September 2016
Volume 57, Issue 11
Open Access
Lens  |   September 2016
The Immunoproteasome in Human Lens Epithelial Cells During Oxidative Stress
Author Affiliations & Notes
  • Anne Petersen
    Department of Clinical Neuroscience, Institute of Neuroscience and Physiology, the Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden
  • Madeleine Zetterberg
    Department of Clinical Neuroscience, Institute of Neuroscience and Physiology, the Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden
  • Correspondence: Anne Petersen, Institute of Neuroscience and Physiology, Department of Clinical Neuroscience, The Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden; anne.petersen@gu.se
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 5038-5045. doi:10.1167/iovs.16-19536
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      Anne Petersen, Madeleine Zetterberg; The Immunoproteasome in Human Lens Epithelial Cells During Oxidative Stress. Invest. Ophthalmol. Vis. Sci. 2016;57(11):5038-5045. doi: 10.1167/iovs.16-19536.

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Abstract

Purpose: The immunoproteasome is known to generate peptides for antigen presentation. However, it has also been proposed to have additional functions such as stress response. The propensity of the immunoproteasome for degradation of oxidatively damaged proteins and peptides makes it interesting in the context of cataract formation and prevention. This study hypothesized that the immunoproteasome is present in human cataractous lenses and that oxidative stress will induce its expression, affect its proteolytic activity and its intracellular location in native cultured human lens epithelial cells (HLECs).

Methods: The expression of the immunoproteasome and the constitutive proteasome subunits β1i/β1, β2i/β2, and β5i/β5 were studied by using Western blotting. The chymotrypsin-like activity was investigated for possible oxidative stress response. Inhibitors specific for the immuno- and constitutive proteasome, ONX-0914 and MG-132, respectively, were used to study their relative contributions to total proteasome activity. The intracellular location of the proteasomal subunits β5i and β5 was studied by immunocytochemistry.

Results: Immunoproteasome subunits were detected both in the lens epithelium and in the lens fibers derived from cataract surgery. Oxidative stress to cultured HLECs upregulated the immunoproteasome but not the constitutive proteasome. The chymotrypsin-like activity decreased with increased oxidative stress and the two proteasome types contributed equally to total proteasome activity. Immunocytochemical labeling of subunit β5i showed mainly cytosolic localization, whereas subunit β5 was localized predominantly in the nucleus. H2O2-induced challenge increased the expression of the immunoproteasome.

Conclusions: The present findings indicate a role for the immunoproteasome in oxidative stress management in the lens.

Cataract, which is the leading cause of blindness globally, is a protein conformational disease characterized by aggregation of oxidatively damaged proteins. In this respect, it resembles other protein aggregation diseases, such as amyloidosis, prion disease, or Alzheimer's disease.1 Because of this type of pathogenesis, the proteasome has been implicated as an important player in cataract formation. The proteasomes are important proteolytic complexes for degradation of proteins and peptides in the cell.26 Studies in the lens have shown a preference of the ubiquitin-proteasome pathway for degrading mildly oxidized or glutathiolated lens proteins as opposed to native protein.7,8 
The first recognized proteasomal subtype, the constitutive proteasome, also called 20S, is the core of a larger complex, the 26S proteasome, which also includes the regulatory complex PA700. Together with a small protein, ubiquitin (Ub), and the ubiquitin-conjugating enzymes E1, E2, and E3, they constitute the ubiquitin-proteasome pathway, which is considered the major system for proteolytic degradation of damaged or misfolded proteins.25 However, there are additional subtypes of the proteasome, of which the immunoproteasome is one, with structural, catalytic, and target preference variations.3,914 Like all subtypes of the proteasome, the immunoproteasome, denoted 20Si, is a barrel-shaped hollow complex consisting of a catalytic core complex with a regulatory complex on each side. The core consists of two types of subunits, α and β, that form a cylinder-shaped structure of 4 stacked rings with 7 subunits in each ring.3,4,6,11 In the immunoproteasome, 3 of the catalytic β-subunits, β1 (PSMB6), β2 (MCP-165), and β5 (PSMB5) are exchanged for 3 homologous subunits, β1i (LMP2), β2i (MECL1), and β5i (LMP7).3,1113,15 
The constitutive proteasome has three different catalytic activities: caspase-like activity for β1, trypsin-like activity for β2, and chymotrypsin-like activity for β5 subunits. However, in the immunoproteasome, β1i has reduced caspase-like activity and increased chymotrypsin-like activity, whereas β2i and β5i exhibit trypsin-like and chymotrypsin-like activity, respectively, as do their counterparts β2 and β5 in the constitutive proteasome.11,13,1618 
Because the immunoproteasome is expressed in immune-privileged tissues such as the brain1921 and the retina,20,2225 it has been ascribed additional functions other than generation of peptides for antigen presentation, such as response to stress and maintenance of cellular homeostasis. Because the immunoproteasome has been found to be upregulated in oxidatively stressed cells in culture23,26 and in aging21,23 and because immunoproteasome subunits have been detected in mouse lens,27 the immunoproteasome may be of importance in cataractogenesis. Oxidative stress is a major risk factor in cataract.2830 It is well known that the 20S proteasome degrades oxidatively modified proteins.3135 However, recent studies show that the immunoproteasome is induced by oxidative stress23,26,36,37 and that it may be more efficient at degrading oxidatively damaged proteins and peptides than the 20S or 26S proteasome.37 There are only a few studies of the immunoproteasome in the lens27,38 and to our knowledge none in the human lens. 
In the present study, we aimed to test the hypothesis that the immunoproteasome is expressed in human cataractous lenses and that its expression can be induced by oxidative stress in cultured native human lens epithelial cells. We also hypothesized that oxidative stress affects the proteolytic activity of the immunoproteasome and that it may influence its subcellular distribution. 
Materials and Methods
Human Lens Epithelial Cell Culture
Human lens epithelium specimens were obtained from lenses through an anterior continuous curvilinear capsulorhexis during phacoemulsification cataract surgery at the Eye Clinic, Sahlgrenska University Hospital, Mölndal, after informed consent. The study was approved by The University of Gothenburg Ethics Committee, and the tenets of the Declaration of Helsinki were followed. Using Dulbecco's modified Eagle medium (Sigma Chemical, St Louis, MO, USA) as culture medium, we cultured human lens epithelium specimens, usually 5 mm in diameter, as described previously.39 For protein expression and proteolytic activity experiments, cells were cultured in 6-well culture dishes (TPP, Trasadingen, Switzerland) and collected with cell scrapers before further analyses in all experiments, except for visualization with immunofluorescence, wherein cells were cultured in 8-well chamber slides (Lab-Tek; Nalge Nunc International, Rochester, NY, USA). For each experiment, human lens epithelial cells (HLEC) from three or more native cell lines were used, each cell line containing cells from one individual, passage V–X. 
Protein Expression in Human Lens Epithelium Specimens
Human lens epithelium specimens obtained from lenses during cataract surgery were immediately put in a solution of 60 μl NuPage 0.5% lithium dodecyl sulfate sample buffer (Life Technologies, Carlsbad, CA, USA) supplemented with protease inhibitors, cOmplete Mini (Roche Diagnostics Scandinavia AB, Bromma, Sweden), for inhibition of serine, cysteine, and metalloproteases. After subsequent freezing and thawing, the lysates were heated at 70°C for 10 minutes and sonicated for 20 seconds at 50% amplitude (Branson Ultrasonic Corp., Danbury, CT, USA). All lysates were handled on ice. 
To detect constitutive- and immunoproteasome subunits, Western blotting was performed. Reducing agent (50 μM Dithiothreitol; Invitrogen, Carlsbad, CA, USA) was added to the lysate, followed by protein separation on precast bis-tris 4%–12% gradient minigels (NuPage MES; Life Technologies) as running buffer. For Western blot analyses, proteins were transferred to nitrocellulose membranes, followed by blocking in 5% nonfat milk powder in 20 mM Tris-HCl (pH 7.5) for 1 hour. 
Primary antibodies used for Western blot detection of constitutive- and immunoproteasome subunits included mouse monoclonal anti-PSMB6 (β1 subunit; 1:100 dilution), anti-LMP2 (β1i subunit; 1:100 dilution), anti-MCP-165 (β2 subunit; 1:100 dilution), anti-LMP7 (β5i subunit; 1:100 dilution), goat polyclonal anti-PLMP2 (β5 subunit; 1:100 dilution), and anti-MECL1 (β2i subunit; 1:100 dilution) (Santa Cruz Biotechnology, Dallas, TX, USA). Monoclonal mouse anti-β-actin (1:2000 dilution) was used as loading control (Santa Cruz Biotechnology). 
Secondary antibodies were conjugated to horseradish peroxidase (Sigma). Protein bands were visualized with Luminata Forte Western horseradish peroxidase substrate (Millipore Corp., Billerica, MA, USA) in ImageQuant LAS 500 (GE Healthcare, Piscataway, NJ, USA). 
Protein Expression in Oxidatively Stressed Human Lens Epithelial Cells
Western blotting was performed to study protein expression in oxidatively stressed HLECs. HLEC exposed to 0, 100, or 1000 μM H2O2 in serum-free culture medium for 1 hour were rinsed in ice-cold phosphate-buffered saline (PBS), after which 200 μl of ice-cold PBS supplemented with protease inhibitor, cOmplete Mini, was added. The H2O2 concentration used for induction of oxidative stress was chosen after dose response experiments with H2O2 (not shown). 
The cells were harvested using a cell scraper and immediately frozen in −80° C. 
Protein concentration of the samples was determined using the BCA protein assay reagent (Pierce Perbio Science UK Ltd., Cheshire, UK) with bovine serum albumin as standard. Following sample preparation, Western blotting was performed as described above. Equal amounts of protein (18 μg) were separated on the gel. The same antibodies used to detect constitutive- and immunoproteasome subunits were also used in human lens epithelium specimens. Densitometric analysis was performed using ImageJ version 1.37 software (provided in the public domain by the National Institutes of Health, Bethesda, MD, USA). 
Protein Expression in Lens Homogenate in Irrigation Solution From Phacoemulsification
To study the presence of the immunoproteasome subunit LMP2 (β1i) and constitutive proteasome subunit PSMB6 (β1) in lens homogenate in irrigation solution from phacoemulsification, the lens homogenate was centrifuged at 580 g for 5 min in +4°C. The pellet was resuspended in 500 μl of PBS containing protease inhibitors, cOmplete Mini, and was sonicated for 4 × 10 seconds at amplitude of 50%. 
The supernatant was centrifuged at 3000 g for 55 minutes at +4°C in Amicon Ultra-15 centrifugal filter unit 30 molecular weight cut-off (MWCO) (Millipore). To further concentrate and capture the immuno- and constitutive proteasomes in the supernatant, immunoprecipitation was performed using Dynabead protein G immunoprecipitation kit (Novex; Life Technologies) according to the manufacturer's protocol. As antibodies, mouse monoclonal anti-LMP2, 10 μg, and anti-PSMB6, 10 μg, were used. Electrophoresis and Western blotting for pellet and immunoprecipitated supernatant with labeling of LMP2 and PSMB6 subunits were carried out, followed by protein band expression and densitometric analysis. Protein concentrations for both the pellet and the supernatant were determined using the BCA protein assay reagent. 
Proteasome Activity
The chymotrypsin-like activities of the immuno- and constitutive proteasomes were assayed to study susceptibility to oxidative stress. Triplicates of confluent cells were rinsed in PBS, followed by exposure to 0, 100, or 1000 μM H2O2 in serum-free culture medium for 1 hour. The medium was removed, and the cells were immediately frozen at −80° C for at least 30 min. 
The chymotrypsin-like activity was measured using the synthetic substrate Suc-Leu-Leu-Val-Tyr-AMC (LLVY, at a final concentration of 50 μM; Bachem AG, Bubendorf, Switzerland). The activity was measured continuously at emission wavelength of 460 nm and excitation wavelength of 380 nm for 2 hours at 37°C, and Vmax was determined in the linear interval by using a microplate reader (Infinite M200 PRO; Tecan Group Ltd., Männedorf, Switzerland). Proteolytic activity was expressed as the increase in relative fluorescence units per second and gram of protein (RFU s−1 g−1). Protein determination was made using the BCA protein assay reagent (Pierce) with bovine serum albumin as standard. Absorption was measured at 570 nm. 
Relative Immuno- and Constitutive Proteasomes' Contribution of Chymotrypsin-like Activity
To study the relative contribution of the immunoproteasome and constitutive proteasomes to total proteasome activity during oxidative stress, specific inhibitors against the immunoproteasome, ONX-0914 (200 μM), and against all forms of proteasomes, MG-132 (10 μM), were used. Confluent cells in triplicate were rinsed in PBS, after which the cells were preincubated with the inhibitors for immunoproteasome and all forms, ONX-0914 and MG-132, respectively, for 30 minutes, followed by exposure to 0, 100, or 1000 μM H2O2 in serum-free culture medium for 1 hour with or without respective proteasome inhibitor. The chymotrypsin-like activity assay was performed as previously described. 
Immunocytochemical Labeling of Immuno- and Constitutive Proteasome Subunits
After exposure to 0, 100, or 1000 μM H2O2 for 1 hour, HLECs were rinsed in PBS and fixed in 4% paraformaldehyde (pH 7.4) for 30 minutes. The cells were rinsed again and permeabilized by 0.25% Triton-X in PBS for 10 minutes at room temperature (Sigma-Aldrich). Following standard protocols for immunocytochemistry, the cells were labeled with primary antibodies, mouse monoclonal anti-LMP7 and rabbit polyclonal anti-PSMB5 (Abcam, Cambridge, UK). The labeling was visualized by Alexa Fluor 488 goat anti-rabbit (green), Alexa Fluor 568 goat-anti-rabbit (red), or Alexa Fluor 488 goat-anti-mouse (green) (Molecular Probes, Eugene, OR, USA). Nuclear morphology was viewed using cell-permeable Hoechst 33342 dye (blue; Sigma-Aldrich) at final concentration of 10 μg/ml, and Alexa Fluor 568 phalloidin (red) was used for labeling of F-actin (Molecular Probes). Both single- and double-labeling were performed. 
Statistical Analysis
Densitometric analysis of Western blots used β-actin for normalization, and results are area under the curve (AUC). Western blotting and proteolytic activity experiments were repeated three or more times using cell lines from different individuals. Representative results are shown. Data are mean ± SEM values from triplicate samples. Statistical analysis was performed using ANOVA with Dunnett post hoc, using SPSS version 22 software (IBM Corp., Armonk, NY, USA). P values ≤0.05 were considered statistically significant. Calculations of colocalization were not performed. 
Results
Effect of Oxidative Stress on Protein Expression in HLEC
Human lens epithelial cells oxidatively stressed by 0, 100, or 1000 μM H2O2 exhibited increased expression of the immunoproteasome subunits LMP2, MECL1, and LMP7 in a dose-dependent manner. LMP2 expression levels in HLEC exposed to 1000 μM H2O2 and LMP7 expression in HLEC exposed to 100 and 1000 μM H2O2 were significantly upregulated (Figs. 1A–C). In contrast, the subunits PSMB6, MCP-165, and PSMB5 of the constitutive proteasome were not significantly affected by oxidative stress (Figs. 1D–F). 
Figure 1
 
Effects of H2O2 on proteasome subunit protein expression in HLECs. Human lens epithelial cells were challenged by 0, 100, or 1000 μM H2O2 for 1 hour, after which protein expression was determined. Expression levels of the immunoproteasome subunits LMP2, MECL1, and LMP7 were upregulated by H2O2 (AC). The constitutive proteasome subunits PPSMB6, MCP-165, and PSMB5 showed no significant change in protein expression (DF). Data from densitometric analyses of Western blot bands were normalized to β-actin. Experiments were performed three times in triplicate (n = 3), and one representative experimental run is shown. Mean ± SEM values are shown. *P < 0.05 and **P < 0.01 compared to control (no H2O2 exposure).
Figure 1
 
Effects of H2O2 on proteasome subunit protein expression in HLECs. Human lens epithelial cells were challenged by 0, 100, or 1000 μM H2O2 for 1 hour, after which protein expression was determined. Expression levels of the immunoproteasome subunits LMP2, MECL1, and LMP7 were upregulated by H2O2 (AC). The constitutive proteasome subunits PPSMB6, MCP-165, and PSMB5 showed no significant change in protein expression (DF). Data from densitometric analyses of Western blot bands were normalized to β-actin. Experiments were performed three times in triplicate (n = 3), and one representative experimental run is shown. Mean ± SEM values are shown. *P < 0.05 and **P < 0.01 compared to control (no H2O2 exposure).
Immuno- and Constitutive Proteasome Expression in Human Lens Epithelium Specimens and in Lens Homogenate From Cataract Surgery
All immunoproteasome subunits, LMP2, MECL1, and LMP7 and the constitutive subunits PSMB6, MCP-165, and PSMB5, were expressed in human lens epithelium specimens obtained from human lenses during cataract surgery (Fig. 2A). The immunoproteasome subunit LMP2 and the constitutive proteasome subunit PSMB6 were detected in both the soluble and insoluble fractions of human lens homogenate from phacoemulsification (Fig. 2B). 
Figure 2
 
Protein expression of immune- and constitutive proteasome subunits in human lens epithelium and lens homogenate. Human lens epithelium specimens obtained from lenses during cataract surgery showed expression of both the immunoproteasome subunits LMP2, MECL1, and LMP7 and the constitutive proteasome subunits PSMB6, MCP-165, and PSMB5 (A). (B) Lens homogenate from phacoemulsification irrigation solution was examined for detection of the immunoproteasome subunit LMP2 and the constitutive proteasome subunit PSMB6, both of which were present in the soluble and insoluble fractions.
Figure 2
 
Protein expression of immune- and constitutive proteasome subunits in human lens epithelium and lens homogenate. Human lens epithelium specimens obtained from lenses during cataract surgery showed expression of both the immunoproteasome subunits LMP2, MECL1, and LMP7 and the constitutive proteasome subunits PSMB6, MCP-165, and PSMB5 (A). (B) Lens homogenate from phacoemulsification irrigation solution was examined for detection of the immunoproteasome subunit LMP2 and the constitutive proteasome subunit PSMB6, both of which were present in the soluble and insoluble fractions.
Chymotrypsin-like Activity in Oxidatively Stressed HLEC
The chymotrypsin-like activity of the proteasome was decreased with increased oxidative stress (Fig. 3A). Most of the activity was inhibited by the general proteasome inhibitor MG-132. Using the immunoproteasome-specific inhibitor ONX-0914 in parallel cell cultures, we demonstrated that the relative contributions of the immunoproteasome and the constitutive proteasome to total chymotrypsin-like activity were approximately equal (Fig. 3B). 
Figure 3
 
Chymotrypsin-like activity of the proteasome during increased oxidative stress. (A) Human lens epithelial cells exposed to increased oxidative stress by H2O2 at 0, 100, and 1000 μM for 1 hour showed a decrease in chymotrypsin-like activity. The activity was significantly decreased at 1000 μM H2O2. The chymotrypsin-like activity is expressed as RFU/s/g protein. (B) Relative contribution of the immunoproteasome and the constitutive proteasome. The chymotrypsin-like activity of the immunoproteasome in cultured HLEC was partially inhibited by the immunoproteasome-specific inhibitor ONX-0914, and most of the activity was inhibited by MG-132. The relative contributions of the immunoproteasome and the constitutive proteasome were approximately equal in terms of total proteasome activity. Experiments were performed three times in triplicate (n = 3), and one representative experimental run is shown. Values are mean ± SEM. ***P < 0.001 compared to control.
Figure 3
 
Chymotrypsin-like activity of the proteasome during increased oxidative stress. (A) Human lens epithelial cells exposed to increased oxidative stress by H2O2 at 0, 100, and 1000 μM for 1 hour showed a decrease in chymotrypsin-like activity. The activity was significantly decreased at 1000 μM H2O2. The chymotrypsin-like activity is expressed as RFU/s/g protein. (B) Relative contribution of the immunoproteasome and the constitutive proteasome. The chymotrypsin-like activity of the immunoproteasome in cultured HLEC was partially inhibited by the immunoproteasome-specific inhibitor ONX-0914, and most of the activity was inhibited by MG-132. The relative contributions of the immunoproteasome and the constitutive proteasome were approximately equal in terms of total proteasome activity. Experiments were performed three times in triplicate (n = 3), and one representative experimental run is shown. Values are mean ± SEM. ***P < 0.001 compared to control.
Intracellular Localization of the Constitutive- and Immunoproteasome
Immunocytochemical labeling of the immunoproteasome subunit LMP7 showed that the immunoproteasome was localized mainly to the cytosol of the cell (Fig. 4A). The constitutive proteasome subunit PSMB5 was localized predominantly to the nucleus (Fig. 4B). When challenged by increasing H2O2 concentrations, the observed immunolabeling of immunoproteasome subunit LMP7 increased (Fig. 4A). Double-labeling against LMP7 and PSMB5 showed some colocalization (Fig. 5). 
Figure 4
 
Immunolocalization of the proteasome subunits LMP7 and PSMB5 in HLECs after exposure to H2O2. The immunoproteasome subunit LMP7 was localized mainly in the cytosol of the cell (A), in contrast to the constitutive proteasome subunit PSMB5, which was localized predominantly in the nucleus (B), as shown by immunocytochemistry. Higher oxidative stress levels showed increased immunolabelling of the immunoproteasome (A). Green: LMP7 and PSMB5; red: F-actin (phalloidin); blue: Hoechst dye (nuclear morphology). Scale bar: 10 μm.
Figure 4
 
Immunolocalization of the proteasome subunits LMP7 and PSMB5 in HLECs after exposure to H2O2. The immunoproteasome subunit LMP7 was localized mainly in the cytosol of the cell (A), in contrast to the constitutive proteasome subunit PSMB5, which was localized predominantly in the nucleus (B), as shown by immunocytochemistry. Higher oxidative stress levels showed increased immunolabelling of the immunoproteasome (A). Green: LMP7 and PSMB5; red: F-actin (phalloidin); blue: Hoechst dye (nuclear morphology). Scale bar: 10 μm.
Figure 5
 
Colocalization of the proteasome subunits LMP7 and PSMB5 in HLECs. Colocalization (yellow) of LMP7 and PSMB5 was evident as shown by double-labeling with anti-LMP7 (green) and anti-PSMB5 (red). Scale bar: 10 μm.
Figure 5
 
Colocalization of the proteasome subunits LMP7 and PSMB5 in HLECs. Colocalization (yellow) of LMP7 and PSMB5 was evident as shown by double-labeling with anti-LMP7 (green) and anti-PSMB5 (red). Scale bar: 10 μm.
Discussion
Oxidation of lens proteins is a major risk factor in cataract formation. Insufficient degradation of damaged proteins leads to formation of light-scattering aggregates that cause visual impairment.28,40 The proteasome and immunoproteasome are therefore important players in cell maintenance. The basal level of immunoproteasomes is low in nonimmune tissue but can be induced by interferon-γ or different types of stress, such as oxidative stress.11,23 In the present study, we found that immunoproteasome subunits were expressed both in human lens epithelium specimens obtained from lenses during cataract surgery and in cataractous lens homogenate derived from phacoemulsification cataract surgery. This might indicate an activation of the immunoproteasome in cataract due to oxidative stress or other stressors. Interestingly, other studies have suggested that the immunoproteasome might protect against oxidative stress due to its efficiency in degrading oxidatively damaged proteins and peptides.11,41,42 
To examine whether immunoproteasome expression was induced by oxidative stress, we subjected cultured HLEC to increasing levels of oxidative stress. This stress significantly upregulated the expression of the immunoproteasome subunits LMP2 and LMP7 and showed an increasing trend for subunit MECL1. A corresponding upregulation was not seen for the constitutive proteasome subunits, indicating that the immunoproteasome is the major proteasomal subtype involved in removal of oxidatively damaged proteins. 
However, when we examined the chymotrypsin-like activity of the proteasome, activity was decreased after exposure to increasing levels of oxidative stress, in this case by H2O2. This finding was in contrast to the expected, as immunoproteasome expression was upregulated by increased oxidative stress and the proteasome prefers oxidatively modified proteins for degradation. However, other studies have shown that the proteasome is susceptible to oxidative modifications including carbonylation, HNE modification, and S-glutathionylation.4347 Demasi et al.45 also showed that GSH treatment of 20S proteasomes inhibited the chymotrypsin-like and trypsin-like activities. Hussong et al.23 found a time-dependent decrease in chymotrypsin-like activity after H2O2 exposure that correlated with increased immunoproteasome expression in retinal pigment epithelial mouse cells, which is well in line with the findings in our study. 
An interesting question is whether the immuno- or constitutive proteasome chymotrypsin-like activity is differentially sensitive to oxidative challenge. Our results indicate that the activity of both proteasome types is equally affected by oxidative stress, that is, they both contribute to proteolytic degradation to approximately the same extent. These findings raise the question of why the effect on activity is comparable when immunoproteasome expression is upregulated by oxidative stress whereas the constitutive proteasome is not. As mentioned earlier, this may depend of oxidative changes of the proteasome but further studies are needed to fully understand the relationship between immunoproteasome expression and catalytic activity during oxidative stress. 
It is known that the 20S proteasome is localized both in the cytoplasm and in the nucleus,4852 and it has also been established that the immunoproteasome is localized in close proximity to the endoplasmic reticulum.49,53,54 We found that the immunoproteasome subunit LMP7 (β5i) is localized mainly to the cytosol as expected and that the subunit PSMB5 (β5) of the constitutive proteasome is localized mainly to the nucleus. As the constitutive proteasome also degrades proteins in the cytosol, it must be transported from the nucleus to the cytosol on demand. Wojcik and DeMartino51 suggested that the intracellular localization of proteasomes changes depending on the stage of the cell cycle.51 
Studies have shown that the constitutive proteasome resides in the nucleus in dividing cells,12,5557 whereas they are accumulated in dot-like clusters in the cytoplasm near the nucleus in quiescent cells.58 Transport into and out of the nucleus has also been confirmed.51,57,59 Our data also demonstrated increased labeling of the immunoproteasome subunit LMP7 by increased oxidative challenge, confirming the presented upregulation of immunoproteasome expression after oxidative stress in HLECs as determined by Western blot analysis. 
The present study is the first to demonstrate the presence of the immunoproteasome in human lenses. This is not unexpected as a few studies have shown its expression in mouse lens.27,38 The major significance of the present findings, however, is that several subunits of the immunoproteasome in cultured native HLECs are upregulated by oxidative stress, whereas subunits of the constitutive proteasome are not. This clearly demonstrates that the immunoproteasome is the more important player in oxidative stress response involved in protein quality control. Because oxidative insults to lens proteins leading to loss of normal protein conformation with subsequent protein aggregation and light scattering are acknowledged features of cataract formation,28,40 these results are important for understanding of protective mechanisms in the lens. In addition, this study demonstrates that the immunoproteasome is retained not only in the lens epithelium but also in human lens fibers from cataractous lenses, a finding that suggests a role of the immunoproteasome even at older age and during cataract development. A limitation of the study is the lack of clear human lenses, preferably from donors of a wide age range. Mishto et al.21 demonstrated higher expression of the immunoproteasome in brain tissue from patients with Alzheimer's disease than that in cognitively healthy individuals, and both the brain and the retina display higher levels of immunoproteasome upon aging.21,23 Another limitation of the study is the lack of information regarding the type of cataract that the patients were suffering from. It is well recognized that different types of cataract are associated with specific risk factors which may in turn be associated with different pathogenic mechanisms, such as oxidative stress.60 
In summary, the present study shows that the immunoproteasome is present in the human lens and that it is upregulated by oxidative stress whereas the constitutive proteasome is not. The chymotrypsin-like activity is decreased by oxidative stress and to this respect, the immuno- and constitutive proteasome are equally affected. As there are only a few studies of 20Si in the lens and, to our knowledge, none using human lens tissue, this study is of great value in increasing knowledge of how 20Si is expressed and activated during oxidative stress in human lens epithelial cells. 
Acknowledgments
Supported by Sahlgrenska University Hospital (“Agreement concerning research and education of doctors”) Grant ALFGBG-441721, the Göteborg Medical Society, the Marianne and Marcus Wallenberg Foundation, the Dr Reinhard Marcuses Foundation, the Konung Gustaf V:s och Drottning Victorias Frimurarestiftelse, the Hjalmar Svensson Foundation, the Greta Andersson Foundation, the Herman Svensson Foundation, Ögonfonden, De Blindas Vänner, and the Kronprinsessan Margaretas Arbetsnämnd för Synskadade. 
Disclosure: A. Petersen, None; M. Zetterberg None 
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Figure 1
 
Effects of H2O2 on proteasome subunit protein expression in HLECs. Human lens epithelial cells were challenged by 0, 100, or 1000 μM H2O2 for 1 hour, after which protein expression was determined. Expression levels of the immunoproteasome subunits LMP2, MECL1, and LMP7 were upregulated by H2O2 (AC). The constitutive proteasome subunits PPSMB6, MCP-165, and PSMB5 showed no significant change in protein expression (DF). Data from densitometric analyses of Western blot bands were normalized to β-actin. Experiments were performed three times in triplicate (n = 3), and one representative experimental run is shown. Mean ± SEM values are shown. *P < 0.05 and **P < 0.01 compared to control (no H2O2 exposure).
Figure 1
 
Effects of H2O2 on proteasome subunit protein expression in HLECs. Human lens epithelial cells were challenged by 0, 100, or 1000 μM H2O2 for 1 hour, after which protein expression was determined. Expression levels of the immunoproteasome subunits LMP2, MECL1, and LMP7 were upregulated by H2O2 (AC). The constitutive proteasome subunits PPSMB6, MCP-165, and PSMB5 showed no significant change in protein expression (DF). Data from densitometric analyses of Western blot bands were normalized to β-actin. Experiments were performed three times in triplicate (n = 3), and one representative experimental run is shown. Mean ± SEM values are shown. *P < 0.05 and **P < 0.01 compared to control (no H2O2 exposure).
Figure 2
 
Protein expression of immune- and constitutive proteasome subunits in human lens epithelium and lens homogenate. Human lens epithelium specimens obtained from lenses during cataract surgery showed expression of both the immunoproteasome subunits LMP2, MECL1, and LMP7 and the constitutive proteasome subunits PSMB6, MCP-165, and PSMB5 (A). (B) Lens homogenate from phacoemulsification irrigation solution was examined for detection of the immunoproteasome subunit LMP2 and the constitutive proteasome subunit PSMB6, both of which were present in the soluble and insoluble fractions.
Figure 2
 
Protein expression of immune- and constitutive proteasome subunits in human lens epithelium and lens homogenate. Human lens epithelium specimens obtained from lenses during cataract surgery showed expression of both the immunoproteasome subunits LMP2, MECL1, and LMP7 and the constitutive proteasome subunits PSMB6, MCP-165, and PSMB5 (A). (B) Lens homogenate from phacoemulsification irrigation solution was examined for detection of the immunoproteasome subunit LMP2 and the constitutive proteasome subunit PSMB6, both of which were present in the soluble and insoluble fractions.
Figure 3
 
Chymotrypsin-like activity of the proteasome during increased oxidative stress. (A) Human lens epithelial cells exposed to increased oxidative stress by H2O2 at 0, 100, and 1000 μM for 1 hour showed a decrease in chymotrypsin-like activity. The activity was significantly decreased at 1000 μM H2O2. The chymotrypsin-like activity is expressed as RFU/s/g protein. (B) Relative contribution of the immunoproteasome and the constitutive proteasome. The chymotrypsin-like activity of the immunoproteasome in cultured HLEC was partially inhibited by the immunoproteasome-specific inhibitor ONX-0914, and most of the activity was inhibited by MG-132. The relative contributions of the immunoproteasome and the constitutive proteasome were approximately equal in terms of total proteasome activity. Experiments were performed three times in triplicate (n = 3), and one representative experimental run is shown. Values are mean ± SEM. ***P < 0.001 compared to control.
Figure 3
 
Chymotrypsin-like activity of the proteasome during increased oxidative stress. (A) Human lens epithelial cells exposed to increased oxidative stress by H2O2 at 0, 100, and 1000 μM for 1 hour showed a decrease in chymotrypsin-like activity. The activity was significantly decreased at 1000 μM H2O2. The chymotrypsin-like activity is expressed as RFU/s/g protein. (B) Relative contribution of the immunoproteasome and the constitutive proteasome. The chymotrypsin-like activity of the immunoproteasome in cultured HLEC was partially inhibited by the immunoproteasome-specific inhibitor ONX-0914, and most of the activity was inhibited by MG-132. The relative contributions of the immunoproteasome and the constitutive proteasome were approximately equal in terms of total proteasome activity. Experiments were performed three times in triplicate (n = 3), and one representative experimental run is shown. Values are mean ± SEM. ***P < 0.001 compared to control.
Figure 4
 
Immunolocalization of the proteasome subunits LMP7 and PSMB5 in HLECs after exposure to H2O2. The immunoproteasome subunit LMP7 was localized mainly in the cytosol of the cell (A), in contrast to the constitutive proteasome subunit PSMB5, which was localized predominantly in the nucleus (B), as shown by immunocytochemistry. Higher oxidative stress levels showed increased immunolabelling of the immunoproteasome (A). Green: LMP7 and PSMB5; red: F-actin (phalloidin); blue: Hoechst dye (nuclear morphology). Scale bar: 10 μm.
Figure 4
 
Immunolocalization of the proteasome subunits LMP7 and PSMB5 in HLECs after exposure to H2O2. The immunoproteasome subunit LMP7 was localized mainly in the cytosol of the cell (A), in contrast to the constitutive proteasome subunit PSMB5, which was localized predominantly in the nucleus (B), as shown by immunocytochemistry. Higher oxidative stress levels showed increased immunolabelling of the immunoproteasome (A). Green: LMP7 and PSMB5; red: F-actin (phalloidin); blue: Hoechst dye (nuclear morphology). Scale bar: 10 μm.
Figure 5
 
Colocalization of the proteasome subunits LMP7 and PSMB5 in HLECs. Colocalization (yellow) of LMP7 and PSMB5 was evident as shown by double-labeling with anti-LMP7 (green) and anti-PSMB5 (red). Scale bar: 10 μm.
Figure 5
 
Colocalization of the proteasome subunits LMP7 and PSMB5 in HLECs. Colocalization (yellow) of LMP7 and PSMB5 was evident as shown by double-labeling with anti-LMP7 (green) and anti-PSMB5 (red). Scale bar: 10 μm.
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