was used. This is the approximate level of H2O2 in the aqueous humor, the fluid that brings nutrients to the lens epithelial cells. 2. Inhibition of ubiquitin-mediated proteolysis potentiates H2O2-mediated cytotoxicity
Figure 1. Oxidatively damaged proteins are preferred substrates for ubiquitination. Cells were infected with recombinant replication deficient adenoviruses that express RGS(His)6K6W-Ub (lanes 1– 4) or "control" virus (lanes 5– 8). Cells were (lanes 3, 4, 7, 8) or were not (1, 2, 5, 6) treated with 500 M H2O2 for 1 h. (His)6-ubiquitinated proteins retrieved using Ni chromatography. A) RGS(His)6-containing ubiquitin conjugates in the samples were visualized after the samples were resolved by Western blot analysis using an antibody to RGS(His)4. All experiments (all figures) were repeated at least twice each time in triplicate. B) An identical blot was probed with a polyclonal anti-ubiquitin antibody. C) To visualize protein carbonyls, the same samples were reacted with DNPH, and blots probed with a polyclonal antibody to DNP. D) Coomassie blue-stained gel of the same samples as those shown above. Identical amounts of cell lysates were used as indicated by the indistinguishable amounts of protein in virtually every protein band in the B or FT fractions from each of the lysates. E) Oxidatively modified proteins are ubiquitinated. HLE cells were treated with a constant level of 60 M H2O2 for 4 h, then lysed. The supernatant was divided into two portions. One portion was derivitized with Biotin-labeled hydrazide and the other portion was mock derivitized. Reaction mixtures were passed through mono-avidin columns. The avidin-bound proteins were eluted with D-biotin. Equal amounts of proteins were resolved by SDS-PAGE and silver stained (lanes 1, 2) or transferred to nitrocellulose for detection of ubiquitin conjugates (lanes 3, 4). Lanes 1 and 3 show nonspecifically-bound proteins (not derivitized with 1708
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It is believed that the accumulation of endogenous high MW protein aggregates is cytotoxic. To determine if this includes cytotoxicity due to elevated levels of high MW ubiquitin conjugates, particularly of ubiquitin conjugates that are enriched in carbonylated proteins, we determined H2O2-mediated cytotoxicity. In exponentially growing RLE cells treated with 0-2000 M H2O2 and allowed to recover for 24 h (Fig. 2) expression of K6W-Ub clearly enhanced H2O2-induced cytotoxicity as indicated by decreased mitochondrial function. Whereas decreased mitochondrial function was observed only at 2000 M in cells infected with control virus, decreased MTS was observed at ⬃25% this level of H2O2 in cells in which the K6W-Ub variant was expressed. Enhanced H2O2-mediated cytotoxicity with K6W-Ub expression was also observed in RLE cells and in human lens cells. The relatively constant MTS activities in uninfected cells, and in cells that were infected with control or wt-Ub demonstrates that: 1) the viral vectors caused little, if any, cytotoxicity under these conditions (Fig. 2A, right panel); and 2) ubiquitin was probably not limiting and ubiquitin function was probably not compromised modified during the experiment. Similar results were obtained using COS and human lens epithelial cells. Taken together, the above results are consistent with the hypothesis that: 1) accumulation of oxidatively modified proteins is cytotoxic; 2) clearance of these damaged proteins from cells is mediated at least in part by the UPP; 3) clearance of oxidized proteins by the UPP is associated with prolonged cell viability; and 4) delayed processing of ubiquitin conjugates of oxidatively modified cellular proteins (i.e., due to the K6WUb) is associated with enhanced cell death. The data further argue for an essential role for the UPP, at least in part, in protein quality-control and, more specifically, in removal of oxidatively modified proteins and restoration of homeostasis in response to oxidative stress. CONCLUSIONS AND SIGNIFICANCE Using direct affinity-based screens, we demonstrated that oxidatively modified proteins are preferred subbiotin-labeled hydrazide) and lanes 2 and 4 show carbonylcontaining proteins (derivitized with biotin labeled hydrazide) eluted from the avidin column.
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DUDEK ET AL.
strates for ubiquitination. Using a physiologically relevant type and amount of oxidative stress we also show that upon such stress, inhibition of the UPP is associated with cytotoxicity. What are the ramifications of having a functional or dysfunctional UPP with respect to age-related diseases? The current data are consistent with the hypothesis that the UPP is a general pathway by which oxidatively modified proteins - either directly or due to associated changes in conformation - are recognized for ubiquitination and degradation, and suggests that the UPP plays an essential role in the clearance of these proteins upon stress and aging. The converse is also probably true: that age-related or stress-induced compromises in function of the UPP result in the accumulation of damaged proteins and with the associated cellular and perhaps, organellar dysfunction. However, as depicted in Fig. 3, the situation may be more complex. In this scheme we posit that compromises of the fidelity of ubiquitination or diminished function of the proteasome would result in enhanced cytotoxicity because of a failure to remove damaged proteins. Inappropriate ubiquitination and/or failure to remove the ubiquitin-protein conjugates would exacerbate the situation because addition of multiple ubiquitins, as occurs in the UPP, renders even a small substrate a much higher MW moiety. Coupled with further modification (i.e., due to oxidation, glycation, glycoxidation, and deamination), age-related proteasome inactivation, and inhibition of the proteasome by aggregates, or by processes associated with generation of the aggregates, these proteins are prone to accumulate at accelerated rates. Rather than being cleared, they aggregate, crosslink and precipitate, disrupting
Figure 2. Cell viability during H2O2-mediated oxidative stress requires proteolysis-competent ubiquitin. RLE cells were infected at ⬃80% confluence with equivalent titers of control adenovirus (filled bars) or adenovirus that encodes K6W-Ub (open bars) for 48 h, then treated with a single bolus of the indicated (final) concentration of H2O2 for 1 h, rinsed with PBS, and allowed to recover for 24 h. After recovery, cell cultures were examined for mitochondrial activity (expressed as % untreated control, 0 M H2O2) using the MTS assay. *P ⬍ 0.01 for cells treated with H2O2 vs. untreated control. **P ⬍ 0.001 for K6W-Ub virus infected cells compared with cells infected with control virus treated at the same H2O2 concentration.
OXIDIZED PROTEINS: SUBSTRATES FOR THE UPP
Figure 3. Predicted consequences of UPP dysfunction during stress and/or aging. The UPP functions in young tissues to degrade oxidatively damaged proteins, thus avoiding cytotoxic effects due to their accumulation. In older or stressed tissues, damaged or inappropriately ubiquitinated proteins may accumulate in cytotoxic aggregates. This may be due to a surfeit of damaged proteins and/or reduced activity of UPP components. When these proteins accumulate and exceed the cell’s degradative capacity, cytotoxicity ensues. The situation may be exacerbated since the aggregates, or stresses associated with aggregate formation, can inhibit the proteasome, further limiting cellular abilities to execute protein quality control. This would result in a self-propagating cycle of enhanced cytotoxicity.
cellular function in the process. Such an accumulation is particularly obvious in nonrenewable tissues such as neurons or the aged and cataractous lens, where levels of ubiquitin-protein conjugates in the insoluble fraction are higher. As evidence of this “autocatalytic” decrement in maintenance of protein quality, it is observed that whereas initiation of lens early opacities takes decades, progression to debilitating cataracts frequently occurs during just a few years. This age-related accumulation and acceleration toward lesion is consistent with the age-related decline in UPP capability, particularly the proteolytic component of the UPP. The accumulation of protein aggregates that contain oxidatively modified proteins with ubiquitin conjugates also disrupt function in many other cell types including premature aging syndromes, age-related diseases such as age-related maculopathy, the amyloid diseases including Alzheimer’s, Parkinson’s and ALS, ataxias, and other neurodegenerative disorders, diabetes, and some cancers. We demonstrate that when functional, the UPP is part of the cellular proteolytic quality control machinery that targets oxidatively modified proteins, and that this capability is associated with extended cell function. However, upon stress, components of the UPP, like other cellular proteins, have their structures and their activities compromised, resulting in several age-related and stress-related debilities and diseases.
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