IFN-gamma-induced immune adaptation of the proteasome system is an accelerated and transient response.
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.
- SourceAvailable from: Peter-Michael Kloetzel[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.PLoS ONE 01/2014; 9(4):e95977. · 3.53 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Significance: Atherosclerosis is a vascular disease of worldwide significance with fatal complications such as myocardial infarction, stroke and peripheral artery disease. Atherosclerosis is recognized as a chronic inflammatory disease leading to arterial plaque formation and vessel narrowing in different vascular beds. Besides the strong inflammatory nature of atherosclerosis, it is also characterized by proliferation, apoptosis and enhanced oxidative stress. The ubiquitin-proteasome system (UPS) is the major intracellular degradation system in eukaryotic cells. Besides its essential role in the degradation of dysfunctional and oxidatively damaged proteins, it is involved in many processes which influence disease progression in atherosclerosis. Hence, it is logical to ask whether targeting the proteasome is a reasonable and feasible option for the treatment of atherosclerosis. Recent advances: Several lines of evidence suggest stage-specific dysfunction of the UPS in atherogenesis. Regulation of key processes by the proteasome in atherosclerosis, as well as the modulation of these processes by proteasome inhibitors in vascular cells, is outlined in this review. Treatment of atherosclerotic animal models with proteasome inhibitors yielded partly opposing results; the potentially underlying reasons of which are discussed here. Critical issues and Future directions: Targeting UPS function in atherosclerosis is a promising but challenging option. Limitations of current proteasome inhibitors, dose-dependency and the cell-specificity of effects, as well as the potential of future therapeutics are discussed. Stage-specific in depth exploration of UPS function in atherosclerosis in the future will help identify targets and windows for beneficial intervention.Antioxidants & Redox Signaling 02/2014; · 8.20 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Protein degradation is essential for maintaining cellular homeostasis. The proteasome is the central enzyme responsible for non-lysosomal protein degradation in eukaryotic cells. Although proteasome assembly is not yet completely understood, a number of cofactors required for proper assembly and maturation have been identified. Ump is a short-lived maturation factor required for the efficient biogenesis of the 20S proteasome. Upon the association of the two precursor complexes, Ump is encased and is rapidly degraded after the proteolytic sites in the interior of the nascent proteasome are activated. In order to further understand the mechanisms behind proteasomal maturation, we expressed and purified yeast Ump in E. coli for biophysical and structural analysis. We show that recombinant Ump is purified as a mixture of different oligomeric species and that oligomerization is mediated by intermolecular disulfide bond formation involving the only cysteine residue present in the protein. Furthermore, a combination of bioinformatic, biochemical and structural analysis revealed that Ump shows characteristics of an intrinsically disordered protein, which might become structured only upon interaction with the proteasome subunits.Computational and structural biotechnology journal. 01/2013; 7:e201304006.
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-
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,
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 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
? subunits. The hydrolyzing activities 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 vertebrates, specific catalytically active proteasome subunits,
collectively referred to as immunosubunits (4), have evolved that
improve proteasome-dependent antigen processing (5–7). IFN-?
induces the synthesis 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
Both c20S and i20S are exclusively formed de novo following a
sophisticated and not yet fully understood biogenesis program. We
a multistep process that occurs via the formation of distinct
proteasome precursor complexes with different ? subunit compo-
he proteasome is the key enzyme in the proteolytic cascade
required for the generation of peptides presented to cytotoxic
sitions. Active-site ? subunits are synthesized 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-
the ? subunit propeptides liberates the active-site threonines of the
now fully active 20S core proteasome (8, 10, 12–16).
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 human?mouseUmp1 according to their yeast homologue Ump1
the fully assembled and activated 20S proteasome. Thus, degrada-
tion of Ump1p?POMP 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).
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
evasion strategies (24).
limits the assembly of MHC I complexes in the endoplasmic
reticulum (25). Moreover, priming of CD8?T cells upon IFN-?
to be a transient response (26). Thus, all known data indicate that
rapid adaptation of the antigen processing machinery to the im-
mechanisms of this proteasomal immune adaptation, however,
remain purely defined.
The present studies were therefore undertaken to investigate the
molecular basis and kinetics of i20S biogenesis and to analyze the
Our experiments demonstrate that both POMP and ?5i?LMP7 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
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,
†To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
© 2005 by The National Academy of Sciences of the USA
June 28, 2005 ?
vol. 102 ?
no. 26 ?
Materials and Methods
standard conditions in RPMI medium 1640 (DLD-1, HeLa, and
2 mM L-glutamine, 100 units?ml penicillin, and 100 ?g?ml strep-
tomycin. Stable transfectants of T2 cells have been described (11,
27). For induction of i20S, cells were incubated with 150 units?ml
human IFN-? up to 24 h.
Northern and Western Blotting. For Northern blots, 5 ?g of total
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 proteasomes
(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 pIVEX2.3?MCS 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 NdeI?BamHI. 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 NotI?XhoI. The chi-
meric ? subunits were constructed by PCR amplification of the
coding sequence of the A. fulgidus proteasome ? subunit and the
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 Biosciences).
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 assessed 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-3?7 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
Angeles) 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 TRAN35S-Label (ICN) in reticulocyte lysate (Pro-
mega). Pull-down assays were done under stringent washing con-
ditions (0.5% Tween 20?500 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).
300 mM imidazole.
Metabolic [35S] Labeling and Immunoprecipitation. For standard
pulse–chase experiments, cells were cultured for 24 h with or
without IFN-?, pulsed with TRAN35S-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-
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%.
POMP Is Up-Regulated by IFN-?, and Its Levels Reciprocally Correlate
with the Presence of Immunosubunits. TostudytheroleofPOMPin
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-
now detected a significant up-regulation of POMP also in HeLa
or reduced expression of immunosubunits. To test this, HeLa,
DLD-1, and SW-480 cells were analyzed with regard to their
expression of LMP2 and LMP7. HeLa, DLD-1, and SW-480 cells
revealed normal IFN-? induction of the two immunosubunits (Fig.
in T2 cells. These experiments therefore showed that POMP levels
led us to hypothesize that INF-? treatment of HeLa cells and the
www.pnas.org?cgi?doi?10.1073?pnas.0501711102Heink et al.
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
We therefore compared the maturation kinetics of c20S with that
of i20S. Because degradation of POMP signals completion of
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
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 processing. Comparison of the maturation kinetics of the
accelerated maturation of ?5i?LMP7 (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
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
expressing 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
of a proliferation assay (not shown).
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
Increased POMP levels after IFN-? stimulation correlate with the
(?) 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.
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
i20S formation is accelerated and indepen-
Heink et al.PNAS ?
June 28, 2005 ?
vol. 102 ?
no. 26 ?
In agreement with the reported half-life of 5 days of c20S (35),
IFN-? showed no effect 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-?, strikingly 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 ?5i?LMP7 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 destabilization requires the presence of an active LMP7
subunit or whether the LMP7 structure itself is sufficient to signal
POMP degradation, we used T2 cells expressing mutated LMP7
derivatives. These mutated and proteolytically inactive forms of
proteasome complex (17). Inactivation of LMP7 by deletion of the
prosequence (T2 ?proLMP7) or by substitution of the active-site
to that observed in untransfected T2 cells (Fig. 4b). Thus, the
amount of POMP depends on the efficient maturation and activity
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
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
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
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
POMP. Untagged POMP did not bind to the nickel beads (7, negative control)
POMP interacts directly with LMP7. (a) Yeast two-hybrid assay for
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
the mean of five independent experiments. A representative experiment is
i20S are less stable than c20S independent of IFN-?. c20S (T2 cells) or
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-
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.
www.pnas.org?cgi?doi?10.1073?pnas.0501711102Heink et al.
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 tested 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-
(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 subtypes pulled down
untagged POMP (Fig. 5c, lanes 1 and 4). In contrast, the wild-type
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-
as well as chimeric ? subunit constructs expressing either the ?5 or
the ?5i propeptide fused to the archaebacterial ? subunit of A.
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
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 propeptides interacted with
POMP (Fig. 5c, lanes 2 and 5), suggesting 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
expression of POMP in HeLa cells was silenced by using short
interfering RNAs (siRNAs). The siRNA targeting POMP mRNA
led to silencing of POMP expression in the absence or presence of
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-
expression 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.
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
i20S biogenesis. In fact, POMP stabilization in the presence of an
inactive LPM7 subunit demonstrates that LMP7 essentially drives
the rapid degradation of POMP.
the faster maturation of LMP7 (compared with ?5) and, in con-
sequence, with the ?4-fold faster formation of i20S. This rapid
of POMP led to a strong reduction of incorporated ?5 and ?5i?LMP7 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 3?7 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
(Left). Mean fluorescence levels are indicated as geometric (Geo) mean.
Heink et al.PNAS ?
June 28, 2005 ?
vol. 102 ?
no. 26 ?
LMP7 into nascent proteasomes (40). Thus, the kinetics of protea-
some biogenesis 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
require any additional IFN-?-induced factors. Consequently, the
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-
comes apparent by the altered chromatographic properties of
The key position of POMP and LMP7 in the accelerated
formation of i20S is supported by their direct interaction. In
subunit but not with LMP7 was recently reported based on a yeast
two-hybrid interaction screen (37). This negative result was inter-
into the respective 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
complexes 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
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 peptides also is reflected by the
reduction of MHC class I surface expression in POMP-depleted
cells (25). These data therefore demonstrate that POMP possesses
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.
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-
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
presentation, following the hypothesis that most antigenic peptides
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 assistance, 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 427?8-5?SFB 421).
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www.pnas.org?cgi?doi?10.1073?pnas.0501711102Heink et al.