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(Received for publication, September 12, 1996, and in revised form, December 30, 1996)
From the ABSTRACT The proteasomal system consists of a proteolytic
core, the 20 S proteasome, which associates in an
ATP-dependent reaction with the 19 S regulatory complex to
form the functional 26 S proteasome. In the absence of ATP, the 20 S
proteasome forms a complex with the INTRODUCTION The 26 S proteasome (or 26 S protease) is a component of the
ubiquitin (Ub)1 pathway involved in cell
cycle and in transcriptional regulation (1, 2). The 11 S regulator (3)
or PA28 (4) stimulates 20 S proteasomal peptidase activities, and
because it is inducible by Cellular data on the effects of HIV-1 Tat are accumulating rapidly. In
addition to its transcriptional functions (7), genetic evidence has
been provided that Tat might have independent effects in determining
infectivity and cytopathicity in a developing HIV-1 infection (8). Tat
is produced in large quantities in HIV-1-infected cells. It is secreted
and can be taken up rapidly by other cells (9). Exogenous Tat
accelerates CD95-mediated, activation-induced T cell apoptosis (10).
This mechanism may lead to a depletion of noninfected CD4+
T cells. It has been reported that HIV-1 Tat potentiates TNF-induced NF- Our interest in Tat was stimulated by the fact that two ATPase subunits
of the 19 S regulatory complex, MSS1 and TBP1 (14), directly or
indirectly influence Tat action. MSS1 (mammalian suppressor of sgv1) is
a modulator of Tat-mediated transactivation (15), and TBP1 (Tat-binding
protein 1) was identified by direct protein-protein interaction with
Tat (16) and hence could be a potential Tat-binding subunit of the 26 S
proteasome. We reasoned that HIV-1 Tat could have an effect on the
proteasomal system and performed experiments to test this
hypothesis.
EXPERIMENTAL PROCEDURES The components of the proteasomal system, the 20 S and 26 S
proteasomes and the 11 S regulator, were purified from human
erythrocytes as described (17, 18).
SucLLVY-AMC cleavage assays were conducted with the electrophoretically
homogenous protein complexes in a final volume of 100 µl. Final
concentrations of the substrate, HIV-1 Tat, and the proteasomal
complexes are indicated in the figures. Fluorescence was measured at
37 °C with a microtiter plate reader (Fluoroscan II, Labsystems) at
355 nm excitation and 460 nm emission after a 5-min lag time over a
60-min period in 5-min intervals. During this time period the reactions
were linear.
The degradation of Ub-[125I]lysozyme conjugates was
measured in the absence and in the presence of ATP. After 30 min of
incubation in the presence of 50 µl of
Ub-[125I]lysozyme conjugates (~4 × 103 cpm), the reaction was stopped, and the percentage of
degradation was determined as described (19).
For Western blotting purified 26 S proteasome (2 µg) was separated by
nondenaturing electrophoresis on a 4-15% Phast gel (Pharmacia Biotech
Inc.) at 300 volt hours. Under these conditions some of the complex
disassembles into the 20 S proteasome and the 19 S regulatory complex.
20 S and 26 S proteasome bands were visualized by substrate overlay
using sucLLVY-AMC (17, 20) or detected by immunoblotting with a
polyclonal antibody directed against the 20 S proteasome (17). The
antibody against subunit 4 of the 19 S regulatory complex (gift from M. Rechsteiner) detects the 26 S proteasome as well as the 19 S regulatory
complex.
To show Tat binding to the protein complexes, blots were incubated with
a Tat solution (0.1 µg/ml) for 60 min. Subsequently the blots were
washed with PBS and developed with an polyclonal anti-Tat antibody. The
antibody against Tat was made with synthesized protein in rabbits using
standard techniques.
The two-exon Tat used in most of the experiments was synthesized as
described previously (21). Recombinant Tat was purchased from
AGMED.
RESULTS AND DISCUSSION As an initial experiment we investigated whether HIV-1 Tat could
affect peptide hydrolysis by the 20 S proteasome. To our surprise the
degradation of a fluorogenic peptide, sucLLVY-AMC, by the 20 S
proteasome was inhibited by Tat with an average 50% inhibition value
(Ki50) of 5 × 10 Kinetics of Tat interactions with the
proteasomal system. A, effect of Tat concentration on the 20 S proteasome sucLLVY-AMC cleavage activity. Increasing amounts of Tat
were added to 100 ng of isolated 20 S proteasome in the presence of 50 (
Tat decreases antigen-induced lymphocyte proliferation (12), possibly
because antigens are not processed and thus not presented. Therefore,
it was intriguing to test its effect on the 20 S proteasome-11 S
regulator complex, which is involved in the processing of antigens presented by MHC class I (6). The kinetic data shown in Fig. 1B demonstrate that Tat competes with the 11 S regulator for
binding sites on the 20 S proteasome. There is a linear increase of the Ki50 values at increasing 11 S regulator
amounts (see Fig. 1B, inset). Sigmoidal kinetics
indicate that Tat must displace the 11 S regulator from both of its
binding sites, the two
Next we examined the effect of Tat on the 26 S proteasome. In the
presence of ATP, the cleavage of sucLLVY-AMC is activated by Tat about
3-5-fold with an average 50% activation value
(Ka50) of approximately 5 × 10 We tested the effect of Tat on a more physiologically relevant function
of the 26 S proteasome, the degradation of Ub conjugates (Fig.
2). Tat activated ATP-dependent conjugate
degradation to the same extent as it stimulated peptide cleavage
activity of the 26 S proteasome. In samples without exogenous ATP (Fig.
2,
Although the kinetic data argue strongly that Tat binds to components
of the proteasomal system, it is demonstrated directly in Fig.
3. The 26 S proteasome was separated by nondenaturing electrophoresis and localized by substrate overlay and specific antibodies, which also identified the 19 S and the 20 S complexes (Fig.
3A). The occurrence of all three complexes reflects partial dissociation of the 26 S proteasome during electrophoresis. As expected
on the basis of our kinetic studies, the lane labeled Tat in Fig. 3A shows binding of Tat to the 26 S
proteasome and the 19 S regulatory complex as well as to the 20 S
proteasome. Binding to the 19 S complex supports our conclusion of Tat
binding site(s) on the 26 S proteasome, independent of that on the 20 S
proteasome. Preliminary experiments using immunological probing of
streptavidin precipitated biotin-Tat peptide indicate that Tat does not
bind the 11 S regulator (data not shown).
To illustrate the competition between Tat and the 11 S regulator for
the proteasomal binding sites, isolated 11 S regulator was used to
displace Tat bound to immobilized 20 S proteasome (Fig.
3B).
The specific Tat-binding subunits of the 20 S proteasome are currently
unknown. However, because the 20 S proteasome subunit C2 is involved in
11 S regulator binding (24), it could also participate in the
interaction with Tat.
It is not unique that a protein such as Tat interacts with the
proteasomal system. Two other viral proteins, Hbx (25) and Tax (26),
also bind to the 20 S proteasome. The consequences of these
interactions for the 20 S and 26 S enzymes activities, however, remain
speculations.
While inhibiting the 20 S proteasome, Tat stimulates the 26 S
proteasome needed for cell cycle progression and transcriptional regulation (1, 2). In this context it is interesting to note that the
26 S enzyme activates NF- The cartoon in Fig. 4 summarizes Tat effects on the
proteasomal system based on the data shown in this paper. The in
vivo consequence of 11 S regulator replacement and 20 S proteasome inhibition by Tat should be a decrease in antigen processing and subsequently, a reduced MHC class I presentation by infected and perhaps noninfected cells. The role of the 26 S proteasome in antigen
processing is unclear. It has been shown that ubiquitination supports
the presentation of antigens derived from ovalbumin (5), and the
initial degradation of antigens by the 26 S proteasome can be assumed.
Whether the enzyme itself produces peptides that can be presented by
MHC class I molecules or whether it makes intermediates that are
further processed by the free 20 S proteasome and/or the 20 S
proteasome-11 S regulator complex is not yet known. The latter pathway
could be inhibited by Tat.
Although the highly basic Tat protein has been reported to bind to a
number of different proteins, the cellular data demonstrating the
effects of exogenous Tat are consistent with our in vitro effects upon the proteasomal system. The immunosuppressive activity of
Tat has been demonstrated with lymphocytes. Tetanus toxoid-induced lymphocyte proliferation was inhibited by exogenous Tat with the same
Ki50 value (12) of 50 nM as
obtained in our studies for 20 S proteasome inhibition. In concert with
its effects on CD4+ T cells (10), Tat inhibition of antigen
processing may at least partially contribute to the profound
immunodeficiency in AIDS patients.
FOOTNOTES ACKNOWLEDGEMENTS We thank Martin Rechsteiner for the
anti-20 S proteasome and anti-S 4 rabbit sera and for preparation of
Ub-[125I]lysozyme conjugates. We also thank Markus
Groettrup for critical reading of the manuscript.
REFERENCES
Volume 272, Number 13,
Issue of March 28, 1997
pp. 8145-8148
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
COMMUNICATION:
,,¶
Institute of Biochemistry,
Humboldt-University, Medical Faculty (Charité), Monbijoustrasse
2A, 10117 Berlin, Germany and the § Zentrum für
Molekulare Biologie Heidelberg, University Heidelberg, Im Neuenheimer
Feld 282, 69120 Heidelberg, Germany
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-interferon-inducible 11 S
regulator. Both the 20 S proteasome and the 11 S regulator have been
implied in the generation of antigenic peptides. The human
immunodeficiency virus (HIV)-1 Tat protein causes a number of
different effects during acquired immunodeficiency syndrome (AIDS).
Here we show that HIV-1 Tat protein strongly inhibits the peptidase
activity of the 20 S proteasome and that it interferes with formation
of the 20 S proteasome-11 S regulator complex. In addition, it slightly
increases the activity of purified 26 S proteasome. These results may
explain the mechanism by which HIV-1-infected cells escape cytotoxic T
lymphocyte response and at least in part immunodeficiency in AIDS
patients.
-interferon, we suggested that it would
be involved in antigen processing (3). Recently it has been
demonstrated that the 20 S proteasome generates antigens presented by
MHC class I molecules (5) and is regulated by the 11 S complex in
vivo (6).
B activation, which stimulates the replication of HIV-1 (11). Moreover, HIV-1 Tat inhibits antigen-induced lymphocyte proliferation (12) possibly by interfering with either antigen processing or
presentation. Both NF-B activation and antigen processing require
the proteasomal system (5, 13).
8
M (Fig. 1A). The inhibition is
almost complete with a remaining 5-10% activity that differs slightly
between preparations. Because the Ki50
values were independent of substrate concentrations (see Fig.
1A), Tat does not compete with the fluorogenic peptide for
binding to the active centers in the lumen of the 20 S proteasome. This
is in accordance with earlier results showing that a folded protein
cannot penetrate into the inner compartment of the 20 S proteasome
(22). To prove whether the effect was restricted to synthesized Tat, we
tested a Tat protein expressed in and isolated from Escherichia
coli that also caused 20 S proteasome inhibition (data not
shown).
Fig. 1.
), 100 (
), and 200 µM (
) peptide substrate in a
final volume of 100 µl. An average 50% inhibition value
(Ki50) of 5 × 108
M, which was independent of the substrate concentration,
can be estimated. The data are representative for three (50 and 100 µM of substrate) and twelve (200 µM of
substrate) independent experiments. B, dependence of 20 S
proteasome inhibition by Tat on 11 S regulator amount. Increasing
amounts of Tat were added to 30 ng of isolated 20 S proteasome
preincubated for 10 min with 0 (), 0.8 (), and 1.6 µl () of
isolated 11 S regulator (200 µg/ml). Increasing amounts of 11 S
regulator caused a linear increase of
Ki50 values (inset)
indicating competition. The final concentration of sucLLVY-AMC was 200 µM. The data are representative for four independent
experiments. C, effect of Tat on the sucLLVY-AMC cleavage
activity of the 26 S proteasome in the presence of ATP. Increasing
amounts of Tat were added to 100 ng of isolated 26 S proteasome in the
presence of 200 µM substrate, 2 mM ATP, and 5 mM MgCl2. The samples were preincubated for 30 min at 37 °C before the fluorescence was measured. An
average 50% activation value (Ka50) of
5 × 107 M was estimated. The data are
representative for four independent experiments.
[View Larger Version of this Image (19K GIF file)]
-rings of the 20 S proteasome (23), to exert
inhibition. The same kinetic profile, although as an activation, was
obtained when the 20 S proteasome was incubated with Tat protein prior
to the addition of different 11 S regulator amounts (data not shown).
Moreover, Western blot analysis demonstrates that the 11 S regulator
displaces Tat from its proteasomal binding sites (Fig.
3B).
Fig. 3.
Interaction of Tat with components of the
proteasomal system. A, Tat binding to immobilized 20 S
proteasome, 19 S regulatory complex, and 26 S proteasome. Purified 26 S
proteasome (2 µg) was separated by nondenaturing electrophoresis.
Under these conditions the complex partially disassembles into the 20 S
proteasome and the 19 S regulatory complex. Immobilized complexes were
stained with Ponceau S (Ponceau). 20 S (20S) and 26 S
(26S) proteasome bands were visualized by substrate overlay
(Overlay) or detected by immunoblotting with a polyclonal antibody
directed against the 20 S proteasome (Anti 20S). The
antibody against subunit 4 of the 19 S regulatory complex (Anti
S4) was used to stain the 26 S proteasome as well as the 19 S
regulatory complex (19S RC). To show Tat binding to the
protein complexes, blots were developed with an anti-Tat antibody
(Tat). B, displacement of Tat from 20 S
proteasome by 11 S regulator. Purified 20 S proteasome (2 µg) was
transferred to nitrocellulose from a nondenaturing gel. Immobilized enzyme was incubated with Tat (0.1 µg/ml) in PBS. Nitrocellulose strips were washed in PBS containing 0.1% Tween 20 and further incubated with PBS (Tat) or with 2 mg/ml 11 S regulator
(Tat + 11S Reg). The blots were then stained with an
anti-Tat antibody using the ECL system (Amersham Corp.). The
Control lane is immobilized 20 S proteasome without Tat or
11 S regulator treatment, showing that the anti-Tat antibody does not
cross-react with the 20 S proteasome.
[View Larger Version of this Image (34K GIF file)]
7 M (Fig. 1C). In experiments
with disassembled 26 S proteasome, Tat activation was observed only
after 30 min in the presence of ATP (data not shown). This time period
corresponds with the ATP-dependent assembly process of the
26 S proteasome from the 19 S regulatory complex and the 20 S
proteasome. It is likely that the 19 S regulatory complex displaces Tat
from the 20 S proteasome, because it likewise displaces the 11 S
regulator (17). Presumably Tat binds to TBP1 and perhaps other ATPases
of the 19 S regulatory complex, which leads to this moderate activation
of the 26 S proteasome.
MgATP) we observed a slight stimulation of proteolysis
in the presence of Tat, probably due to residual ATP in the preparation (18).
Fig. 2.
Tat stimulates the Ub-lysozyme conjugate
degradation by the 26 S proteasome. Samples containing 2.5 µg 26 S proteasome were preincubated in the absence (Control) and
presence of 50 µg/ml Tat protein (Tat) for 30 min at
37 °C in 50 µl of buffer with (white columns) and
without (black columns) 2 mM ATP and 5 mM MgCl2. Tat increased the
ATP-dependent cleavage of Ub-lysozyme conjugates
approximately 3-fold. Data are means ± S.E. (n = 4).
[View Larger Version of this Image (14K GIF file)]
B (13). It has been shown with different
cell lines that Tat enhances tumor necrosis factor-induced activation
of NF-B, which leads to a stimulation of HIV-1 replication (11).
Fig. 4.
Summary of the interconversion of proteasomal
complexes, their reported in vivo functions, and the
effects of Tat shown in this paper. The 11 S regulator (11S
Reg) is presented as a heptameric ring. Its exact symmetry,
however, is unknown. The presentation of the 19 S regulatory complex
(19S RC) is based on earlier structural data (27).
[View Larger Version of this Image (27K GIF file)]
¶
To whom correspondence should be addressed. Tel.:
4930-28026510; Fax: 4930-28026615.
1
The abbreviations used are: Ub, ubiquitin; MHC,
major histocompatibility complex; SucLLVY-AMC,
succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin; PBS,
phosphate-buffered saline; HIV, human immunodeficiency virus.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
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