©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Amyloid -Protein Inhibits Ubiquitin-dependent Protein Degradation in Vitro(*)

(Received for publication, May 11, 1995)

Luisa Gregori (§) Chana Fuchs Maria E. Figueiredo-Pereira (1) William E. Van Nostrand (2) Dmitry Goldgaber

From the  (1)Department of Psychiatry and Behavioral Science, School of Medicine, State University of New York, Stony Brook, New York 11794, the Department of Pharmacology, Mt. Sinai Medical School, City University of New York, New York, New York 10029, and the (2)Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine, California 92717

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Intraneuronal accumulation of ubiquitin conjugates in inclusion bodies and neurofibrillary tangles is a pathological feature of neurodegenerative disorders such as Alzheimer's disease and Down's syndrome and of normal aging of the brain. Amyloid beta-protein (Abeta) and its precursor are found in neurofibrillary tangle-containing neurons. Abeta is the major component of extracellular plaques. We showed that Abeta acts as an inhibitor of the ubiquitin-dependent protein degradation in vitro. We examined the effect of Abeta on the steps of this proteolytic pathway that contribute to the level of ubiquitin conjugates in the cell. Neither conjugate formation nor conjugate deubiquitination was affected by the presence of Abeta. However, Abeta significantly reduced the rate of conjugate degradation. Our results indicate that Abeta interacts with the proteolytic step of the ubiquitin degradative pathway. Since this step is performed by the 26 S proteasome, the effect of Abeta on the catalytic core of this proteolytic complex, the 20 S proteasome, was determined. We found that Abeta selectively inhibits the chymotrypsin-like activity of the 20 S proteasome. Under pathological conditions in the affected neuron, Abeta could interfere with ubiquitin-dependent degradation by inhibiting the 26 S proteasome activity. This finding may explain the origin of the accumulation of ubiquitin conjugates.


INTRODUCTION

Alzheimer's disease (AD) (^1)is a progressive neurodegenerative disorder characterized by specific lesions in the brain. Some of the neuropathological features of this disease are found in Down's syndrome, in hereditary cerebral hemorrhage with amyloidoses of the Dutch type, and to a lesser extent in normal aging of the brain(1, 2) . The affected brains show intraneuronal accumulation of paired helical filaments(3, 4) , which form neurofibrillary tangles and extracellular amyloid deposition in senile plaques(5, 6) . The amyloid core of senile plaques contains predominantly a 4-kDa peptide, amyloid beta-protein (Abeta)(7) . Abeta is the proteolytic product of a longer protein called amyloid beta-protein precursor (APP)(8, 9) . APP is found as a transmembrane protein(9, 10) and, in the cell, is associated with the cytoskeletal network(11, 12) . The membrane-bound APP is oriented with a short cytoplasmic C-terminal sequence and a large extracellular N-terminal domain. In the nonamyloidogenic pathway, transmembrane APP is cleaved within the Abeta sequence, and the N-terminal-containing product is released extracellularly (sAPP)(10, 13) . Potentially amyloidogenic forms of APP have been reported both inside and outside of the cell(14, 15, 16) . Intracellular as well as extracellular Abeta has been detected by several laboratories(17, 18, 19) . Internalization of Abeta from cell culture media into normal human fibroblasts by the lysosomal/endosomal pathway was also reported(20) . Cellular Abeta immunoreactivity is specifically associated with intraneuronal neurofibrillary tangle-bearing bodies(21, 22, 23) . These findings indicate that under pathological conditions in affected neurons, Abeta is in the cell where it is available for interaction with cellular pathways.

Biochemical and immunohistochemical studies have identified ubiquitin (24, 25) and abnormal tau (26, 27, 28) as the major components of neurofibrillary tangles. Multiubiquitinated forms of abnormal tau have been isolated from these structures(29) . Accumulation of ubiquitin and ubiquitin conjugates is also observed in inclusion bodies(30) . The affected nerve cell develops intracellular fibrous structures and inclusion bodies incorporating material of neuronal cytoskeletal origin. Since ubiquitin is not normally found associated with the cytoskeletal proteins from which the inclusion bodies are derived, its accumulation could be related to neurodegeneration. These observations are consistent with a role for ubiquitin-dependent degradation in the turnover of abnormal proteins under pathological conditions.

Ubiquitin is a highly conserved protein found in the cytoplasm, in the nucleus, and in the membrane of all eukaryotic cells(31) . The conjugation of ubiquitin to proteins is a posttranslational modification in which the carboxyl terminus of ubiquitin is linked to the -amino group of a lysine residue in the target protein. Ubiquitin protein conjugates are formed by the addition of several ubiquitin molecules to the same protein. In the conjugate, the ubiquitin moieties are linked by ubiquitin-ubiquitin isopeptide bonds forming a multiubiquitin chain(32, 33) . This particular multiubiquitin chain structure is recognized by the ATP-dependent 26 S proteasome complex that degrades the protein while ubiquitin is recycled(32, 33, 34) . Abnormal and short lived proteins are natural substrates of the ubiquitin-dependent proteolytic pathway (31) . Ubiquitin conjugates can also undergo disassembly by the action of ubiquitin carboxyl-terminal hydrolases(35) . In this ATP-independent process, ubiquitin moieties are removed from the conjugate, releasing free substrate and ubiquitin(31) . Thus, in vivo, the level of ubiquitin conjugates results from a balance between the rates of conjugate formation, conjugate deubiquitination, and conjugate degradation. A breakdown in this balance appears to occur in the affected neurons of AD brains, as suggested by the abnormally high levels of ubiquitinated proteins(29, 30, 36, 37, 38) . As a consequence, proteolytically stable ubiquitinated forms of abnormal proteins accumulate. Abnormal tau is found multiubiquitinated in paired helical filaments, but the ubiquitin-tau conjugates are not degraded(29) . This suggests that in AD, ubiquitin-dependent protein degradation is reduced. There are no studies that specifically address this question in AD. However, studies on aging, which shares several aspects of the AD pathology, document a decrease in protein turnover (39, 40) .

In this report we present evidence that Abeta inhibits the ubiquitin-dependent degradation of proteins in vitro. We propose that in the affected cells, Abeta interacts with the proteolytic pathway. This interaction may contribute to certain pathological manifestations of AD, such as inclusion bodies and neurofibrillary tangle formation.


EXPERIMENTAL PROCEDURES

Material

Rabbit reticulocytes were purchased from Pel-Freez Biologicals (Rogers, AK). The proteins and reagents were obtained from Sigma except lysozyme, which was purchased from Boehringer-Mannheim. Ubiquitin-aldehyde was a gift of Dr. R. Cohen (University of Iowa, IO). Abeta peptides were purchased from Bachem California (Torrance, CA) reconstituted in water at 10 mg/ml, aliquoted, and stored at -20 °C. Abeta was reconstituted in 5% acetic acid to a final concentration of 5 mg/ml. Abeta was reconstituted in dimethyl sulfoxide to a final concentration of 10 mg/ml. Under these conditions, no aggregation of Abeta was detected over a 1-month period, as determined by gel electrophoresis analysis. Mouse monoclonal antibodies 6E10 recognizing the Abeta sequence 1-16 (41) were a gift of Dr. M. Vitek (The Picower Institute, NY). Human recombinant sAPP was produced in the baculovirus expression system and purified to homogeneity as described previously(42, 43) .

Preparation of the Ubiquitin-dependent Degradative System

Rabbit reticulocytes were used as the source of enzymes participating in the ubiquitin degradative pathway. ATP- and ubiquitin-depleted fraction II was prepared from rabbit reticulocytes essentially as described by Hershko et al.(44) .

Protein Degradation

Ubiquitin-dependent degradation of iodinated substrates was performed in 50 µl of reaction volume containing 50 mM Tris, pH 7.6, 10 mM MgCl(2), 2 mM ATP, 2 mM dithiothreitol, 10 µM ubiquitin, and an ATP regeneration system (10 mM creatine phosphate, 0.5 µg of creatine phosphokinase). The final substrate concentration was approximately 5 µM for each protein tested except for sAPP used at 60 nM. Abeta peptide was added at the indicated concentration. The reaction was started by the addition of 20 µg of fraction II proteins and incubated at 37 °C for the indicated times. The degradation reaction was stopped by the addition of 400 µl of 5% cold trichloroacetic acid and 0.1 mg of bovine serum albumin as the carrier. During degradation, the digested protein is reduced to amino acids and small peptides, which are acid-soluble. The acid-soluble fraction was separated from the precipitated protein by centrifugation. The radioactivity in the supernatant was measured on a -counter and reported as the percentage of total radioactivity.

Ubiquitin Conjugate Formation

Ubiquitin conjugates were formed during the degradation reaction as described above. The reaction was stopped by the addition of gel electrophoresis sample buffer containing 1% SDS and 0.5% beta-mercaptoethanol, boiled for 3 min, and subjected to gel electrophoresis. The gel was dried, and ubiquitin conjugates were visualized by autoradiography.

Pulse-Chase Analysis of Conjugate Deubiquitination

Labeled lysozyme conjugates were formed in 50 µl of reaction as described above in the presence of 5 µM lysozyme (1.7 10^7 cpm) and 20 µg of fraction II. In this experiment, the ATP regeneration system was omitted. The reaction was incubated at 37 °C for 20 min. To deplete ATP, 2 milliunits/µl of apyrase were added to the reaction and incubated for 5 min at room temperature. The sample was divided into two parts; to one, 50 µM Abeta was added, and to the other, water was added as the control (see Fig. 4A). In Fig. 4B, isolated ubiquitin conjugates, prepared as described below, were analyzed. The conjugates were incubated with 20 µg of fraction II in the presence or in the absence of Abeta. At the indicated times, aliquots were withdrawn and analyzed by gel electrophoresis.


Figure 4: Effect of Abeta on conjugate deubiquitination. A, radiolabeled lysozyme was conjugated under standard conditions (see ``Experimental Procedures'') for 20 min. Apyrase was added to the reaction mixture for 5 min at room temperature. The sample was divided into two aliquots: to one, 50 µM Abeta was added (+Abeta), and the other was analyzed as the control (-Abeta). Samples of the reaction mixture were withdrawn at the indicated times, analyzed on a 12% gel electrophoresis and the conjugates were detected by autoradiography. The arrow indicates the position of free labeled lysozyme. B, fraction II proteins were conjugated to ubiquitin under standard conditions, and the ubiquitin conjugates were isolated as described under ``Experimental Procedures.'' The isolated conjugates were incubated with 20 µg of fresh fraction II without ATP, in the absence (-Abeta) or in the presence (+Abeta) of 50 µM Abeta. The decay of the conjugates by the action of the deubiquitination enzymes was followed with time. Aliquots were withdrawn, analyzed by gel electrophoresis, and autoradiographed. The arrow indicates the position of free labeled ubiquitin. Molecular weight markers positions are indicated on the right.



Ubiquitin Conjugates Isolation

Forty µg of radioiodinated ubiquitin (2.3 10^7 cpm) were used in the reaction mixture described above. The final volume of the reaction was increased to 500 µl. After 20 min, 5 mMN-ethylmaleimide was added for 5 min to inactivate the enzymes of the ubiquitin pathway(45) . The sample was mixed with 700 µl of DEAE-Sepharose CL-6B and 300 µl of CM-Sepharose CL-6B resins equilibrated with 10 mM potassium phosphate buffer, pH 7.0. Free ubiquitin does not bind either resins, but the conjugates are retained, presumably due to the substrate portion of the conjugate. The resins were washed with 25 mM Tris, pH 7.2. Conjugates were eluted with 2 ml of 0.5 M NaCl in the same buffer. The eluted proteins were dialyzed against the washing buffer and concentrated by ultrafiltration on Centricon concentrators (Amicon, MA) to 10^3 cpm/µl. Although this procedure was repeated twice, the final purified conjugate preparations showed the presence of free ubiquitin.

Pulse-Chase Analysis of Conjugate Degradation

Fraction II was pretreated for 5 min at 37 °C with 0.5 µM ubiquitin-aldehyde. This step is necessary to inactivate most of the ubiquitin carboxyl-terminal hydrolases present in fraction II(46) . Iodinated lysozyme conjugates were formed using the pretreated fraction II for 20 min as described above. 30-fold excess of unlabeled lysozyme was added together with 50 µM Abeta or water as the control. At the indicated times, two sets of samples were withdrawn and frozen until use. Trichloroacetic acid was added to one set of samples to determine the acid-soluble radioactivity released during the chase. Gel electrophoresis sample buffer was added to the second set of samples for gel electrophoresis analysis.

Western Blot Analysis

Electrophoretic analysis of Abeta was performed using a 14% Tris/Tricine gel electrophoresis buffer system as described previously(47) . The proteins were electrotransferred onto polyvinylidene difluoride membrane. The Western blots were probed with mouse monoclonal antibody 6E10 recognizing the amino acid sequence 1-16 of Abeta(47) . Antibodies bound to the antigen were detected with goat anti-mouse horseradish peroxidase conjugate and visualized with ECL (Amersham Corp.) according to the manufacturer's instructions.

Protein Concentration

Protein concentration was determined using BCA (Pierce) with bovine serum albumin as the standard.

Protein Iodination

Iodination of proteins was performed using 25 µg of Iodogen (Pierce) according to the manufacturer's instructions. Approximately 150 µg of proteins or 80 µg of APP were iodinated using 0.5 mCi of carrier-free I (DuPont NEN). The reaction was allowed to proceed for 10 min at room temperature following the removal of unbound iodine on a 10-ml prepacked desalting column (Bio-Rad). Fractions containing the radiolabeled protein were stored at 4 °C. The specific radioactivities for the iodinated proteins were 5.7 10^5 cpm/µg for ubiquitin, 5 10^5 cpm/µg for lysozyme, 10^6 cpm/µg for beta-lactoglobulin, 8 10^5 cpm/µg for bovine serum albumin, and 2 10^5 cpm/µg for sAPP.

Assay for the 20 S Proteasome Catalytic Activities

Purified 20 S proteasome was prepared from bovine pituitaries(48) . 20 S proteasome (2 µg) and 400 µM of each substrate were incubated at 37 °C for 1 h with the peptides in water (Abeta and Abeta), dimethyl sulfoxide (Abeta), or with vehicle alone (control) in a total volume of 100 µl. The peptide final concentration was 11.4 µM (see Fig. 6A). In Fig. 6B, the indicated peptide concentrations were used. The activities were assayed colorimetrically as described previously(49) .


Figure 6: Effect of Abeta on purified 20 S proteasome. A, effect of Abeta fragments on the catalytic activities of the 20 S proteasome. Activities were determined with 2 µg of purified 20 S proteasome and the respective substrates (400 µM) as described under ``Experimental Procedures.'' The indicated Abeta fragments were added to the reaction mixture without preincubation at a final concentration of 15 µM, except for Abeta, which was added at the final concentration of 11.5 µM. Data are shown as the percentage of the activity measured with no added peptide (control) and are mean values of at least three experiments. *, data presented in B. B, effect of Abeta on the chymotrypsin-like activity of the 20 S proteasome. Two µg of purified 20 S proteasome were assayed with Z-LLL-AMC (400 µM) as the substrate. Data are shown as the percentage inhibition of the activity measured with no added peptide and are mean ± S.E. (values) of three experiments.



Assay for the Protease Activities

The proteolytic activity of the proteases was tested as described previously(50) . Briefly, in each assay, a final concentration of 10 nM protease was incubated with 10 µM Abeta for 30 min at room temperature. The corresponding synthetic peptides, used to test the proteases' activity, were added at a final concentration of 0.5 mM. The hydrolytic activity of each protease was measured as the change in absorbence at 405 nm in a kinetic microtiter plate reader (data not shown).


RESULTS

AbetaInhibits Ubiquitin-dependent Protein Degradation

The ability of Abeta to affect the degradation of known substrates of the ubiquitin-dependent degradation pathway was investigated using rabbit reticulocyte lysate fraction II. Abeta was added to the degradation reaction of iodinated sAPP, lysozyme, beta-lactoglobulin, or bovine serum albumin. Ubiquitin-dependent degradation was measured by counting the radioactivity in the acid-soluble fraction containing the products of the hydrolyzed proteins. Degradation of these proteins was linear during the time tested (data not shown). Table 1shows the results of this analysis. We have previously shown that reticulocyte lysate fraction II contains a background ATP- and ubiquitin-independent proteolytic activity in addition to the ubiquitin-dependent activity determined in our assay(42) . The level of this background activity, marked (-Ub), was small and varied according to the specific radioactivity of the analyzed substrate. In the presence of ubiquitin, degradation of each analyzed substrate increased 2-5 times. This activity was reduced to background levels by the addition of 50 µM Abeta.



Effect of Abeta Concentration and Length

The inhibitory phenomenon was further characterized by testing the effect of Abeta, Abeta, and the reverse peptide Abeta on ubiquitin-dependent degradation. Inhibition of lysozyme degradation was determined as a function of peptide concentration. The results are presented as the percentage of inhibition of degradation (Fig. 1). Ubiquitin-dependent protein degradation was also inhibited by the shorter peptides Abeta and Abeta. The control peptide Abeta did not have any effect. The peptide concentration required to achieve 50% inhibition of lysozyme degradation (IC) was also calculated from Fig. 1. We found that IC decreases with the elongation of the C-terminal sequence of Abeta. The value of IC for Abeta, Abeta, and Abeta are 9.3, 28.6, and 47 µM, respectively.


Figure 1: Concentration-dependent inhibition curves. Lysozyme degradation was performed as described under ``Experimental Procedures,'' in the presence of Abeta, Abeta, Abeta, and the reverse peptide Abeta at the indicated final concentrations. The percentage of inhibition was calculated as the ratio between inhibition in the absence and in the presence of each peptide. The mean ± S.E. (values) of three experiments is indicated.



Effect of Abeta Fragments

To identify regions of Abeta that are critical for inhibition, different fragments of Abeta were tested as inhibitors of ubiquitin-dependent degradation (Fig. 2). Total inhibition of lysozyme degradation was observed with 170 µM Abeta (100%). Fragments Abeta, Abeta, and Abeta, when used at the same final concentration, have a reduced, but appreciable inhibitory effect (40, 42, and 25%, respectively). In contrast, Abeta and Abeta did not have a significant effect and caused only 10 and 7% inhibition of degradation, respectively.


Figure 2: Effect of Abeta fragments on ubiquitin-dependent degradation. Lysozyme degradation was performed as described under ``Experimental Procedures,'' and the indicated Abeta fragments were added at 170 µM final concentration. The percentage of inhibition was calculated as in Fig. 1. The results reported represent the mean ± S.E. (values) of three independent experiments.



Abeta Does Not Affect Protein Conjugation

Inhibition of ubiquitin-dependent degradation by Abeta could result from Abeta interfering with the step of conjugate formation as well as with conjugate degradation and/or deubiquitination. To identify which step is affected by Abeta, we first determined the effect of Abeta on protein conjugation. If Abeta inhibits the enzymes involved in the conjugation process, formation of the conjugates in the presence of Abeta would be drastically reduced. Conjugation of radiolabeled lysozyme was performed in the presence or in the absence of Abeta. The formation of conjugates was analyzed by gel electrophoresis (Fig. 3). Fig. 3A, lane2, shows conjugation of iodinated lysozyme to unlabeled ubiquitin. An increasing number of ubiquitin moieties attached to lysozyme generates a ladder of radiolabeled conjugate bands (Lys-Ub conjugates). When Abeta was present in the reaction (Fig. 3A, lane3), ubiquitin conjugation proceeded as in the control (Fig. 3A, lane2), with only a slight decrease in the intensity of lysozyme conjugates. This small decrease cannot account for the total inhibition of lysozyme degradation under the same conditions (Table 1). The inability of Abeta to inhibit conjugation was further confirmed when we analyzed the conjugation of radiolabeled ubiquitin to the endogenous proteins present in fraction II (Fig. 3B). The reaction was performed in the presence (Fig. 3B, lane2) or in the absence (Fig. 3B, lane3) of Abeta. The conjugates were subjected to gel electrophoresis and visualized by autoradiography. No decrease in protein conjugation was observed. In fact, we found that certain radioactive bands that were also detected in the absence of the peptide (Fig. 3B, lane3) showed an enhanced intensity with Abeta (Fig. 3B, lane2). These results clearly indicate that Abeta does not inhibit the enzymes involved in the step of ubiquitin conjugation, and normal conjugate formation occurs.


Figure 3: Effect of Abeta on conjugate formation. A, autoradiography of a 12% gel electrophoresis. Radiolabeled lysozyme was used to form conjugates either in the absence (lane2) or in the presence (lane3) of 50 µM Abeta. In lane1, ubiquitin and ATP were omitted from the reaction mixture. B, autoradiography of a 15% gel electrophoresis. Radiolabeled ubiquitin was conjugated to the endogenous proteins present in fraction II. The conjugates were formed in the presence (lane2) or in the absence (lane3) of 50 µM Abeta. In lane1, no ATP was added. The reactions were stopped after 20 min by the addition of gel electrophoresis sample buffer. The position of the molecular weight markers is indicated on the right. The arrows on the left point to the radioactive band corresponding to unconjugated radiolabeled lysozyme (A) or ubiquitin (B). C, Western blot analysis of Abeta incubated with fraction II. Aliquots of the reaction mixture described under ``Experimental Procedures'' containing 50 µM Abeta were withdrawn at different times 0, 20, and 60 min (lanes2, 3, and 4, respectively). Lane1, no Abeta. Lane5, approximately 0.8 µg of Abeta alone. The proteins were subjected to 14% Tris/Tricine gel electrophoresis analysis, transferred on polyvinylidene difluoride membrane, and probed with mouse monoclonal antibody 6E10. Antibodies bound to the membrane were detected with ECL (Amersham Corp.). The position of the molecular weight markers is indicated on the right.



Next, we determined whether Abeta itself could be a substrate of ubiquitin-dependent degradation when incubated with reticulocyte lysate fraction II, since this could deplete available ubiquitin. Initially, iodinated Abeta was used for these studies. However, variability in the results, presumably due to an enhanced tendency of the labeled peptide to aggregate in solution, made this analysis very difficult (data not shown). As an alternative, unlabeled Abeta was used and was detected by Western blot analysis using a monoclonal anti-Abeta antibody (Fig. 3C). We observed no significant decrease in the intensity of the Abeta immunoreactive band throughout the time course of our experiment, suggesting that Abeta is not degraded in the presence of fraction II and that the inhibitory phenomenon does not involve the degradation of Abeta.

Abeta Does Not Affect Conjugate Deubiquitination

Since normal conjugate formation occurs in the presence of Abeta, inhibition of protein degradation could result from Abeta affecting either the proteolytic or the deubiquitination processes. We examined the effect of Abeta on both mechanisms. Deubiquitination is an ATP-independent reaction and can be distinguished from the degradation process under conditions of ATP depletion. Radiolabeled lysozyme was conjugated to ubiquitin under standard conditions. The reaction mixture was incubated with apyrase to deplete ATP and thereby prevent both the formation of new conjugates and the degradation of the formed lysozyme conjugates. 50 µM Abeta were added to start the chase of the conjugates. Aliquots of the reaction were withdrawn at different times and analyzed by gel electrophoresis (Fig. 4A). The addition of Abeta did not affect the rate of conjugate deubiquitination. No release of acid-soluble radioactivity was associated with this process (data not shown). This result indicates that the peptide, under these experimental conditions, did not interfere with the deubiquitination reaction. In order to confirm this observation and to rule out alternative explanations, we performed the same analysis using isolated labeled ubiquitin conjugates. Endogenous proteins present in fraction II were conjugated to radioiodinated ubiquitin. The conjugates were isolated and incubated with fresh fraction II without adding ATP (Fig. 4B). Despite the double chromatography on ionic exchange resins, we always observed some unconjugated ubiquitin in the final isolated conjugate preparations. However, since no ATP was added, we can exclude a de novo formation of conjugates during the incubation with fresh fraction II. In the absence of Abeta, ubiquitin was removed by the hydrolases and the radioactivity associated with the conjugates decreased. Correspondingly, an increase of radioactivity migrating with unconjugated ubiquitin was observed. When Abeta was added during the chase period, we observed no difference in the rate of decay of the radioactive bands. The results presented in Fig. 4, A and B, indicate that Abeta does not affect the deubiquitination process.

Abeta Inhibits Conjugate Degradation

Next, we determined the effect of Abeta on the degradation of preformed lysozyme conjugates. Fraction II was pretreated with ubiquitin-aldehyde to inhibit most of the deubiquitination activities(52) . In experiments without this treatment, radiolabeled lysozyme conjugates were removed very rapidly. 30-fold excess of unlabeled lysozyme was added to the lysozyme conjugates, and their decay was followed with time (Fig. 5). PanelA represents the time course of lysozyme conjugate degradation as determined by the release of radioactive acid-soluble material. In the reaction without Abeta, we observed an increase in radioactivity, indicating that the conjugates are degraded. Only a slight increase in radioactivity was observed in the presence of Abeta. This increase is due to a ubiquitin-independent degradation that takes place under these conditions (Table 1). Aliquots of the reaction were withdrawn at the same times as in A and analyzed by gel electrophoresis. The conjugates were visualized by autoradiography (Fig. 5B). When Abeta was omitted, we observed a decrease in amount of lysozyme conjugates and a parallel increase in acid-soluble material (AS) as detected after a shorter exposure time (Fig. 5C). In the presence of Abeta, we observed a rapid disappearance of the high molecular weight conjugates (>28 kDa). However, 12% of the radioactive signal was found to be associated with a new band migrating at approximately 21 kDa (Fig. 5D, lowerpanel). The electrophoretic position of this band corresponds to that of the monoubiquitinated lysozyme conjugate. Quantitative analysis of the radioactivity associated with the conjugates shows two distinct patterns of conjugate decay. Without Abeta, conjugate degradation follows a linear kinetics, with a half-life of approximately 42 min. With Abeta, the conjugates are removed with a nonlinear kinetics, and their half-life is about 19 min (Fig. 5D, toppanel). Furthermore, a new band corresponding to the monoubiquitinated conjugates is formed in the presence of Abeta, suggesting the action of a deubiquitinating activity. This band is not formed when Abeta is absent and degradation of conjugates takes place. Because only a small percentage of lysozyme is conjugated (3-5%), we could not detect the increase in free lysozyme radioactivity associated with the deubiquitination activity. The results in Fig. 5suggest that Abeta blocks the normal degradation of the conjugates. Under these conditions, deubiquitination removes the conjugated ubiquitins without degradation of the substrate at a rate that appears to be higher than in the absence of Abeta. This deubiquitination activity is not inactivated by the pretreatment of fraction II with ubiquitin-aldehyde. A ubiquitin-aldehyde-insensitive and ATP-dependent ubiquitin carboxyl-terminal hydrolase activity was reported as part of the 26 S proteasome complex(51) . Since degradation of the conjugate is performed by the 26 S proteasome complex, our results suggest that Abeta affects this proteolytic complex causing inhibition of conjugate degradation.


Figure 5: Effect of Abeta on conjugate degradation. Iodinated-lysozyme conjugates were formed for 20 min using ubiquitin-aldehyde-treated fraction II (see ``Experimental Procedures''). 30-fold excess of unlabeled lysozyme was added to start the chase. Abeta was added to a final concentration of 50 µM. The reaction was stopped at the indicated times, and duplicates were withdrawn, one to determine the acid-soluble radioactivity (panelA) and the other for gel electrophoresis analysis (panelsB and C). Lysozyme-ubiquitin conjugates were resolved on 12% gel electrophoresis and subjected to autoradiography. Conjugates can be seen on longer exposure (B), while the degradation products are better visualized on shorter exposure (C). The arrowhead on the right indicates the monoubiquitinated lysozyme formed by deubiquitination of the higher molecular weight conjugates when the incubation is performed in the presence of Abeta. The arrows on the left indicate the position of free labeled lysozyme (I-Lys) and the acid-soluble material released as a result of degradation. Molecular weight markers positions are indicated on the right. PanelD, quantitative analysis on a PhosphorImager (Bio-Rad) of the radioactivity associated with the conjugate bands shown in panelB. The two panels represent the kinetics of conjugate decay for higher (top) and lower (bottom) molecular weight conjugates (for details, see text). Both panels show the results in the presence or in the absence of Abeta. The radioactivity was calculated as the percentage of radioactivity at time zero.



Abeta Inhibits the Chymotrypsin-like Activity of the 20 S Proteasome

To ascertain if this interpretation is correct, we determined the effect of Abeta on the proteolytic activity of the 20 S proteasome. This multisubunit complex, known also as the multicatalytic proteinase complex, represents the proteolytic core of the 26 S proteasome(52) . At least three distinct catalytic activities have been shown for the 20 S proteasome: chymotrypsin-like, trypsin-like, and peptidyl-glutamylpeptide hydrolase(48) . We tested these activities using synthetic chromatogenic peptide substrates. 20 S proteasome was incubated with 400 µM of the respective substrate in the presence of Abeta, Abeta, and Abeta. As shown in Fig. 6A, Abeta selectively inhibited the chymotrypsin-like activity of the purified 20 S proteasome. The inhibition of the chymotrypsin-like activity by Abeta was dose-dependent (Fig. 6B). The IC was calculated to be between 3 and 6 µM, which is similar to the IC determined for this peptide in reticulocyte lysates (Fig. 1). To test whether Abeta can directly inhibit proteases, we measured the effect of 10 µM Abeta on the hydrolytic activity of trypsin, chymotrypsin, factor XIa, thrombin, and cathepsin G. Abeta did not affect any of these proteolytic activities, including chymotrypsin (data not shown).


DISCUSSION

We have shown that Abeta inhibits the ubiquitin-dependent degradation of proteins in vitro. Our results suggest a biochemical mechanism, inhibition of the 26 S proteasome, by which Abeta may exert its cell-damaging effect in AD brains.

The detection of ubiquitin-conjugated proteins within abnormal intraneuronal structures and the occurrence of morphologically altered lysosomes (53) suggest that protein degradation might be compromised in AD. Lysosomes eventually became leaky, and active lysosomal proteases are found outside of these structures(54) . Although the two proteolytic pathways are distinct, ubiquitination has been proposed as a signal for protein up-take into the lysosomes(55, 56) . Therefore, a situation where inhibition of ubiquitin-dependent degradation by Abeta overloads the lysosomal system to a point in which it is unable to remove the abnormal and damaged proteins is not excluded.

We previously reported that sAPP and full-length APP have a different susceptibility to ubiquitin-dependent degradation(42) . sAPP, which contains the amino acids 1-15 of the Abeta sequence(10, 13) , is degraded by the ubiquitin proteolytic pathway. Full-length APP, which contains the entire Abeta sequence, is not a substrate of the same degradative system. Our current findings suggest two possible explanations for this difference. Intact Abeta is generated during the incubation of APP with the reticulocyte lysate. Only a small percentage of APP, not detected by our assay, may be degraded to produce free Abeta. The in situ generated Abeta would inhibit a further degradation of APP. Alternatively, the conformation of full-length APP allows exposure of intact Abeta to interaction with the 26 S proteasome, causing inhibition of APP degradation. On the other hand, sAPP conformation makes the Abeta sequence 1-15 unavailable for the inhibitory interaction with the proteasome, and, therefore, it does not prevent degradation of sAPP. Furthermore, Abeta, corresponding to the Abeta sequence present in sAPP, when tested as an inhibitor of protein degradation showed a much reduced inhibitory effect than Abeta (Fig. 2). Thus, our results suggest that the presence of intact Abeta in APP influences the turnover of the protein. On its own, the same Abeta sequence is able to inhibit the degradation of other proteins by the ubiquitin degradative system. Evidence for a domain within the substrate that regulates the ubiquitin proteolytic pathway was recently reported by Treier et al. (57) . They observed that two highly similar proteins, c-Jun and v-Jun, have a completely different susceptibility to ubiquitin-dependent degradation. The results indicated that the sequence of the domain in c-Jun, which is not present in v-Jun, is responsible for the in vivo ubiquitin-mediated degradation of c-Jun.

Using fragments of Abeta, we established that there are at least two regions of Abeta that are important for inhibition: the N-terminal region (Fig. 2, Abeta and Abeta) and a portion of the hydrophobic sequence of the transmembrane domain (Fig. 2, Abeta). However, the inhibitory effect of Abeta was much greater than the sum of the inhibitory effects of Abeta and Abeta combined (data not shown), which suggests a cooperative effect of the two sequences within the intact Abeta molecule for complete inhibition. We also showed that in addition to the amino acid sequence, the length of the peptide appears to be a critical factor in the inhibitory mechanism. Although additional studies are required, it seems possible that a spatial separation of the two determined inhibitory regions of Abeta is necessary for inhibition.

We found that Abeta does not affect protein conjugation, nor is it degraded by the proteolytic process. Although we cannot totally exclude that Abeta is conjugated to ubiquitin during the incubation with fraction II, our results strongly suggest that Abeta conjugation is not involved in the inhibitory process. First, conjugation of lysozyme as well as of endogenous protein was not affected by the presence of Abeta (Fig. 3, A and B). This finding indicates that Abeta conjugation, if it occurs, does not compete with the conjugation of other proteins. Second, inhibition of ubiquitin-dependent degradation is independent from Abeta conjugation. In fact, Abeta, which has no lysine residues and thereby cannot be ubiquitinated, showed a robust inhibitory effect on ubiquitin-dependent protein degradation (Fig. 2). Third, Abeta is not degraded during incubation with fraction II (Fig. 3C), suggesting that the peptide is not a substrate of the ubiquitin proteolytic pathway. Pulse-chase analysis of lysozyme conjugate degradation showed that Abeta does not inhibit deubiquitination but reduces the rate of conjugate degradation. However, this inhibitory mechanism did not produce an in vitro accumulation of ubiquitin conjugates as is observed in neurons of AD patients. One possible explanation is that in vivo, the increase in conjugates due to inhibition of their degradation causes the overloading of the brain hydrolases, which eventually became trapped into the conjugates-containing inclusion bodies. This possibility is supported by the presence of the neuronal-specific ubiquitin carboxyl-terminal hydrolase in selected ubiquitinated inclusion bodies in neurodegenerative diseases(58) . Alternatively, neuronal cells may lack the fraction II-specific hydrolase that removes the conjugates under our experimental conditions. In either case, the triggering cause of conjugates accumulation is the inhibition of the ubiquitin degradative pathway by Abeta, while a failure of the deubiquitination enzymes may be a contributing factor to the final increase of conjugates.

Our experiments with the reticulocyte lysate fraction II suggested an inhibitory mechanism that involves at least one of the proteolytic activities associated with the 26 S proteasome. The results were supported by experiments with isolated 20 S proteasome, the catalytic core of the larger protease complex. Of the three catalytic activities tested, only the chymotrypsin-like activity was affected by Abeta. Our finding is also consistent with previously reported accumulation of ubiquitin conjugates upon inhibition or inactivation of the chymotrypsin-like activity of the 26 S proteasome(59, 60) . Whether this is the only mechanism by which Abeta affects conjugate degradation is not known at the present. The 26 S proteasome complex is a barrel-shaped structure, that consists of the 20 S proteasome and two additional components at both ends with presumably regulatory functions (61) . In the current model, the proteolytically active sites of the catalytic subunits are located in the inner space of the barrel-shaped structure and are accessible only to completely unfolded peptides(62, 63) . Abeta inhibition of the 26 S proteasome activity could be caused by the interaction of the peptide with the catalytic core of the proteolytic complex. Abeta may contain the conformational prerequisites of a substrate but may not have the correct degradation signals. Alternatively, Abeta interaction with the proteasome may obstruct the passage of other substrates to the inner proteolytic compartment of the 20 S proteasome. We showed that Abeta specifically inhibits the chymotrypsin-like activity but has no effect on the proteolytic activity of the protease chymotrypsin, suggesting that Abeta does not interact with the active site of the proteasome subunit. These observations favor a model in which Abeta allosteric effects induce conformational changes in the proteasome that prevent the substrate from interacting with the subunit active site. However, other possibilities are not excluded since degradation of ubiquitin conjugates is performed by the 26 S proteasome for which a model of action has not been established.

We have demonstrated that Abeta inhibits ubiquitin-dependent degradation in vitro. The current findings extend our understanding of this phenomenon by identifying the specific step in the ubiquitin-dependent degradation pathway that is compromised by Abeta. The ubiquitin proteolytic pathway is not only responsible for the degradation of damaged and abnormal proteins, but also of regulatory and short lived proteins. Therefore, it affects many cellular mechanisms that are dependent on protein degradation. Our in vitro results suggest a model for the breakdown in cellular metabolism that may lead to neuronal damage observed in AD. Although the contribution of this inhibition in AD and aging is yet to be determined, our findings represent an important step toward elucidating the cascade of events leading to neuronal degeneration.


FOOTNOTES

*
This work was supported by Alzheimer's Association Grant ZEN-91-017 (to D. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Dept. of Psychiatry and Behavioral Science, Health Science Center T-10, School of Medicine, SUNY, Stony Brook, NY 11794-8101.

(^1)
The abbreviations used are: AD, Alzheimer's disease; Abeta, amyloid beta-protein; APP, amyloid beta-protein precursor; sAPP, secreted APP; Ub, ubiquitin; Tricine, N-[2-hydroxy-1,1,bis(hydroxymethyl)ethyl]glycine.


ACKNOWLEDGEMENTS

We thank Drs. Mike Maurizi and Amit Banerjee for advice and suggestions during the preparation of this manuscript.


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