(Received for publication, May 11, 1995)
From the
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
-protein (A
) and its precursor are found in neurofibrillary
tangle-containing neurons. A
is the major component of
extracellular plaques. We showed that A
acts as an inhibitor of
the ubiquitin-dependent protein degradation in vitro. We
examined the effect of A
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 A
. However, A
significantly reduced the
rate of conjugate degradation. Our results indicate that A
interacts with the proteolytic step of the ubiquitin degradative
pathway. Since this step is performed by the 26 S proteasome, the
effect of A
on the catalytic core of this proteolytic complex, the
20 S proteasome, was determined. We found that A
selectively
inhibits the chymotrypsin-like activity of the 20 S proteasome. Under
pathological conditions in the affected neuron, A
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.
Alzheimer's disease (AD) ()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
-protein
(A
)(7) . A
is the proteolytic product of a longer
protein called amyloid
-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 A
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 A
has been detected by several
laboratories(17, 18, 19) . Internalization of
A
from cell culture media into normal human fibroblasts by the
lysosomal/endosomal pathway was also reported(20) . Cellular
A
immunoreactivity is specifically associated with intraneuronal
neurofibrillary tangle-bearing
bodies(21, 22, 23) . These findings indicate
that under pathological conditions in affected neurons, A
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 A inhibits the ubiquitin-dependent degradation of
proteins in vitro. We propose that in the affected cells,
A
interacts with the proteolytic pathway. This interaction may
contribute to certain pathological manifestations of AD, such as
inclusion bodies and neurofibrillary tangle formation.
Figure 4:
Effect of A 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 A
was
added (+A
), and the other was analyzed as the
control (-A
). 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 (-A
) or in the presence
(+A
) of 50 µM A
. 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.
Figure 6:
Effect of A on purified 20 S
proteasome. A, effect of A
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 A
fragments were added to the
reaction mixture without preincubation at a final concentration of 15
µM, except for A
, 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
A
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.
Figure 1:
Concentration-dependent inhibition
curves. Lysozyme degradation was performed as described under
``Experimental Procedures,'' in the presence of
A, A
,
A
, and the reverse peptide
A
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.
Figure 2:
Effect of A fragments on
ubiquitin-dependent degradation. Lysozyme degradation was performed as
described under ``Experimental Procedures,'' and the
indicated A
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.
Figure 3:
Effect of A 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
A
. 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 A
. 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
A
incubated with fraction II. Aliquots of the
reaction mixture described under ``Experimental Procedures''
containing 50 µM A
were
withdrawn at different times 0, 20, and 60 min (lanes2, 3, and 4, respectively). Lane1, no A
. Lane5, approximately 0.8
µg of A
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 A itself could be a
substrate of ubiquitin-dependent degradation when incubated with
reticulocyte lysate fraction II, since this could deplete available
ubiquitin. Initially, iodinated A
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
A
was used and was detected by Western blot analysis using a
monoclonal anti-A
antibody (Fig. 3C). We observed
no significant decrease in the intensity of the A
immunoreactive
band throughout the time course of our experiment, suggesting that
A
is not degraded in the presence of fraction II and that the
inhibitory phenomenon does not involve the degradation of A
.
Figure 5:
Effect of A 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. A
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 A
.
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
A
. The radioactivity was calculated as the
percentage of radioactivity at time zero.
We have shown that A inhibits the ubiquitin-dependent
degradation of proteins in vitro. Our results suggest a
biochemical mechanism, inhibition of the 26 S proteasome, by which
A
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 A 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 A
sequence(10, 13) , is degraded by the ubiquitin
proteolytic pathway. Full-length APP, which contains the entire A
sequence, is not a substrate of the same degradative system. Our
current findings suggest two possible explanations for this difference.
Intact A
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 A
. The in situ generated A
would inhibit a further degradation of APP.
Alternatively, the conformation of full-length APP allows exposure of
intact A
to interaction with the 26 S proteasome, causing
inhibition of APP degradation. On the other hand, sAPP conformation
makes the A
sequence 1-15 unavailable for the inhibitory
interaction with the proteasome, and, therefore, it does not prevent
degradation of sAPP. Furthermore, A
,
corresponding to the A
sequence present in sAPP, when tested as an
inhibitor of protein degradation showed a much reduced inhibitory
effect than A
(Fig. 2). Thus, our
results suggest that the presence of intact A
in APP influences
the turnover of the protein. On its own, the same A
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
A, we established that there are at least two regions of A
that are important for inhibition: the N-terminal region (Fig. 2, A
and A
)
and a portion of the hydrophobic sequence of the transmembrane domain (Fig. 2, A
). However, the
inhibitory effect of A
was much greater than
the sum of the inhibitory effects of A
and
A
combined (data not shown), which suggests
a cooperative effect of the two sequences within the intact A
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 A
is necessary for
inhibition.
We found that A does not affect protein
conjugation, nor is it degraded by the proteolytic process. Although we
cannot totally exclude that A
is conjugated to ubiquitin during
the incubation with fraction II, our results strongly suggest that
A
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 A
(Fig. 3, A and B). This finding indicates that A
conjugation, if it
occurs, does not compete with the conjugation of other proteins.
Second, inhibition of ubiquitin-dependent degradation is independent
from A
conjugation. In fact, A
, which
has no lysine residues and thereby cannot be ubiquitinated, showed a
robust inhibitory effect on ubiquitin-dependent protein degradation (Fig. 2). Third, A
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 A
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
A
, 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 A. 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 A
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) . A
inhibition of the 26 S
proteasome activity could be caused by the interaction of the peptide
with the catalytic core of the proteolytic complex. A
may contain
the conformational prerequisites of a substrate but may not have the
correct degradation signals. Alternatively, A
interaction with the
proteasome may obstruct the passage of other substrates to the inner
proteolytic compartment of the 20 S proteasome. We showed that A
specifically inhibits the chymotrypsin-like activity but has no effect
on the proteolytic activity of the protease chymotrypsin, suggesting
that A
does not interact with the active site of the proteasome
subunit. These observations favor a model in which A
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 A
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 A
. 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.