Proteolytic Mechanisms Underlying Mitochondrial ... - CiteSeerX

Proteolytic Mechanisms Underlying Mitochondrial Degradation in the Ocular Lens Anna J. Zandy1 and Steven Bassnett1,2 PURPOSE. To remove light-scattering structures from the visual axis, all intracellular organelles are eliminated from cells in the center of the developing ocular lens. Organelle degradation is accompanied by an increase in VEIDase (caspase-6-like) activity, but data from caspase-null mice suggest that the lens VEIDase is not caspase-6. The goal of the present work was to identify the lens VEIDase and determine whether it plays a role in organelle breakdown. METHODS. The approximate molecular mass of the lens VEIDase was determined by size-exclusion chromatography. Three proteasome inhibitors (NLVS, MG132, and clasto-lactacystin betalactone) were tested for their ability to inhibit lens VEIDase activity. Lens lysates were immunodepleted of proteasomes using an antibody against the 20S proteasome. To inhibit the ubiquitin–proteasome pathway (UPP) in vivo, lactacystin was injected into the vitreous humor of the developing chicken eye. The effect of lactacystin on mitochondrial degradation was assessed by examining the disappearance of succinate– ubiquinone oxidoreductase, an integral protein of the inner mitochondrial membrane. RESULTS. The lens VEIDase eluted at approximately 700 kDa from a size-exclusion column and was inhibited by the proteasome inhibitors NLVS, MG132, and clasto-lactacystin beta-lactone. In vivo, the trypsin-like activity of the proteasome was reduced by 60% to 70% after lactacystin injection. Proteasome inhibition was associated with the accumulation of ubiquitinated proteins and reversible opacification of the lens cortex. In lactacystin-injected eyes, the programmed degradation of succinate– ubiquinone oxidoreductase was inhibited in the central lens fiber cells. CONCLUSIONS. These data suggest that lens VEIDase activity is attributable to the proteasome and that the UPP may function in the removal of organelle components during lens fiber cell differentiation. (Invest Ophthalmol Vis Sci. 2007;48:293–302) DOI:10.1167/iovs.06-0656

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he lens of the eye is composed of two distinct cell types, epithelial cells that form an anterior monolayer and fiber cells that constitute the bulk of the tissue. Near the lens

From the Departments of 1Ophthalmology and Visual Sciences and 2Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri. Supported by National Institutes of Health Grants R01EY009852 and EY02687 (Core grant for vision research) and by an unrestricted grant to the Department of Ophthalmology and Visual Sciences from Research to Prevent Blindness (RPB). SB is a William and Mary Greve RPB scholar. Submitted for publication June 14, 2006; revised August 1, 2006; accepted October 25, 2006. Disclosure: A.J. Zandy, None; S. Bassnett, None The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact. Corresponding author: Steven Bassnett, Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8096, St. Louis, MO 63110; [email protected]. Investigative Ophthalmology & Visual Science, January 2007, Vol. 48, No. 1 Copyright © Association for Research in Vision and Ophthalmology

equator, epithelial cells withdraw from the cell cycle and differentiate into fiber cells. The lens grows continuously throughout life by the addition of fiber cells at the surface. Cell turnover does not occur. Consequently, the age of a fiber cell can be inferred from its radial position: the oldest cells are located in the core of the lens and the youngest are located near the surface. Late in the fiber cell differentiation process, all intracellular organelles (including nuclei, endoplasmic reticulum [ER], and mitochondria) are degraded.1 Organelle breakdown eliminates light-scattering structures from the optical axis and thereby ensures the transparency of the tissue. Failure to properly degrade organelles is associated with cataracts in humans and mouse models.2–10 Organelle degradation begins in the center of the lens during embryonic development. On or about embryonic day (E) 12 in the chicken lens or E18 in the mouse lens, structural changes that presage organelle breakdown become evident. Fiber cell nuclei change shape from elongated to spherical. This is accompanied by clumping and marginalization of DNA and perforation of the nuclear envelope. The nuclear envelope subsequently disintegrates into membrane vesicles, the remaining chromatin condenses, and low-molecular–weight DNA is released into the cytoplasm.11–13 The degradation of mitochondria and ER appears to be synchronized with that of the nuclei. Mitochondria lose the ability to incorporate rhodamine 123, indicating a loss in membrane potential (⌬⌿), and mitochondrial proteins, including succinic dehydrogenase, BAP37, and prohibitin, are degraded.14,15 Similarly, protein disulfide isomerase (a luminal ER protein) is degraded at the onset of nuclear breakdown.16 Removal of all organelle structures may be accomplished in as few as 4 hours.16 The degradation of organelles and their proteinaceous contents implies the presence and activity of one or more proteases. However, little is known about the proteolytic systems underlying organelle breakdown. Among many endogenous substrates that disappear during this process are poly(ADPribose) polymerase,17,18 nuclear lamins,11 and DNA fragmentation factor.18 In apoptotic cells, the cleavage of these proteins is accomplished by caspase proteases. This has led to the suggestion that lens organelle breakdown represents an “incomplete” or “attenuated” form of apoptosis.19 –21 Recently, we examined lenses from mice in which each of the executioner caspases was deleted, individually or in combination. Inactivation of the executioner caspases had no discernible effect on organelle loss, indicating that organelle breakdown and apoptosis may be distinct cellular processes.22 In the course of that study, we observed that cytosolic extracts of the lens contained strong VEIDase activity. The ability to cleave a VEID substrate is usually taken as a measure of cellular caspase-6 activity. However, the VEIDase activity was not diminished in lenses from caspase-6 null animals, indicating that the activity was not caused by caspase-6 itself. Strong lens VEIDase activity has been reported previously and, significantly, has been shown to increase sixfold immediately before organelle breakdown.23 To date, lens VEIDase activity is the only proteolytic activity known to correlate with organelle breakdown. In the present study, therefore, we sought to 293

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determine the identity of the lens VEIDase and to test its role in organelle degradation.

MATERIALS

AND

METHODS

IOVS, January 2007, Vol. 48, No. 1 the original lens lysate along with the final proteasome-depleted supernatant were assayed for ARRase activity, VEIDase activity, and DEVDase activities, as described.22 For negative controls, beads incubated with either nonspecific IgG or no antibody were included in the assay.

Animals

Chicken Embryo Injections

The generation and characterization of ␣A- and ␣B-crystallin knockout mice has been described.24,25 Wild-type mice (C57-BL6) were obtained from the Jackson Laboratory (Bar Harbor, ME). Mice were killed by CO2 inhalation. Fertilized eggs from White Leghorn chickens (CBT Farms, Chestertown, MD) were incubated at 38°C until E9 to E19, at which time embryos were removed from the eggs and decapitated. Eyes from either species were enucleated and lenses were removed using fine forceps through an incision in the posterior of the globe. All dissections were performed in warm DMEM-F12 medium supplemented with insulin, transferrin, and selenium (ITS), penicillin/streptomycin, and fungizone (Gibco, Grand Island, NY). The procedures described herein were approved by the Washington University Animal Studies Committee and are in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

On E3, a small hole was made in the eggshell, and 4 mL albumin was removed with an 18-gauge needle. The hole was sealed with tape, and a 2-cm2 window was made on the side of the shell. After “windowing,” eggs were sealed with tape and returned to the incubator. On E10, eggs were opened and a hole was made in the chorioallantoic membrane, with care taken to avoid major blood vessels. A small spatula was inserted to support the head of the embryo while injections were made. The injection needle was inserted through the posterior sclera, and 3 ␮L drug or vehicle control was injected into the vitreous humor through a hand-held 30-gauge needle attached to a Hamilton syringe. The spatula was removed, the shell resealed, and the egg returned to the incubator. Chickens were killed 3, 24, or 48 hours after injection, and lenses were removed for analysis.

Inhibitors Caspase and proteasome inhibitors were used at the concentrations stated in the figure legends. Z-VAD-FMK, Boc-D-FMK, VEID-CHO, MG132, and NLVS were purchased (Calbiochem, La Jolla, CA), as were lactacystin and clasto-lactacystin ␤-lactone (Sigma, St. Louis, MO).

Protease Activity Assays Fluorogenic enzyme assays were performed, as described.22 The effect of proteasome inhibitors on lens protease activity was measured in vitro, ex vivo, and in vivo. During the in vitro enzyme assays, lens lysates were added to the microplate and treated with an inhibitor for 10 minutes at room temperature before a fluorogenic substrate was added. In the ex vivo enzyme assays, whole lenses were incubated in vehicle- or inhibitor-containing medium at 37°C for periods of 0 to 24 hours before they were removed, homogenized, and assayed for activity. For in vivo analyses, eyes were injected with inhibitor, and lenses were removed at intervals after injection, homogenized, and assayed for activity. In some cases, the lens fiber mass was dissected into outer (cortical) and inner (core) regions before homogenization.

Fast-Performance Liquid Chromatography P30 mouse lenses (n ⫽ 60) were homogenized in 500 ␮L lysis buffer (10 mM Tris pH 7.5, 100 mM NaCl, 1 mM EDTA, and 0.01% Triton X-100) and centrifuged at 14,000g at 4°C for 3 minutes. The supernatant was passed through a 0.8-␮m/0.2-␮m combination filter before injection on an equilibrated column (Superdex 200; GE Healthcare, Piscataway, NJ). Lysis buffer (lacking Triton X-100) was run through the column at a flow rate of 1 mL/min. In-line ultraviolet (UV) detection was performed at 280 nm. One-milliliter fractions were collected, and 50 ␮L from each fraction was added to the enzymatic assay (with VEID-AMC or LLE-AMC as substrates). Enzyme activity data were collected over a 24-hour period after incubation at 37°C in a fluorescence microplate reader.

Immunoprecipitation Immunoprecipitation of the proteasome was achieved through a “cycling” approach. Protein A agarose beads were incubated with 1 ␮L of 20S proteasome core subunit polyclonal antibody (Calbiochem) for 2 hours at room temperature with constant mixing. Beads were spun at 1100g for 30 seconds and were washed with 12 vol of 100 mM Tris pH 8, followed by a 12-vol wash of 10 mM Tris pH 8. Beads were incubated overnight at 4°C with lysates prepared from P21 lenses. The beads were then centrifuged, and the supernatant was removed. The supernatant was incubated with a fresh set of antibody-conjugated beads and again incubated overnight at 4°C. After five cycles, equal volumes of

Immunofluorescence and Immunoblotting Lens tissue was analyzed by immunofluorescence or Western blotting, as described.22 Primary antibodies used for immunoblotting were a monoclonal antibody against succinate– ubiquinone oxidoreductase (Molecular Probes, Eugene, OR) and a monoclonal anti– ubiquitin antibody (Sigma). Primary antibodies were detected by a horseradish peroxidase (HRP)– conjugated secondary antibody (Pierce, Rockford, IL) followed by chemiluminescence detection (Supersignal; Pierce). The chemiluminescent signal was recorded using a 16-bit CCD gel imaging system (GeneGnome; Synoptics Inc, Frederick, MD).

Statistical Analysis Data were compared using an unpaired two-tailed t-test where P ⬍ 0.05 was considered significant.

RESULTS Effects of Pan-Caspase Inhibitors on Lens VEIDase Activity The goal of this study was to identify the lens VEIDase and determine whether it contributes to organelle breakdown. We have shown previously that caspase-6 is not required for lens VEIDase activity.22 It was possible, however, that another caspase expressed in the lens might be responsible. Although VEID-AMC is a preferred caspase-6 substrate, other caspases can also cleave this peptide, albeit less efficiently.26 To determine whether lens VEIDase activity might be attributable to a caspase other than caspase-6, lens lysates were treated with the pan-caspase inhibitor Z-VAD-FMK or Boc-D-FMK. Neither drug inhibited lens VEIDase activity (Fig. 1A). As a control, the VEIDase activity of recombinant caspase-3, -6, -7, or -8 was also measured. In each case, the VEIDase activity of the recombinant caspase was abolished after incubation with 100 ␮M Z-VAD-FMK or Boc-D-FMK (Fig. 1B), confirming the potency of the inhibitors against a range of caspase proteases. To exclude the possibility that factors in the lysate inactivated the pancaspase inhibitors, some lysates were spiked with a recombinant caspase (caspase-8). The VEIDase activity of exogenous caspase-8 in the lysate was abolished by treatment with pan-caspase inhibitors (Supplementary Fig. S1, online at http://www.iovs.org/cgi/content/full/48/1/293/DC1), demonstrating that the inhibitors retained their potency in the lysate. Taken together, these data suggested that the lens VEIDase activity was not caused by a caspase.

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Lens Mitochondrial Degradation

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FIGURE 1. Lens VEIDase activity is not caused by a caspase. (A) Lysates were prepared from mouse lenses and assayed for VEIDase activity using a fluorogenic assay. The pan-caspase inhibitors Z-VAD-FMK (100 ␮M) and Boc-D-FMK (100 ␮M) have no significant effect on the VEIDase activity in mouse lens lysates. Dimethyl sulfoxide (DMSO) is the vehicle control. In each case, data represent the mean ⫾ SD (n ⫽ 6 independent determinations). (B) VEIDase activity of recombinant caspase-3 (100 ng), -6 (2 U), -7 (400 ng), or -8 (100 ng) was measured after preincubation with Z-VAD-FMK, Boc-D-FMK, or DMSO. In each case, the pan-caspase inhibitors have a significant inhibitory effect and reduce VEIDase activity to near background levels. Data represent mean ⫾ SD (n ⫽ 4).

Determining the Molecular Mass of the Lens VEIDase The approximate molecular mass of the lens VEIDase was determined by size-exclusion chromatography (Fig. 2A). Surprisingly, the lens VEIDase eluted at a molecular mass of approximately 700 kDa, much greater than that expected for caspase proteases (20 – 40 kDa). The lens VEIDase coeluted with the ␣-crystallin fraction; ␣A- and ␣B-crystallin are mem-

FIGURE 2. Lens VEIDase activity coelutes with ␣-crystallin and LLEase activity. (A) P30 mouse lenses (60) were homogenized and passed through a size-exclusion column. Major peaks (high-molecular–weight complexes [HMWCs], ␣, ␤H, ␤L, and ␥) on the A280 protein trace correspond to classes of lens crystallin proteins. Lens VEIDase activity coelutes with ␣-crystallin at approximately 700 kDa. Note that the VEIDase activity is reduced after treatment with VEID-CHO, a caspase-6 inhibitor. (B) In lysates prepared from wild-type (WT) or ␣A/␣B double knockout (dKO) mouse lenses, VEIDase activity is retained by a 100-kDa cutoff filter. No VEIDase activity is observed in the filtrate. Data represent mean ⫾ SD (n ⫽ 7 determinations for wild-type lenses; n ⫽ 5 for dKO lenses). (C) Fractions from the sizeexclusion column were also assayed for LLEase activity (a measure of the PGPH domain activity of the 20S proteasome). Lens lysates contain abundant LLEase activity that elutes in the same fraction as the VEIDase activity (compare Figs. 2A and 2C).

bers of the small heat shock protein family. Together, they constitute approximately 35% of the lens protein and play a critical role in the maintenance of lens clarity.27 ␣-Crystallins function as molecular chaperones, binding a variety of proteins, including caspase-3.28 We considered the possibility that an interaction between ␣-crystallin and the lens VEIDase might explain the large apparent size of the latter. To test this, we compared the molecular mass and activity of the VEIDase in

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wild-type lenses and lenses from mice in which both ␣-crystallin genes had been knocked out (Fig. 2B). It was impractical to collect the large number of ␣A–/–;␣B–/– lenses required for fast-performance liquid chromatography (FPLC) analysis. Instead, we passed lens lysates through 100-kDa molecular weight cutoff filters to separate the proteins into high- and low-molecular–weight fractions. Consistent with the FPLC data, the VEIDase activity in samples from wild-type lenses was retained by the 100-kDa cutoff filter, and there was little or no activity in the filtrate. Similarly, in lens homogenates from ␣-crystallin double-knockout mice, VEIDase was retained by the filter, and no activity was detected in the filtrate. These data indicated that neither the apparent molecular mass of the VEIDase nor its activity depended on the presence of ␣-crystallin, though the possibility that the VEIDase might be complexed with other members of the small heat shock family was not excluded.

Evidence That Lens VEIDase Activity Was Caused by the Proteasome The large apparent size of the VEIDase prompted us to test the possibility that it represented the 20S proteasome. The proteasome is a large (700 kDa), multisubunit, cylindrical complex responsible for most nonlysosomal protein degradation in cells, and it has three characteristic peptidase activities: chymotrypsin-like (which cleaves after hydrophobic side chains), trypsin-like (which cleaves after basic side chains), and postglutamyl peptide– hydrolyzing (PGPH) activities (which cleaves after acidic side chains).29,30 We assayed column fractions for LLEase activity (Fig. 2C), a measure of the activity of the PGPH domain of the proteasome. Lens lysates contained a strong LLEase activity that coeluted with the VEIDase activity (compare Figs. 2A and 2C), suggesting that both activities might be caused by the proteasome.

IOVS, January 2007, Vol. 48, No. 1 We next tested three structurally unrelated proteasome inhibitors—MG132 (Z-LLL-CHO), NLVS (4-hydroxy-5-iodo-3-nitrophenylacetyl-Leu-Leu-Leu-vinylsulfone), and clasto-lactacystin ␤-lactone (the active analog of lactacystin)—for their ability to inhibit lens VEIDase activity. All three inhibited lens VEIDase activity in a dose-dependent fashion (Figs. 3A–C). Importantly, the three inhibitors have different modes of action. MG132 is a reversible aldehyde inhibitor, NLVS is a peptide vinyl sulfone that covalently interacts with the proteasome, and clasto-lactacystin ␤-lactone is a nonpeptide inhibitor.31 To confirm that the lens VEIDase activity was caused by the proteasome, lens lysates were depleted of proteasomes by immunoprecipitation. Immunodepletion of proteasomes from lens lysates was associated with an approximately 85% reduction in proteasome ARRase (trypsin-like) activity (Fig. 4). Activity lost from the lens lysates was recovered from the antibody-conjugated agarose beads (data not shown). Lens VEIDase activity was depleted in parallel with the ARRase activity. Neither VEIDase nor ARRase activity was reduced after treatment with antibody-free beads or beads conjugated to an irrelevant IgG. Interestingly, DEVDase (caspase-3-like) activity was also depleted after treatment with the proteasome antibody (data not shown).

Inhibiting the Proteasome In Vivo To test the hypothesis that proteasome activity is required for organelle breakdown, we developed an in vivo model using the embryonic chicken eye, which, unlike the mouse eye, is accessible at those stages of development (E9-E12) during which organelle degradation commences. Before attempting to inhibit the organelle degradation process in vivo, we used an ex vivo organ culture technique to test the efficacy of proteasome inhibitors in the intact lens (Fig. 5). Lenses from E10 chickens were isolated and cultured in medium containing 100 ␮M

FIGURE 3. Mouse lens VEIDase activity is inhibited by proteasome inhibitors. Increasing concentrations of (A) MG132, (B) NLVS, and (C) clasto-lactacystin ␤-lactone inhibit lens lysate VEIDase activity in a dosedependent manner. Negative controls, consisting of fluorogenic substrate dissolved in lysis buffer, are also included. Data represent mean ⫾ SD (n ⫽ 3).


FIGURE 4. Immunoprecipitation of proteasomes depletes lens lysates of VEIDase activity. Mouse lens lysates were incubated with a polyclonal proteasome antibody conjugated to protein A agarose beads. After a series of centrifugation steps, “depleted” lysates were assayed for ARRase and VEIDase activities. In lysates depleted of proteasomes, ARRase and VEIDase activities are reduced in parallel and to a significantly greater degree (P ⬍ 0.05) than control samples prepared with unconjugated beads or beads conjugated to an irrelevant antibody. Data represent mean ⫾ SD (n ⫽ 3).

lactacystin or an equal volume of vehicle control (water). In control lenses, proteasome activity was constant over the 24hour time course of the experiment. In contrast, all three proteasome activities were significantly inhibited in lactacystin-treated cultures. Lactacystin most potently inhibited the LLVYase (chymotrypsin-like) activity. After 1 hour in culture,

FIGURE 5. Lactacystin inhibits all three activities of the proteasome in embryonic chicken lenses ex vivo. Lenses were removed from E10 embryos and placed in organ culture with 100 ␮M lactacystin or vehicle (H2O) control. Lenses were harvested at intervals and assayed for (A) LLEase, (B) LLVYase, and (C) ARRase activities corresponding, respectively, to the PGPH, chymotrypsin-like, and trypsin-like domains of the 20S proteasome. Composite data (D). Lactacystin strongly inhibits each activity of the proteasome, though LLVYase activity is most rapidly and profoundly inhibited. Inhibition is maximal by 12 hours. Data represent the mean ⫾ SD of three lenses for each time point, and activity assays were run in duplicate.


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87% of LLVYase activity was inhibited. After 1 hour, ARRase and LLEase activities were also inhibited, but to lesser extents (54% and 55%, respectively). By 24 hours, 79% to 96% of all three activities were inhibited. The ex vivo system was useful for testing the efficacy of inhibitors, but, because fiber cell differentiation ceases in organ culture,32 we were unable to use this approach to determine the effect of proteasome inhibitors on organelle breakdown. To inhibit the activity of the proteasome in vivo, 3 ␮L of 10 mM lactacystin or water (vehicle control) was injected into the vitreous humor of the eye on E10, immediately before the establishment of the organelle-free zone.14 Lenses were harvested 3, 24, and 48 hours after injection, and proteasome (ARRase) activity was measured (Fig. 6A). After 3 hours, ARRase activity was significantly (approximately 60%) inhibited. Inhibition was maintained for at least 24 hours; by 48 hours, the activity of the proteasome had recovered to normal levels. Intravitreal injection of water had no effect on lens ARRase activity. Thus, a single intravitreal injection of lactacystin caused a significant but transient inhibition of proteasome activity. Organelle breakdown is initiated in the central fiber cells.14 To test whether intravitreal injection of lactacystin resulted in proteasome inhibition in this sequestered cell population, ARRase activity was measured in dissected cortical and core lens samples from lactacystin-injected eyes (Fig. 6B). ARRase activity was equally and potently (70%– 80%) inhibited in both fractions after lactacystin injection. Most proteasome substrates are covalently modified by the addition of ubiquitin chains before their degradation. Ubiquitinated substrates can be visualized as a high-molecular–weight smear on Western blots. To further verify that the proteasome had been inhibited in vivo, we tested whether treatment with

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FIGURE 6. Lactacystin inhibits lens proteasome activity in embryonic chicken lenses in vivo. A single injection of lactacystin or water (control) was made into the vitreous humor of the E10 chicken embryo eye. Lenses were removed from the eye 3, 24, or 48 hours later and assayed for ARRase activity using a fluorogenic assay. (A) Lactacystin significantly inhibits lens ARRase activity at 3 hours and 24 hours after injection compared with water-injected eyes. By 48 hours, ARRase activity recovers to control levels. (B) Twenty-four hours after injection of lactacystin or water, lenses were removed from the eye and dissected into cortical and core fractions. ARRase activity was measured independently in the two fractions. ARRase activity in the core of water-injected samples is significantly lower than in the cortex of water-injected control samples. Lactacystin inhibits ARRase activity in core and cortical fractions to approximately the same degree. Data represent the mean ⫾ SD of at least three lenses (or parts thereof), and each assay was run in duplicate. *Statistically significant differences (P ⬍ 0.05) between lactacystin- and water-injected samples.

lactacystin resulted in the accumulation of ubiquitinated substrates in lens fiber cells. At 24 hours after injection (Fig. 7), there was an approximately sixfold increase in the concentration of ubiquitinated proteins in the lens cortex of lactacystininjected eyes compared with vehicle controls and a lesser (approximately 2-fold) increase in the lens core. At 48 hours after injection, the levels of ubiquitinated proteins had declined to those seen in water-injected controls (data not shown). Inhibition of the UPP was associated with marked changes in lens transparency (Fig. 8). Lenses from water-injected eyes were transparent throughout the 2-day postinjection period, indicating that the injection process did not itself cause lens opacification (Figs. 8A, 8C). Similarly, contralateral lenses from water- or lactacystin-injected eyes remained transparent (data not shown). In contrast, after 24 hours, lenses from lactacystininjected eyes developed dense cortical opacities (Fig. 8B) that largely, but not completely, resolved by 48 hours (Fig. 8D). Thus, the loss and subsequent recovery of lens transparency after lactacystin injection closely paralleled the inhibition and recovery of proteasome activity (Fig. 6A).

Effects of Proteasome Inhibition on Degradation of a Mitochondrial Membrane Protein Before measuring the effect of lactacystin-injection on organelle breakdown, the normal pattern of degradation of an organelle marker was established (Fig. 9). Succinate– ubiquinone oxidoreductase is part of complex II of the oxidative phosphorylation pathway and is an integral protein of the inner mitochondrial membrane.33 Previous studies in the chicken embryo have established that, at E8, mitochondria in the central lens cells begin to fragment and, by E12, have disappeared completely.14 Immunofluorescence analysis of lenses at E13 using an antibody to succinate– ubiquinone oxidoreductase confirmed that mitochondria were absent from the lens core (Fig. 9A). The disappearance of succinate– ubiquinone oxidoreductase from the central region of the lens during the period E9 to E19 was measured by Western blotting. At E9, the succinate– ubiquinone oxidoreductase signal was strong but, over the next 4 days, declined sharply. By E13, this marker was barely detectable, and by E15 it was completely absent (Figs. 9B, 9C). Finally, we tested whether in vivo inhibition of the UPP affected the breakdown of succinate– ubiquinone oxidoreduc-

tase in core fiber cells between E10 and E11. A single intravitreal injection of lactacystin (or water control) was made at E10. Twenty-four hours after injection, lenses were removed from the eye and dissected into core or cortical fractions. The level of succinate– ubiquinone oxidoreductase in the samples was measured by Western blot (Fig. 10). In lenses from waterinjected eyes, a marked difference in the levels of succinate– ubiquinone oxidoreductase developed between cortical and core samples between E10 and E11 because of the rapid degradation of mitochondrial proteins in the lens core that occurred during this period (see also Fig. 9C). In contrast, in lactacystin-injected eyes, the level of succinate– ubiquinone oxidoreductase in the lens core remained close to that of the cortex (Fig. 10B), indicating that the proteolytic degradation of the marker was inhibited in the core. These data suggest that the degradation of the mitochondrial membrane marker, succinate– ubiquinone oxidoreductase, depends on the UPP.

DISCUSSION Programmed elimination of nuclei and other organelles occurs in only three cell types: differentiating lens fiber cells, erythroblasts, and keratinocytes. In no case is the process well understood. Recent studies have focused on the potential involvement of the apoptotic machinery in these processes and, in particular, the role of caspase proteases. Activated caspases are observed in differentiating keratinocytes in vivo, and pancaspase inhibitors perturb the denucleation process in vitro.34 Treatment of differentiating erythroid cells with caspase-3 siRNA blocks cells at the proerythroblast stage, suggesting a role for caspases in the early stages of erythropoiesis,35 but caspases appear not to function during terminal erythroid differentiation (when enucleation occurs).36 In lens cells, the widely held notion that caspases play a critical role in organelle degradation is based largely on the observation that characteristic caspase substrates are cleaved during organelle breakdown11,17,18 and that caspase-like activity can be recovered from lens lysates.23 However, we have shown previously that cleavage of endogenous substrates can be an unreliable indicator of the presence or activity of a given protease.22 Furthermore, knockout of caspase-6, the caspase most strongly implicated in lens fiber organelle breakdown, does not inhibit organelle loss.22



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activity were measured in mouse lens extracts, even in the presence of lactacystin. The reason for the discrepancy is unclear. Foley et al.23 state that proteasome activity was completely inhibited in the presence of 100 ␮M lactacystin. If this were so, the substantial VEIDase activity measured subsequently in those experiments could not have been caused by the proteasome. However, proteasome activity and its inhibition by lactacystin were measured with LLVY-AMC, a substrate for the chymotrypsin-like domain. It is well documented that compounds such as lactacystin inhibit the different domains of the proteasome to varying degrees. Of the three intrinsic proteolytic activities of the 20S proteasome, the chymotrypsin-like domain is the most sensitive to inhibition by lactacystin (see Fig. 5D and Kisselev et al.38). In contrast, the PGPH domain of the proteasome (the domain most likely responsible for the VEIDase activity of the proteasome; see below) is relatively resistant to lactacystin treatment and, therefore, might not have been fully inhibited under the conditions used in the earlier study. Studies using purified proteasomes have shown that the PGPH domain can cleave synthetic substrates containing aspartic residues in the P1 position, including several standard caspase substrates.39,40 This has led to the suggestion that the PGPH domain be renamed the caspase-like domain.40 It seems likely that VEIDase activity in the unstressed lens is attributed largely to this domain of the proteasome. Treatment of lens lysates with the caspase-6 inhibitor, VEID-CHO, inhibits LLEase and VEIDase activity (data not shown), suggesting that both may be attributed to the caspase-like domain. It should be noted, however, that authentic caspase-6 is also expressed in the lens. The substantial increase in VEIDase activity observed

FIGURE 7. Ubiquitinated proteins accumulate after lactacystin injection. (A) Lenses were dissected into cortical and core fractions 24 hours after injection of lactacystin or water (control) into the vitreous humor. Western blots were probed with a monoclonal antibody against ubiquitin. Ubiquitinated proteins form a high-molecular– weight smear on the blot. Low levels of ubiquitinated protein are present in cortex and core lens samples from water-injected eyes. In lactacystin-injected eyes, there is a large (sixfold) increase in ubiquitinated protein levels in the lens cortex, and a lesser (twofold) increase in the lens core. (B) Densitometric analysis of three such Western blots. *Statistically significant differences (P ⬍ 0.05) between lactacystin- and water-injected samples.

Some of the confusion over the contribution of caspases to lens organelle degradation has arisen from conflicting data regarding the molecular identity of the lens VEIDase. VEIDase activity, which is reported to increase sixfold when organelle breakdown is initiated, has been attributed to caspase-6.23,37 However, experiments with caspase knockout animals demonstrated that although VEID-AMC was a preferred substrate for caspase-6, the VEIDase activity in the lens could not be attributed to that enzyme. In the present study, the results of sizeexclusion chromatography, pharmacologic inhibition, and immunodepletion experiments supported the notion that lens VEIDase activity resulted from the action of the proteasome. The present data are at odds with findings in a previous study by Foley et al.,23 in which significant levels of VEIDase

FIGURE 8. In vivo inhibition of proteasome activity is associated with loss of lens transparency. Embryonic chickens were injected intravitreally with lactacystin (B, D) or water (A, C) on E10. Lenses were removed and photographed 24 (A, B) or 48 (C, D) hours later. (A) Twenty-four hours after injection, lenses from water-injected eyes remain transparent. (B) Lenses from lactacystin-injected eyes exhibit marked cortical cataracts. (C) At 48 hours, lenses from water-injected eyes remain transparent. (D) By 48 hours, the cortical cataracts seen in lactacystin-injected eyes have largely resolved. Scale bar, 300 ␮m. Images are representative of four independent experiments.

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FIGURE 9. Degradation of succinate– ubiquinone oxidoreductase in the centermost fibers of the embryonic chicken lens. (A) A midsagittal vibratome section from an E13 lens slice was stained with propidium iodide (red) to label lens nuclei and an antibody to the mitochondrial protein, succinate– ubiquinone oxidoreductase (green). Punctate nuclear staining in the central lens cells indicates that denucleation has begun. Note the absence of mitochondrial immunofluorescence from the lens core. The region delineated in blue shows the region of the lens used for Western blot analysis. Ep, epithelium; F, fiber cells. Scale bar, 250 ␮m. (B) Proteins isolated from the centermost region of the lens, delineated in blue (A), were probed with anti-succinate– ubiquinone oxidoreductase. A single band of the expected size (70 kDa) is present at E9. During the succeeding days, the intensity of the band decays, and by E15, succinate– ubiquinone oxidoreductase is undetectable in the center of the lens. (C) Densitometric analysis of data from three Western blots. The concentration of succinate– ubiquinone oxidoreductase in the center of the lens declines sharply from E9 to E13. Data represent mean ⫾ SD (n ⫽ 3 for each time point).

on treating lenses with staurosporine results from the activation of that enzyme.22 Lens fiber cells are highly differentiated and specialized cells, but they have a fully functional UPP.41,42 Indeed, some components of the UPP appear to be required for fiber differentiation.41,43 Ubiquitin-activating enzymes, ubiquitinconjugating enzymes, and 19S and 20S proteasome subunits are distributed throughout the lens fiber mass, even in the oldest cells in the lens core. Recent studies suggest that components of the UPP may translocate to the nucleus late in fiber cell differentiation, potentially implicating the UPP in the degradation of nuclei and, possibly, other organelles.44 It has been suggested previously that the UPP may have a role in lens organelle breakdown,44 but studies to test this hypothesis directly have been hampered by the absence of an appropriate organ culture model. Although cellular integrity and tissue transparency are maintained in organ-cultured rodent lenses, the fiber cell differentiation process ceases abruptly.32 We have examined the fate of pulse-labeled nuclei within organ-cultured lenses and concluded that the organelle degradation process is also inhibited in vitro (data not shown). In the absence of a suitable rodent lens culture system, we developed an in vivo model in which to evaluate the role of the UPP in organelle degradation in the developing chicken lens. Morphologic studies suggest that the organelle degradation process is broadly similar in mice and chickens,1,11,15 though it is unknown whether the underlying biochemical mechanisms are conserved. The injection paradigm developed here allowed us to test the function of the UPP in vivo by manipulating its activity before the establishment of the organelle-free zone. We found that a single intravitreal injection of lactacystin at E10 inhibited

the majority of lens proteasome ARRase (trypsin-like) activity over a 24-hour period. This was probably a conservative estimate, because proteases other than the proteasome may be able to degrade the ARR-AMC substrate. Although lactacystin is considered an irreversible inhibitor, its interaction with the proteasome is slowly hydrolyzed (t1/2 ⬇ 20 hours).31 This may explain why proteasome activity recovered 48 hours after lactacystin injection in vivo. Data obtained from in vivo injection experiments indicated that succinate– ubiquinone oxidoreductase, an integral protein of the inner mitochondrial membrane, was degraded in the lens core from E9 to E13 and that lactacystin significantly inhibited this degradation. The coordinated breakdown of intracellular organelles presumably involves the proteolysis of an assortment of organelle components. Here, we only examined the fate of a single mitochondrial marker. Further studies will be required to determine whether other mitochondrial proteins and, indeed, constituents of other organelles are degraded through the UPP. It is not yet established at which point in the degradation process the UPP acts. It is possible, for example, that the UPP initiates the dismantlement of organelle structures. Such a role has been demonstrated in other systems. For example, the UPP functions in the recognition and elimination of paternal mitochondria after fertilization in mammals.45 Alternatively, the UPP might act to remove organelle components only after they have been released from organelles through the action of another, initiating, agent. The latter possibility seems more likely in the lens, in which preliminary histologic analysis has shown that mitochondrial disintegration is not prevented in the lenses of lactacystin-treated embryos (data not shown).


FIGURE 10. Proteasome inhibition prevents the degradation of succinate– ubiquinone oxidoreductase, a mitochondrial marker, in the center of the developing chicken lens. Chicken eyes were injected intravitreally with lactacystin or water (control) on E10. Lenses were harvested 24 hours after injection and separated into cortical and core fractions. Equal amounts (10 ␮g) of protein were probed on Western blots with an antibody to succinate– ubiquinone oxidoreductase. (A) At E11, the concentration of succinate– ubiquinone oxidoreductase is higher in the lens cortex from water-injected eyes than in the lens core. In samples from lactacystin-injected eyes, the concentration of succinate– ubiquinone oxidoreductase in the two regions of the lens is comparable. (B) Densitometric analysis of three such blots. *Statistically significant differences (P ⬍ 0.05) between lactacystin- and waterinjected samples.

Acknowledgments The authors thank Mark Petrash, Terry Griest, and Kelly Barton for their help with FPLC measurements, David Beebe for advice regarding the chicken injection system, Peggy Winzenburger for animal husbandry, and Alicia De Maria for many helpful discussions. The ␣-crystallin-null mice were originally generated in the laboratory of Eric Wawrousek (National Eye Institute, Bethesda, MD) and were provided to us, with permission, by Usha Andley (Washington University, St. Louis, MO).

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