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

Abstract

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 alphaB-crystallin modified by HNE was ubiquitinated at a faster rate than that of native alphaB-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 alphaB-crystallin was degraded at a faster rate than that of unmodified alphaB-crystallin. Inhibition of the lysosomal activity stabilized HNE-modified alphaB-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-beta-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.

Figure 1.

Formation of HNE-protein adducts. Lane 1 is α-crystallin not treated with HNE. Lanes 2-5 are α-crystallins exposed to 25, 50, 100, and 200 μM HNE, respectively, for 2 h at 37°C. Lower panel) Protein profiles of α-crystallins isolated from bovine lenses after incubation with HNE (Coomassie Blue staining). Upper panel) modification of α-crystallins by HNE as determined by Western blotting with antibodies to HNE-modified proteins.

Figure 2.

Ubiquitination and degradation of HNE-modified αB-crystallin. ¹²⁵I-labeled recombinant αB-crystallin was incubated with or without 100 μM HNE for 2 h at 37°C . After removal of unreacted HNE, ¹²⁵I-labeled αB-crystallins were incubated in proteasome-free fraction II prepared from rabbit reticulocyte lysate in the presence or absence of ubiquitin. A) Ubiquitination of HNE-modified αB-crystallin: Lanes 1, 3, 5, and 7 are unmodified αB-crystallin and lanes 2, 4, 6, and 8 are HNE-modified αB-crystallins. Lanes 1, 2, 5 and 6 show formation of ubiquitin conjugates in the presence of ubiquitin and lanes 3, 4, 7, and 8 are negative control after incubation without ubiquitin. Formation of ubiquitin-¹²⁵I-labeled αB-crystallin conjugates was assessed by autoradiography following SDS-PAGE and transferring to nitrocellulose membrane (lanes 1-4), and the ubiquitin conjugates were confirmed by Western blotting with antibody to ubiquitin (lanes 5-8). B) degradation of HNE-modified αB-crystallin. Percent degradation was calculated from acid-soluble radioactivity recovered in supernatants after 2 h of incubation with reticulocyte lysate at 37°C. Values are means ± SD of 4 independent determinations; each was done in duplicate.

Figure 3.

Effects of different protease inhibitors on the stability of HNE-modified αB-crystallin delivered into lens cells. αB-crystallin was iodinated and modified by HNE as described in Fig. 2. ¹²⁵I-αB-crystallins were delivered into cultured human lens epithelial cells using the BioPORTER protein delivery reagent. Levels of ¹²⁵I-αB-crystallins (middle panel) and the putative mono-ubiquitnated ¹²⁵I-αB-crystallins (upper panel) were determined after incubation in the absence or presence of indicated inhibitors for 4 h. Autoradiograms were scanned, and levels of ¹²⁵I-αB-crystallins in the cells were quantified with a densitometer (lower panel). Data in lower panel are means ± SD of 3 independent experiments.

Figure 4.

Removal of HNE-modified proteins in human lens epithelial cells. Cells were treated with 5 μM HNE for 1 h and allowed to recover in a medium with or without 40 μM MG132 for 0-24 h. Cells were collected, and an equal amount of protein (30 μg) from cell lysate was analyzed by Western blotting with antibodies to HNE-conjugated proteins. Upper panel ) Typical Western blotting result. Lower panel) Densitometry quantification of HNE-modified proteins in the cells. Data are lower panel are means ± SD of 3 independent experiments.

Figure 5.

Effects of protease inhibitor on removal of HNE-modified proteins from lens epithelial cells. Cells were treated with 5 μM HNE for 1 h and allowed to recover in a medium contains different protease inhibitors for 4 h. Cells were then collected and an equal amount of protein (30 μg) from the cell lysate was analyzed by Western blotting with antibodies to HNE-conjugated proteins. Lane 1, untreated cells; lane 2; cells treated with HNE for 1 h; lane 3, recovered without any inhibitor; lane 4, recovered in the presence of MG132; lane 5, recovered in the presence of clasto-lactacystin-β-lactone; lane 6, recovered in the presence of epoxomicin; lane 7, recovered in the presence of chloroquine. Upper panel) Typical Western blotting result. Lower panel) Densitometry quantification of HNE-modified proteins in the cells. Data in the lower panel are means ± SD of 3 independent experiments.

Figure 6.

HNE-modified proteins are ubiquitinated in lens cells. A) ATP-dependent removal of HNE-modified proteins. Human lens epithelial cells were treated with 5 μM HNE for 1 h and allowed to recover in a normal medium or in a medium containing antimycin A and 2-deoxyglucose for 4 h. The cells were collected and an equal amount of protein (30 μg) from cell lysate was analyzed by Western blotting with antibodies to HNE-conjugated proteins (upper panel) or antibodies to ubiquitin (lower panel). B) Ubiquitination of HNE-modified proteins in cells. Cells were infected with a control virus or with a recombinant adenovirus encoding (His)₆-tagged ubiquitin for 48 h, and then cells were treated with HNE for 1 h. Ubiquitin conjugates were isolated from the cell lysate by Ni-NTA resin and resolved by SDS-PAGE. Lanes 1 and 2 were probed with antibodies to ubiquitin, and lanes 3 and 4 were identical samples as lanes 1 and 2 but probed with antibodies to HNE-conjugated proteins.

Figure 7.

Hypothesis of ubiquitin-dependent protein quality control mechanism. We hypothesize that many proteins have internal signal for ubiquitination as indicated by “DEG” in the diagram. Ubiquitination signal is hidden in the native conformation, and therefore native proteins are not recognized by the ubiquitination machinery. However, various modifications and damage may cause protein unfolding and exposure of ubiquitination signal. Thus, the unfolded proteins will be recognized by the combination of specific E2 s and E3 s and be ubiquitinated. Specific modification, such HNE, may serve as the signal for ubiquitination. Poly-ubiquitinated proteins will be recruited to the 26S proteasome for degradation, and mono-ubiquitinated proteins may enter into the lysosome for degradation.