Article

Heink S, Ludwig D, Kloetzel PM, Kruger EIFN-gamma-induced immune adaptation of the proteasome system is an accelerated and transient response. Proc Natl Acad Sci USA 102(26): 9241-9246

Institute of Biochemistry, Charité-Universitätsmedizin Berlin, 10117 Berlin, Germany.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 07/2005; 102(26):9241-6. DOI: 10.1073/pnas.0501711102
Source: PubMed
ABSTRACT
Peptide generation by the proteasome is rate-limiting in MHC class I-restricted antigen presentation in response to IFN-gamma. IFN-gamma-induced de novo formation of immunoproteasomes, therefore, essentially supports the rapid adjustment of the mammalian immune system. Here, we report that the molecular interplay between the proteasome maturation protein (POMP) and the proteasomal beta5i subunit low molecular weight protein 7 (LMP7) has a key position in this immune adaptive program. IFN-gamma-induced coincident biosynthesis of POMP and LMP7 and their direct interaction essentially accelerate immunoproteasome biogenesis compared with constitutive 20S proteasome assembly. The dynamics of this process is determined by rapid LMP7 activation and the immediate LMP7-dependent degradation of POMP. Silencing of POMP expression impairs recruitment of both beta5 subunits into the proteasome complex, resulting in decreased proteasome activity, reduced MHC class I surface expression, and induction of apoptosis. Furthermore, our data reveal that immunoproteasomes exhibit a considerably shortened half-life, compared with constitutive proteasomes. In consequence, our studies demonstrate that the cytokine-induced rapid immune adaptation of the proteasome system is a tightly regulated and transient response allowing cells to return rapidly to a normal situation once immunoproteasome function is no longer required.

Full-text

Available from: Elke Krüger
IFN-
-induced immune adaptation of the proteasome
system is an accelerated and transient response
Sylvia Heink*, Daniela Ludwig, Peter-M. Kloetzel, and Elke Kru
¨
ger
Institute of Biochemistry, Charite´ –Universita¨ tsmedizin Berlin, 10117 Berlin, Germany
Edited by Peter Cresswell, Yale University School of Medicine, New Haven, CT, and approved May 5, 2005 (received for review March 2, 2005)
Peptide generation by the proteasome is rate-limiting in MHC class
I-restricted antigen presentation in response to IFN-
. IFN-
-
induced de novo formation of immunoproteasomes, therefore,
essentially supports the rapid adjustment of the mammalian im-
mune system. Here, we report that the molecular interplay be-
tween the proteasome maturation protein (POMP) and the pro-
teasomal
5i subunit low molecular weight protein 7 (LMP7) has a
key position in this immune adaptive program. IFN-
-induced
coincident biosynthesis of POMP and LMP7 and their direct inter-
action essentially accelerate immunoproteasome biogenesis com-
pared with constitutive 20S proteasome assembly. The dynamics of
this process is determined by rapid LMP7 activation and the
immediate LMP7-dependent degradation of POMP. Silencing of
POMP expression impairs recruitment of both
5 subunits into the
proteasome complex, resulting in decreased proteasome activity,
reduced MHC class I surface expression, and induction of apoptosis.
Furthermore, our data reveal that immunoproteasomes exhibit a
considerably shortened half-life, compared with constitutive pro-
teasomes. In consequence, our studies demonstrate that the cyto-
kine-induced rapid immune adaptation of the proteasome system
is a tightly regulated and transient response allowing cells to
return rapidly to a normal situation once immunoproteasome
function is no longer required.
antigen presentation immunoproteasome MHC class I
T
he proteasome is the key enzyme in the proteolytic cascade
required for the generation of peptide s presented to cytotoxic
T lymphocytes by MHC class I molecules. Within this cascade, the
26S proteasome is responsible for the initial selective degradation
of polyubiquitinated cellular protein substrates. This multisubunit
enzyme is formed by the catalytic 20S core complex and two 19S
regulator complexes that are responsible for the binding and
unfolding of ubiquitinated substrates (1, 2). The 20S proteasome is
composed of 14 nonidentical subunits building four stacked rings of
seven subunits each. Seven different but related
subunits form the
two outer rings, whereas the two inner rings contain seven different
subunits. The hydrolyzing activitie s of the 20S core are conferred
by three of the seven
subunits, i.e., subunits
1,
2, and
5 (3),
located in both of the two inner heptameric
rings.
In vertebrate s, specific catalytically active proteasome subunits,
collectively referred to as immunosubunits (4), have evolved that
improve proteasome-dependent antigen processing (5–7). IFN-
induces the synthe sis of the immunosubunits
1i [also named low
molecular weight protein 2 (LMP2)],
2i [multicatalytic endopep-
tidase complex-like 1 (MECL1)], and
5i (LMP7). These subunits
are cooperatively incorporated into nascent proteasomes, thereby
replacing their constitutive homologues
1,
2, and
5 (8–11).
Thus, there exist two types of proteasomes, i.e., constitutive pro-
teasomes (c20S) that are constitutively expressed in all cells and
immunoproteasomes (i20S) that are formed upon exposure of cells
to IFN-
.
Both c20S and i20S are exclusively formed de novo following a
sophisticated and not yet fully understood biogene sis program. We
previously showed that assembly of mammalian 20S proteasomes is
a multistep process that occurs via the formation of distinct
proteasome precursor complexes with different
subunit compo-
sitions. Active-site
subunits are synthe sized and incorporated as
proproteins that essentially mature by a two-step procedure within
the precursor complexes (12, 13). Final activation of the
subunits
requires the formation of the preholoproteasome assembly inter-
mediate. Concomitantly, the cis- and trans-autocatalytic removal of
the
subunit propeptides liberate s the active-site threonines of the
now fully active 20S core proteasome (8, 10, 12–16).
Eukaryotic proteasome biogene sis requires acce ssory proteins to
promote its assembly and final maturation steps. A protein that is
directly associated with proteasome precursor complexes is pro-
teasome maturation protein (POMP), also named proteassemblin
or humanmouseUmp1 according to their yeast homologue Ump1
(17–20). These factors are proposed to be involved in the coordinate
processing of the
subunits, thereby becoming the first substrate of
the fully assembled and activated 20S proteasome. Thus, degrada-
tion of Ump1pPOMP signals the successful completion of the
proteasome biogenesis program (17, 20). Importantly, in mamma-
lian cells, IFN-
was found to enhance POMP mRNA levels,
suggesting that POMP may play an important role in i20S biogen-
esis (17, 19).
Highlighting the functional importance of i20S, it was shown that
deficiency in the immunosubunits
1i or
5i reduces the cytotoxic
T lymphocyte repertoire and thus the efficiency of the immune
response (5, 6). Impairment of i20S formation has been observed
as consequences of oncogenesis (21–23) and virus-induced immune
evasion strategie s (24).
The availability of peptides for loading of MHC class I molecule s
limits the assembly of MHC I complexe s in the endoplasmic
reticulum (25). Moreover, priming of CD8
T cells upon IFN-
treatment or infection is a surprisingly rapid process and was shown
to be a transient response (26). Thus, all known data indicate that
the timely formation and availability of i20S are decisive steps in the
rapid adaptation of the antigen processing machinery to the im-
munological requirements of a challenged organism. The molecular
mechanisms of this proteasomal immune adaptation, however,
remain purely defined.
The present studie s were therefore undertaken to investigate the
molecular basis and kinetics of i20S biogenesis and to analyze the
role of POMP,
5iLMP7, and proteasome turnover in this process.
Our experiments demonstrate that both POMP and
5iLMP7 are
essential for the accelerated up-regulation of i20S. In combination
with the observed drastically reduced half-life of i20S, our exper-
iments explain that proteasomal immune adaptation is designed to
be a highly dynamic and transient response that permits the rapid
return to the constitutive situation once i20S function is no longer
required.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: POMP, proteasome maturation protein; LMPn, low molecular weight pro-
tein n; siRNA, short interfering RNA.
See Commentary on page 9089.
*Present address: Institute of Immunology, Friedrich-Schiller University, 07740 Jena,
Germany.
To whom correspondence should be addressed. E-mail: elke.krueger@charite.de.
© 2005 by The National Academy of Sciences of the USA
www.pnas.orgcgidoi10.1073pnas.0501711102 PNAS
June 28, 2005
vol. 102
no. 26
9241–9246
IMMUNOLOGY SEE COMMENTARY
Page 1
Materials and Methods
Cell Culture and Transfection. Human cell lines were cultivated under
standard conditions in RPMI medium 1640 (DLD-1, HeLa, and
T2) or Basal Iscove’s medium (SW-480), each containing 10% FCS,
2mM
L-glutamine, 100 unitsml penicillin, and 100
gml strep-
tomycin. Stable transfectants of T2 cells have been described (11,
27). For induction of i20S, cells were incubated with 150 unitsml
human IFN-
up to 24 h.
Northern and Western Blotting. For Northern blots, 5
goftotal
RNA per lane were processed and hybridized with digoxygenin-
labeled riboprobes of LMP7
5i or POMP, as described (28).
Equal RNA loading and RNA quality were monitored by ethidium
bromide-stained 28S rRNA.
Equal amounts of protein extracts were separated on SDS-
Laemmli gels, transferred by electroblotting onto poly(vinylidene
difluoride) membranes, and immunodetected for 20S proteasome s
(MP3), LMP7, LMP2,
5 (all laboratory stock), POMP (17), or
GAPDH (Santa Cruz Biotechnology), as indicated. The protein
amount in each lane was equalized by amidoblack staining before
immunodetection and served as a loading control.
Cloning Procedures. DNA manipulations and transformation of
Escherichia coli were performed according to standard protocols
(29). All plasmids were verified by sequencing.
Cloning of both untagged
5 subunits in pIVEX2.3MCS plas-
mid (30) and expression of His-6-POMP have been described (17).
For expression of untagged POMP, the cDNA was subcloned into
pRSETA by NdeIBamHI. The plasmid encoding the Archaeoglo-
bus fulgidus
subunit was kindly provided (31). His-6-tagged
subunit full-length and propeptide constructs were generated by
PCR amplification of the inserts and subcloning in the pIVEX2.4a
plasmid (Roche Molecular Biochemicals) by NotIXhoI. The chi-
meric
subunits were constructed by PCR amplification of the
coding sequence of the A. fulgidus proteasome
subunit and the
fragments encoding the propeptide of the human
5or
5i subunit.
Both fragments were subcloned in pIVEX2.4a. For yeast two-
hybrid studies, the cDNAs of POMP and both
5 subunits were
subcloned into pAS2–1 for binding domain fusion and into pACT2
for activation domain fusion (BD Bioscience s).
Gene Silencing. For silencing of POMP expression with siRNAs, we
used the RNA–oligonucleotide duplex technique (32). Two du-
plexes were designed (1.1 GGACAGUAUUCCAGUUACUd-
TdT; 1.2 GGACAGUAUUCCAGUUACUdTdT) and used in
combination in 100 nM concentration. HeLa cells were transfected
with duplexes following the JetSI-ENDO protocol (Eurogentech,
Brussels). A universal control oligonucleotide duplex (Eurogen-
tech) or mock-transfected cells served as controls. Four hours after
transfection, cells were stimulated either immediately with IFN-
for 24 h or after 24 h, as indicated. Immunoprecipitation was
performed with a monoclonal
2 antibody and immunostained for
5, LMP7, and 20S proteasomes. Chymotryptic activity of the
proteasome was asse ssed by using the peptide substrate Suc-Leu-
Leu-Val-Tyr-aminomethylcoumarin. The onset of apoptosis was
measured by assaying caspase 3 and 7 activity (Apo-ONE Homo-
geneous Caspase-37 assay, Promega). MHC class I surface ex-
pression was determined by FACSCalibur flow cytometry (BD
Biosciences) by using anti-class I antibody (One Lambda, Los
Angele s) staining after 16-h IFN-
stimulation (23).
Protein Interaction. Yeast two-hybrid interaction trap experiments
were performed by using the Saccharomyces cerevisiae strain HF7c
with the auxotrophic markers leu2–3, trp1–901, his3–200, and the
interaction reporters HIS3 and lacZ. Protein interactions were
assessed by His-prototrophy and by a colony-lift filter assay for
-galactosidase expression (MATCHMAKER Yeast Two-Hybrid
assay, BD Biosciences).
For in vitro interaction, His-tagged
subunits and POMP or
untagged
subunits and His-tagged POMP were coexpressed in
the presence of TRAN
35
S-Label (ICN) in reticulocyte lysate (Pro-
mega). Pull-down assays were done under stringent washing con-
ditions (0.5% Tween 20500 mM NaCl) by using magnetic nickel
beads (Qiagen, Valencia, CA). For interaction of His-tagged
subunits with POMP, the interacting proteins were cleaved off the
beads by factor Xa proteolysis (Roche Molecular Biochemicals).
Interacting
subunits of the His-6-POMP pull-down were eluted by
300 mM imidazole.
Metabolic [
35
S] Labeling and Immunoprecipitation. For standard
pulsechase experiments, cells were cultured for 24 h with or
without IFN-
, pulsed with TRAN
35
S-Label (ICN) for 1 h, washed
3-fold, and chased for the times indicated. To determine the
stability of c20S or i20S, T2 or T2 LMP2 7 cells were labeled for
8 h, washed intensively, and grown for 24 h in the presence or
absence of IFN-
in cold medium and further chased up to 4 days
in the absence of cytokines. Radioactivity was determined by liquid
scintillation counting. Equal counts were supplied to immunopre-
cipitation and processed as described (17). The following polyclonal
antisera were used: POMP (17),
5 and LMP7 (all laboratory
stock), or anti-C8, specifically recognizing precursor complexes
(10). The radioactive protein pattern was detected by phosphoim-
aging (Fuji FLA3000) and evaluated by
AIDA software (Raytest,
Straubenhardt, Germany). The turnover of single proteasome
precursor proteins or complete proteasome complexes was calcu-
lated by adjusting the evaluated pixel densities to the background
and integrating to obtain intensity values per area. Logarithmizing
the intensities and bringing them into a function of chase time
resulted in an approximated linear function. The slope of the line
of the calculated linear equation served as a value for protein
turnover, and their half-life values were estimated. Alterations of
turnover rates were calculated from ratios of the slopes. For
proteasome stability, intensity values per area were calculated, and
time-point zero was set to 100%.
Results
POMP Is Up-Regulated by IFN-
, and Its Levels Reciprocally Correlate
with the Presence of Immunosubunits. To study the role of POMP in
i20S formation, POMP expression was analyzed with respect to its
induction by IFN-
. In all cell lines tested, POMP mRNA levels
increased significantly after stimulation with the cytokine (Fig. 1a).
However, despite increased POMP mRNA levels in HeLa, DLD-1
and SW-480 cells immunoblotting revealed no increase in the
amount of POMP (Fig. 1a). In fact, when analyzed under steady-
state conditions, cytokine treatment even resulted in a decrease of
cellular POMP levels (Fig. 1a). In contrast, analysis of the human
lymphoblastoma cell line T2, which lack the immunosubunits
LMP2 and LMP7 (33, 34), gave opposite results and revealed an
up-regulation of POMP upon IFN-
induction (Fig. 1a).
To resolve this apparent contradiction, POMP biosynthesis was
analyzed by pulse labeling and immunoprecipitation before and
after IFN-
stimulation of HeLa cells. In contrast to the steady-
state situation reflected by immunoblotting on total cell extracts, we
now detected a significant up-regulation of POMP also in HeLa
cells (Fig. 1a). We therefore hypothesized that the POMP induction
observed by immunoblotting in T2 cells may be due to an abolished
or reduced expression of immunosubunits. To test this, HeLa,
DLD-1, and SW-480 cells were analyzed with regard to their
expre ssion of LMP2 and LMP7. HeLa, DLD-1, and SW-480 cells
revealed normal IFN-
induction of the two immunosubunits (Fig.
1b). As expected, no expression of LMP2 and LMP7 was detectable
in T2 cells. These experiments therefore showed that POMP levels
reciprocally correlate with the presence of the immunosubunits and
led us to hypothe size that INF-
treatment of HeLa cells and the
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Page 2
concomitant up-regulation of immunosubunits result in an en-
hanced turnover of POMP.
Immunoproteasome Formation Is Accelerated and Independent of
IFN-
Signaling. The above data led us to investigate whether the
reciprocal relationship between POMP turnover and up-regulation
of immunosubunits also influences the dynamics of i20S formation.
We therefore compared the maturation kinetics of c20S with that
of i20S. Because degradation of POMP signals completion of
proteasome maturation (17, 19, 20), the turnover of POMP and the
processing of
5 proproteins served as markers for maturation
progress. Metabolically labeled precursor complexes from un-
treated and IFN-
-treated HeLa cells were specifically immuno-
precipitated (10). Comparison of the POMP half-life in untreated
vs. IFN-
-stimulated cells revealed an 4-fold acceleration of
POMP turnover upon IFN-
exposure (Fig. 2a). This accelerated
degradation of POMP was accompanied by a faster turnover of i20S
precursor complexes (Fig. 2a). Calculation of the turnover rates of
POMP revealed a mean half-life of 82 min in untreated vs. 21 min
in IFN-
-treated HeLa cells. Importantly, comparison of the turn-
over of proteasome precursors in T2 LMP2 7 and T2 cells
revealed that the rapid turnover of i20S precursors is independent
of any cytokine signal and considerably faster than that of precur-
sors of constitutive proteasomes (Fig. 2a). The difference in mat-
uration kinetics was also reflected at the level of individual
5
subunit proce ssing. Comparison of the maturation kinetics of the
constitutive subunit
5 and the immunosubunit
5i also revealed an
accelerated maturation of
5iLMP7 (Fig. 2b).
Thus, taking the turnover kinetics of POMP and POMP-
containing precursor complexes and the processing of both
5
individual subunits as indicative for completion of proteasome
assembly and activation, we conclude that the generation of i20S
occurs 4-fold faster than that of c20S and is independent of other
IFN-
-induced proteins.
Immunoproteasome Formation Is a Transient Response. The accel-
erated de novo formation of i20S raised questions regarding the
influence of INF-
on the fate of c20S as well as i20S. To study this,
we used human T2 cells expressing c20S and T2 LMP2 7 cells
expre ssing i20S independent of INF-
stimulation and pulsed the
cells in the absence of cytokines to label proteasomes. Subse-
quently, the cells were either exposed for 24 h to IFN-
or not and
chased in the absence of IFN-
. For a period of 5 days, no effect of
IFN-
on T2 or T2 LMP2 7 cell viability was observed on the basis
of a proliferation assay (not shown).
Fig. 1. Increased POMP levels after IFN-
stimulation correlate with the
absence of immunosubunits. (a) POMP mRNA levels are increased by 24-h
IFN-
stimulation () in different human cell lines analyzed by Northern
blotting. Ethidium bromide-stained 28S rRNA bands are shown as an internal
control (Top). Cellular POMP levels did not reflect mRNA levels, as shown by
Western blot analysis of total lysates by using a POMP-specific antibody
(Middle). Induction of POMP synthesis in response to IFN-
as visualized by
immunprecipitation of radio-labeled POMP from protein extracts of pulsed
HeLa cells (Bottom). (b) Expression of immunosubunits reciprocally correlates
with the cellular amount of POMP. Western blot analysis of the proforms (p)
and the matured forms (m) of LMP2 (Upper) and LMP7 (Lower) in total cell
lysates of different human cell lines in the presence () or absence () of IFN-
.
Fig. 2. i20S formation is accelerated and indepen-
dent of IFN-
signaling. (a) Turnover of precursor com-
plexes and incorporated POMP is faster in the presence
() than in the absence () of IFN-
in metabolically
labeled HeLa cells (Left). Turnover of i20S precursor
complexes of labeled T2 LMP2 7 cells is accelerated
in comparison to constitutive precursors of T2 inde-
pendent of the cytokine signal (Right). Precursor com-
plexes were specifically immunoprecipitated from to-
tal cell lysates at the different chase times indicated.
Quantification of autoradiograms and calculated half-
lives are shown (Lower). (b) For visualization of faster
individual LMP7 subunit maturation compared with
5, subunits were immunoprecipitated from radiola-
beled HeLa cell lysates ( IFN-
) at different chase
times. The quantifications of POMP, proteasome pre-
cursors, or
5 proprotein turnover by phosphoimag-
ing represent the mean of at least two independent
experiments.
Heink et al. PNAS
June 28, 2005
vol. 102
no. 26
9243
IMMUNOLOGY SEE COMMENTARY
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In agreement with the reported half-life of 5 days of c20S (35),
IFN-
showed no ef fect on the turnover of c20S (mean half-life
133 h) or of i20S (Fig. 3). However, with a calculated mean
half-life of 27 h, i20S displayed a much shorter half-life than c20S
and thus were, independent of IFN-
, strik ingly less stable than
c20S (Fig. 3). Thus, the up-regulation of i20S in response to
IFN-
is a transient response.
Rapid Turnover of POMP Requires the Active
5iLMP7 Subunit. The
presence or absence of immunosubunits seemed to affect the
stability of POMP. Therefore, we analyzed whether POMP levels
were affected in T2 cells stably expressing either LMP2 or LMP7
(Fig. 4a). Nontransfected T2 cells exhibited high levels of POMP.
However, as soon as wild-type LMP7 was expressed, the amount of
POMP dramatically decreased (T2 LMP7 and T2 LMP2 7). In
contrast, expression of LMP2 alone had no effect on the amount of
POMP (T2 LMP2; Fig. 4a). Independent of the type of immuno-
subunit expressed, POMP mRNA levels were not changed in the
different transfected cell lines (not shown). To examine whether
POMP de stabilization require s the pre sence of an active LMP7
subunit or whether the LMP7 structure itself is sufficient to signal
POMP degradation, we used T2 cells expre ssing mutated LMP7
derivatives. These mutated and proteolytically inactive forms of
LMP7 were previously reported to be incorporated into the nascent
proteasome complex (17). Inactivation of LMP7 by deletion of the
prosequence (T2 proLMP7) or by substitution of the active-site
threonine to alanine (T2 LMP7T1A) led to POMP amounts similar
to that observed in untransfected T2 cells (Fig. 4b). Thus, the
amount of POMP depends on the efficient maturation and activity
of LMP7.
The interdependence of POMP stability and LMP7 was further
investigated by immunoprecipitation experiments with protein ex-
tracts of metabolically labeled T2 and T2 LMP2 7 cells (Fig. 4c).
During a 2-hr chase, the amount of precipitated POMP in T2 cells
was only moderately reduced, whereas the amount of POMP in T2
LMP2 7 cells dramatically decreased after 30 min (Fig. 4c).
Together, these experiments demonstrate that the varying stability
Fig. 5. POMP interacts directly with LMP7. (a) Yeast two-hybrid assay for
interaction of POMP with the proforms of
5(Left) or LMP7 (Right). Interaction
is shown by selection for His-prototrophy and
-galactosidase expression as
detected by using activation-domain POMP fusion (AD-POMP) and the binding
domain fused to the
5 subunit (BD-
5 or BD-LMP7) or vice versa. (b) Schematic
representation of untagged
subunits and His-6-tagged POMP (His-tag trian-
gle). Both proforms of the human
5 subunits (1,
5; or 2, LMP7) bind to
His-6-POMP, whereas the A. fulgidus
subunit did not bind (3, AF
; specificity
control). (c) The N-terminal His-6-fusions of human
5 subunits or their chimeras
and the factor Xa site are schematically illustrated. (Upper) Input controls (12%
input). (Lower) His-tagged proforms (1 and 4),
5 subunits without propeptides
(2 and 5), as well as chimeric
subunits containing a human propeptide and the
subunit of A. fulgidus (3,
5-pro AF
; 6, LMP7-pro AF
) pulled down untagged
POMP. Untagged POMP did not bind to the nickel beads (7, negative control)
Fig. 3. i20S are less stable than c20S independent of IFN-
. c20S (T2 cells) or
i20S (T2 LMP2 7 cells) were specifically immunoprecipitated at different time
points during the chase period in the absence (Upper) or presence of 24-h
IFN-
(Lower). The diagram shows percent pixel density of time-point zero
with trend lines. The calculated half-lives of 133 h for c20S and 27 h for i20S are
the mean of five independent experiments. A representative experiment is
shown.
Fig. 4. Rapid turnover of POMP requires the presence of active LMP7. (a) The
presence of LMP7 affected POMP stability as analyzed by Western blots of
POMP, LMP2, and LMP7 in cell lysates of different T2 cell lines, stably express-
ing LMP7 andor LMP2. The proforms (pi, p) and the matured forms (m) of the
subunits LMP7 and LMP2 are indicated. (b) POMP stability is restored by
expression of inactive variants of LMP7. Western blot analyses of POMP and
LMP7 in cell lysates of T2 cell lines stably expressing LMP7 without the
propeptide (pLMP7) and the active-site mutation LMP7T1A. (c) POMP turn-
over in T2 is lower than in T2 LMP2 7 cells independent of the IFN-
signal.
Cells were metabolically labeled, and POMP was immunoprecipitated from
total cell lysates at the different chase times indicated.
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Page 4
of POMP is essentially controlled by the activity of the
5i subunit
LMP7 and is independent of other cytokine-induced proteins.
POMP Directly Interacts with the
5i Subunit LMP7. Supported by
several lines of evidence, it was previously discussed that POMP-
like factors interact with the constitutive
5 subunit (20, 36, 37).
Based on our results, we te sted whether POMP can also bind to the
5i subunit. This appeared to be of particular importance, because
it had been reported that POMP and
5i did not interact (37). Our
yeast two-hybrid screens, however, demonstrated that POMP in-
teracts not only with the constitutive
5 subunit but also with LMP7
(Fig. 5a). To verify this interaction, we performed pull-down assays
by using either His-tagged POMP (His-6-POMP) and untagged
5
and
5i subunits or His-tagged
subunits and untagged POMP
(Fig. 5 b and c). Indeed, His-6-POMP was found to interact with
both untagged proforms of the
5 and
5i subunits (Fig. 5b, lanes
1 and 2). Reversely, the proforms of both
5 subtype s pulled down
untagged POMP (Fig. 5c, lanes 1 and 4). In contrast, the wild-type
form of the A. fulgidus
subunit, serving as control, did not interact
with His-6-POMP, indicating the specificity of the observed inter-
action of POMP with the human
5 subunits (Fig. 5b, lane 3). To
reveal a potential interaction of POMP with the subunit propep-
tides, we tested constructs encoding
5 and
5i without propeptides
as well as chimeric
subunit constructs expre ssing either the
5or
the
5i propeptide fused to the archaebacterial
subunit of A.
fulgidus. These experiments (Fig. 5c, lanes 3 and 6) showed that both
chimeric
5 subunits interacted with POMP, and that the binding
of the chimeric
subunits to POMP was mediated by the propep-
tides of the
5 and
5i subunits, respectively. Strikingly, in all
experimental subsets, the interaction of POMP with the proform of
LMP7 appeared to be stronger than that with the
5 proform,
indicating a higher affinity of POMP to LMP7. Surprisingly, even
those forms of
5 and
5i that lack the propeptide s interacted with
POMP (Fig. 5c, lanes 2 and 5), sugge sting that there exist at least
two interaction sites for POMP, one within the propeptide and
another within the sequence of the matured
5 subunits.
POMP Expression Is Essential for Proteasome Formation. So far, our
experiments revealed an interaction between POMP and LMP7
and a LMP7-mediated rapid degradation of POMP during i20S
formation. To analyze whether, in a reverse relationship, incorpo-
ration and maturation of the LMP7 subunit depend on POMP, the
expre ssion of POMP in HeLa cells was silenced by using short
interfering RNAs (siRNAs). The siRNA targeting POMP mRNA
led to silencing of POMP expre ssion in the absence or presence of
IFN-
, compared with mock-transfected cells (Fig. 6a). As checked
by RT-PCR, mRNA expression of LMP7 and actin was affected
neither by transfection of POMPsi nor by an unspecific control
siRNA (not shown). Silencing of POMP expression abolished
incorporation of
5 into c20S complexes as well as severely im-
paired incorporation of the
5i subunit LMP7 into i20S complexes
(Fig. 6a). As a result, the absence of POMP caused a strongly
decreased total amount of 20S complexes (Fig. 6a).
Thus, knockdown of POMP led to a considerable reduction of
the proteasomal hydrolyzing activity and thereby to an accumula-
tion of ubiquitin conjugates. In fact, after 24 h, proteolytic activity
was reduced to 60% of the respective controls with or without
IFN-
(not shown). A prolonged depletion of POMP for 48 h
resulted in a dramatic decrease of the proteasomal hydrolyzing
activity to 40% of the untransfected control (not shown). Con-
sequently, we found that the cells induced apoptosis under pro-
longed silencing of POMP (Fig. 6b). Moreover, MHC class I surface
expre ssion was reduced by 50% in POMP-depleted cells after
IFN-
(Fig. 6c). Therefore, these experiments reveal not only that
POMP is required for both the incorporation of the LMP7 subunit
and its timely maturation but also that it is essential for proteasome
formation in general and consequently for cell survival.
Discussion
Our study demonstrates that immune adaptation of the proteasome
system caused by infection and concomitant IFN-
production (38,
39) is an extremely rapid and transient response. The dynamics of
this process is controlled by the molecular interplay between the
LMP7 subunit and POMP and the consequent accelerated forma-
tion of i20S.
Challenging the postulated importance of POMP in i20S bio-
genesis, we observed that, despite an up-regulation of POMP
mRNA in IFN-
-stimulated cells (17, 19), POMP levels were
reduced even under steady-state conditions. Up-regulation of
POMP was detected only after a short radioactive pulse or in the
absence of a fully processed functional LMP7 subunit. Our data
therefore extend the present knowledge by revealing a strong
functional interdependence of POMP and the
5i subunit LMP7 in
i20S biogenesis. In fact, POMP stabilization in the pre sence of an
inactive LPM7 subunit demonstrates that LMP7 e ssentially drives
the rapid degradation of POMP.
The accelerated degradation of POMP is directly connected with
the faster maturation of LMP7 (compared with
5) and, in con-
sequence, with the 4-fold faster formation of i20S. This rapid
maturation of
5i may also reflect the preferential incorporation of
Fig. 6. Silencing of POMP gene expression by RNA interference results in a decrease of proteasomes. (a) Transfection of siRNA targeting POMP (POMPsi) silenced
POMP expression in the presence or absence of IFN-
() but not mock transfection of cells (control). GAPDH levels served as loading control (Upper). Silencing
of POMP led to a strong reduction of incorporated
5 and
5iLMP7 and immunoprecipitated 20S proteasome complexes (IP of 20S; Lower). GAPDH levels
represent 15% input. (b) Prolonged knockdown of POMP (POMPsi) up to 48 h caused the induction of apoptosis as measured by caspase 37 activity in the
presence (IFNg) or absence (co) of IFN-
.(c) POMP depletion caused a decrease in MHC class I surface expression as measured by HLA class I fluorescence staining
of mock (black line) or POMPsi RNA-transfected cells (gray line) after IFN-
stimulation. Background staining with the second antibody only (2nd ab; IgG1) is shown
(Left). Mean fluorescence levels are indicated as geometric (Geo) mean.
Heink et al. PNAS
June 28, 2005
vol. 102
no. 26
9245
IMMUNOLOGY SEE COMMENTARY
Page 5
LMP7 into nascent proteasomes (40). Thus, the kinetics of protea-
some biogene sis seems to be differentially determined by the two
5 subunits and their molecular interplay with POMP, allowing a
rapid switch from c20S to i20S function.
Importantly, the rapid degradation of POMP and the strongly
accelerated maturation of i20S do not, as might have been expected,
require any additional IFN-
-induced factors. Consequently, the
dynamics of i20S formation appears to be e ssentially self-controlled
and the result of a functional interdependence of LMP7 and POMP.
Moreover, the up-regulation of i20S is a transient response because,
independent of IFN-
signaling, i20S exhibit a much shorter
half-life than c20S. Thus, both i20S-specific characteristics may be
due to intrinsic properties of the enzyme complex. As shown by
immunological experiments (27), the incorporation of LMP7 can
affect the structural properties of the proteasome. This also be-
come s apparent by the altered chromatographic properties of
immunoproteasomes (41).
The key position of POMP and LMP7 in the accelerated
formation of i20S is supported by their direct interaction. In
contrast with our experiments, an interaction of POMP with the
5
subunit but not with LMP7 was recently reported based on a yeast
two-hybrid interaction screen (37). This negative result was inter-
preted to reflect the observed biased incorporation of
5 and LMP7
into the re spective proteasome complexes due to their different
prosequences (40). As shown here, interaction of POMP with both
mammalian
5 homologues is independent of the prosequences,
suggesting a second interaction site within domains of the matured
subunits. This is in agreement with our previous observation that
the
5i prosequence is not absolutely essential for its incorporation
into 20S proteasomes, and that POMP incorporation into precursor
complexe s is independent of the
5i prosequence (17). Although
the
5i prosequence is not essential for
5i subunit incorporation,
the presence of the correct prosequence strongly supports the
subunit’s incorporation efficiency (17).
In the presence of sufficient amounts of POMP, the availability
of LMP7 seems to be the rate-limiting factor of this process and vice
versa. Our POMP knockdown experiments demonstrate that
POMP determines the recruitment of the LMP7 subunit (and that
of
5) into the proteasome complex. In contrast to the role of the
yeast Ump1p homologue (20), POMP function turns out to be
essential for proteasome biogenesis and consequently also for
mammalian cell viability. As an immunological consequence, the
limited generation of antigenic peptide s also is reflected by the
reduction of MHC class I surface expression in POMP-depleted
cells (25). These data therefore demonstrate that POMP possesse s
an essential coordinative function in proteasome assembly that is
independent of the different prosequences and not directly corre-
lated with the differential incorporation of the two
5 subunits.
IFN-
-induced amounts of POMP therefore will permit an efficient
recruitment of the LMP7 subunit, which concomitantly facilitates
the accelerated formation of i20S.
The effectiveness of the MHC class I immune response of an
attacked organism is largely determined by the rapid and coordi-
nated generation of antigenic peptides and their presentation on the
cell surface. In conclusion, the accelerated formation of i20S forced
by the interplay between POMP and LMP7 meets the demands of
an efficient and rapid answer to an immunological challenge.
Subsequently, the observed strongly reduced half-life of i20S per-
mits cells to return more rapidly to a normal situation once i20S
functions is no longer needed, also supporting the finding of a
transient nature of CD8
T cell priming (26). Thus, our model is in
good agreement with the current knowledge of MHC class I antigen
presentation, following the hypothe sis that most antigenic peptide s
derived from defective ribosomal products, allowing cells to cope
immediately with rapidly replicating viruses (42). Therefore, we
present an immune-adaptation mechanism by the proteasome
system, which is essentially self-controlled and rapid enough to
contribute to an efficient immune response.
We are grateful to J. Monaco (University of Cincinnati, College of
Medicine, Cincinnati) for kindly providing the C8 antiserum, to C. Beier
for excellent technical assist ance, and to the members of the Kloetzel
laboratory for support and helpful discussions and for critically reading
the manuscript. This study was supported by the Deutsche Forschungs-
gemeinschaft (Kl 4278-5SFB 421).
1. Kloetzel, P. M. (2001) Nat . Rev. Mol. Cell. Biol. 2, 179–187.
2. Rock, K. L., York, I. A., Saric, T. & Goldberg, A. L. (2002) Adv. Immunol. 80, 1–70.
3. Groll, M., Ditzel, L., L owe, J., Stock, D., Bochtler, M., Bartunik, H. D. & Huber, R.
(1997) Nature 386, 463–471.
4. Aki, M., Shimbara, N., Takashina, M., Akiyama, K., Kagawa, S., Tamura, T.,
Tanahashi, N., Yoshimura, T., Tanaka, K. & Ichihara, A. (1994) J. Biochem. (Tok yo)
115, 257–269.
5. Chen, W., Norbury, C. C., Cho, Y., Yewdell, J. W. & Bennink, J. R. (2001) J. Exp.
Med. 193, 1319–1326.
6. Toes, R. E., Nussbaum, A. K., Deger mann, S., Schirle, M., Emmerich, N. P., Kraft,
M., Laplace, C., Zw inderman, A., Dick, T. P., Muller, J., et al . (2001) J. Exp. Med.
194, 1–12.
7. Kloetzel, P. M. (2004) Nat . Immunol. 5, 661–669.
8. Frentzel, S., Pesold-Hurt, B., Seelig, A. & Kloetzel, P. M. (1994) J. Mol. Biol . 236,
975–981.
9. Groettrup, M., Standera, S., Stohwasser, R. & Kloetzel, P. M. (1997) Proc. Natl. Acad.
Sci. USA 94, 89708975.
10. Nandi, D., Woodward, E., Ginsburg, D. B. & Monaco, J. J. (1997) EMBO J. 16,
5363–5375.
11. Schmidt, M., Zantopf, D., Kraft, R., Kostka, S., Preissner, R. & Kloetzel, P. M. (1999)
J. Mol. Biol. 288, 117–128.
12. Schmidtke, G., Kraft, R., Kostka, S., Henklein, P., Frommel, C., L owe, J., Huber, R.,
Kloetzel, P. M. & Schmidt, M. (1996) EMBO J. 15, 6887–6898.
13. Schmidtke, G., Schmidt, M. & Kloetzel, P. M. (1997) J. Mol. Biol . 268, 95–106.
14. Chen, P. & Hochstrasser, M. (1996) Cell 86, 961–972.
15. Heinemeyer, W., Fischer, M., Krimmer, T., St achon, U. & Wolf, D. H. (1997) J. Biol.
Chem. 272, 25200–25209.
16. Jager, S., Groll, M., Huber, R., Wolf, D. H. & Heinemeyer, W. (1999) J. Mol. Biol.
291, 997–1013.
17. Witt, E., Zantopf, D., Schmidt, M., Kraft, R., Kloetzel, P. M. & Kru¨ger, E. (2000)
J. Mol. Biol. 301, 1–9.
18. Griffin, T. A., Slack, J. P., McCluskey, T. S., Monaco, J. J. & Colbert, R. A. (2000)
Mol. Cell. Biol. Res. Commun. 3, 212–217.
19. Burri, L., Hockendorff, J., Boehm, U., Klamp, T., Dohmen, R. J. & Levy, F. (2000)
Proc. Natl. Acad. Sci. USA 97, 10348–10353.
20. Ramos, P. C., Hockendorff, J., Johnson, E. S., Varshavsky, A. & Dohmen, R. J. (1998)
Cell 92, 489499.
21. Cerundolo, V., Kelly, A., Elliott, T., Trowsdale, J. & Townsend, A. (1995) Eur.
J. Immunol. 25, 554–562.
22. Seliger, B., Wollscheid, U., Momburg, F., Blankenstein, T. & Huber, C. (2000) Tissue
Antigens 56, 327–336.
23. Sun, Y., Sijts, A. J., Song, M., Janek, K., Nussbaum, A. K., Kral, S., Schirle, M.,
Stevanov ic, S., Paschen, A., Schild, H., Kloetzel, P. M. & Schadendorf, D. (2002)
Cancer Res. 62, 2875–2882.
24. Ehrlich, R. (1997) Hum. Immunol. 54, 104–116.
25. Benham, A. & Neefjes, J. (1997) J. Immunol. 159, 5896–5904.
26. Wong, P. & Pamer, E. (2003) Immunity 18, 499–511.
27. Sijts, A. J., Ruppert, T., Rehermann, B., Schmidt, M., Koszinowski, U. & Kloetzel,
P. M. (2000) J. Exp. Med. 191, 503–514.
28. Kru¨ger, E., Msadek, T. & Hecker, M. (1996) Mol. Microbiol. 20, 713–723.
29. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular Cloning: A Laboratory
Manual (Cold Spring Harbor Lab. Press, Woodbury, NY).
30. Apcher, G. S., Heink, S., Zantopf, D., Kloetzel, P. M., Schmid, H. P., Mayer, R. J.
&Kru¨ger, E. (2003) FEBS Lett . 553, 200–204.
31. Groll, M., Brandstetter, H., Bartunik, H., Bourenkow, G. & Huber, R. (2003) J. Mol.
Biol. 327, 75–83.
32. Elbashir S. M., H. J., Lendeckel W., Yalcin A., Weber K., Tuschl T. (2001) Nature
411, 494498.
33. Salter, R. D. & Cresswell, P. (1986) EMBO J. 5, 943–949.
34. Ortiz-Navarrete, V., Seelig, A., Gernold, M., Frentzel, S., Kloetzel, P. M. &
Hammerling, G. J. (1991) Nature 353, 662–664.
35. Hendil, K. B. (1988) Biochem. Int. 17, 471–477.
36. Cagney, G., Uetz, P. & Fields, S. (2001) Physiol. Genom. 7, 27–34.
37. Jayarapu, K. & Griffin, T. A. (2004) Biochem. Biophys. Res. Commun. 314, 523–528.
38. Khan, S., van den Broek, M., Schwarz, K., de Giuli, R., Diener, P. A. & Groettrup,
M. (2001) J. Immunol. 167, 68596868.
39. Kuckelkorn, U., Ferreira, E. A., Drung, I., Liewer, U., Kloetzel, P. M. & Theobald,
M. (2002) Eur. J. Immunol. 32, 1368–1375.
40. Kingsbury, D. J., Grif fin, T. A. & Colbert, R. A. (2000) J. Biol. Chem. 275,
24156–24162.
41. Dahlmann, B., Ruppert, T., Kuehn, L., Merforth, S. & Kloetzel, P. M. (2000) J. Mol.
Biol. 303, 643–653.
42. Yewdell, J.W., Reits, E. & Neefjes, J. (2003) Nat. Rev. Immunol. 3, 952–961.
9246
www.pnas.orgcgidoi10.1073pnas.0501711102 Heink et al.
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  • Source
    • "We stably transfected U2OS cells to express POMP with its wild-type 3 0 UTR or with a mutant 3 0 UTR that contains four base exchanges at each of the two miR-101-binding sites to eliminate base pairing with the miR- 101 seed sequence (Figure S3). The overexpression of POMP only resulted in enhanced mRNA levels, but not protein levels (Figures 3D and 3E), due to previously described feedback regulation of POMP (Heink et al., 2005). When transfected with miR- 101, the cells containing the wild-type construct showed severely reduced POMP levels and increased amounts of ubiquitinated proteins; in contrast, the mutation in the 3 0 UTR was enough to abolish both effects completely (Figure 3F). "
    [Show abstract] [Hide abstract] ABSTRACT: Proteasome inhibition represents a promising strategy of cancer pharmacotherapy, but resistant tumor cells often emerge. Here we show that the microRNA-101 (miR-101) targets the proteasome maturation protein POMP, leading to impaired proteasome assembly and activity, and resulting in accumulation of p53 and cyclin-dependent kinase inhibitors, cell cycle arrest, and apoptosis. miR-101-resistant POMP restores proper turnover of proteasome substrates and re-enables tumor cell growth. In ERα-positive breast cancers, miR-101 and POMP levels are inversely correlated, and high miR-101 expression or low POMP expression associates with prolonged survival. Mechanistically, miR-101 expression or POMP knockdown attenuated estrogen-driven transcription. Finally, suppressing POMP is sufficient to overcome tumor cell resistance to the proteasome inhibitor bortezomib. Taken together, proteasome activity can not only be manipulated through drugs, but is also subject to endogenous regulation through miR-101, which targets proteasome biogenesis to control overall protein turnover and tumor cell proliferation. Copyright © 2015 Elsevier Inc. All rights reserved.
    Full-text · Article · Jul 2015 · Molecular cell
  • Source
    • "The subunit replacements and the association of the 11S regulator to at least one end of the 20S core alter the cleavage pattern of the proteasome, optimizing the generation of small peptides for loading on the groove of MHC class I molecules [25]–[27]. These changes are also related to increase the production of immunogenic peptides compared to standard proteasome [28], [29]. "
    [Show abstract] [Hide abstract] ABSTRACT: Generally, Trypanosoma cruzi infection in human is persistent and tends to chronicity, suggesting that the parasite evade the immune surveillance by down regulating the intracellular antigen processing routes. Within the MHC class I pathway, the majority of antigenic peptides are generated by the proteasome. However, upon IFN-γ stimulation, the catalytic constitutive subunits of the proteasome are replaced by the subunits β1i/LMP2, β2i/MECL-1 and β5i/LMP7 to form the immunoproteasome. In this scenario, we analyzed whether the expression and activity of the constitutive and the immunoproteasome as well as the expression of other components of the MHC class I pathway are altered during the infection of HeLa cells with T. cruzi. By RT-PCR and two-dimensional gel electrophoresis analysis, we showed that the expression and composition of the constitutive proteasome is not affected by the parasite. In contrast, the biosynthesis of the β1i, β2i, β5i immunosubunits, PA28β, TAP1 and the MHC class I molecule as well as the proteasomal proteolytic activities were down-regulated in infected-IFN-γ-treated cell cultures. Taken together, our results provide evidence that the protozoan T. cruzi specifically modulates its infection through an unknown posttranscriptional mechanism that inhibits the expression of the MHC class I pathway components.
    Full-text · Article · Apr 2014 · PLoS ONE
    • "It is not clear whether the upregulation of immunoproteasome levels reflects a compensatory and homeostatic effect after initial downregulation during bortezomib resistance development. Importantly, increased β5i expression can drive incorporation of immunoproteasome subunits into prototypic immunoproteasomes [32] or facilitate assembly in hybrid types of proteasomes (β1 + β2 + β5i and β1i + β2 + β5i) [33]. Conceivably, these hybrid forms could compensate for impaired catalytic activity of constitutive proteasomes assembled with a mutated β5-subunit. "
    [Show abstract] [Hide abstract] ABSTRACT: Despite encouraging results with the proteasome inhibitor bortezomib in the treatment of hematologic malignancies, emergence of resistance can limit its efficacy, hence calling for novel strategies to overcome bortezomib-resistance. We previously showed that bortezomib-resistant human leukemia cell lines expressed significantly lower levels of immunoproteasome at the expense of constitutive proteasomes, which harbored point mutations in exon 2 of the PSMB5 gene encoding the beta5 subunit. Here we investigated whether up-regulation of immunoproteasomes by exposure to interferon-gamma restores sensitivity to bortezomib in myeloma and leukemia cell lines with acquired resistance to bortezomib. RPMI-8226 myeloma, THP1 monocytic/macrophage and CCRF-CEM (T) parental cells and sub lines with acquired resistance to bortezomib were exposed to Interferon-gamma for 24-48h where after the effects on proteasome subunit expression and activity were measured, next to sensitivity measurements to proteasome inhibitors bortezomib, carfilzomib, and the immunoproteasome selective inhibitor ONX 0914. At last, siRNA knockdown experiments of beta5i and beta1i were performed to identify the contribution of these subunits to sensitivity to proteasome inhibition. Statistical significance of the differences were determined using the Mann-Whitney U test. Interferon-gamma exposure markedly increased immunoproteasome subunit mRNA to a significantly higher level in bortezomib-resistant cells (up to 30-fold, 10-fold, and 6-fold, in beta1i, beta5i, and beta2i, respectively) than in parental cells. These increases were paralleled by elevated immunoproteasome protein levels and catalytic activity, as well as HLA class-I. Moreover, interferon-gamma exposure reinforced sensitization of bortezomib-resistant tumor cells to bortezomib and carfilzomib, but most prominently to ONX 0914, as confirmed by cell growth inhibition studies, proteasome inhibitor-induced apoptosis, activation of PARP cleavage and accumulation of polyubiquitinated proteins. This sensitization was abrogated by siRNA silencing of beta5i but not by beta1i silencing, prior to pulse exposure to interferon-gamma. Downregulation of beta5i subunit expression is a major determinant in acquisition of bortezomib-resistance and enhancement of its proteasomal assembly after induction by interferon-gamma facilitates restoration of sensitivity in bortezomib-resistant leukemia cells towards bortezomib and next generation (immuno) proteasome inhibitors.
    Preview · Article · Jan 2014 · Journal of Hematology & Oncology
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