From the Department of Molecular Biology, Stockholm University, S-10691 Stockholm, Sweden
Received for publication, August 14, 2000, and in revised form, October 4, 2000
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ABSTRACT |
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We report the cloning and characterization of a
Drosophila proteasome 11 S REG The proteasome is the major nonlysosomal protease present in
eukaryotic cells and has a central role in regulating protein levels
within the cell. Proteasomal degradation determines the turnover rate
for a wide range of proteins including abnormal proteins, short lived
regulators, and a significant proportion of cellular proteins (1).
Within the cytoplasm, proteasomes participate in the generation of
peptide antigens for the major histocompatibility complex class
I system (2). Proteasomes are also present in the nucleus and are
assumed to function in degrading a variety of nuclear proteins (3-6).
Nevertheless, a number of nuclear proteins, such as p53, require export
to the cytoplasm for proteasomal degradation (7, 8).
The Saccharomyces cerevisiae 20 S crystal structure reveals
that the proposed entrance sites of the 20 S proteasome are closed (9).
Isolation of the catalytic sites prevents indiscriminate cleavage of
proteins within the cell but requires a mechanism to transfer
substrates efficiently into the cavity of the proteasome. Two protein
complexes have been identified that allow access through the ends of
the proteasome. A 19 S regulatory complex can associate with the 20 S
proteasome and forms the ubiquitin-ATP-dependent 26 S
proteasome (10, 11). A second activating complex, 11 S REG (PA28), has
been identified, and activates the proteasome to hydrolyze short
peptides but cannot independently stimulate the degradation of intact
folded proteins (12, 13). The 11 S REG complex has recently been
proposed to participate in a mixed ternary complex. This complex
consists of 20 S proteasomes associating both with 19 S regulatory and
REG complexes (14). The existence of such complexes has led to the
proposal by Hendil et al. that the REG complex (PA28) may
actually participate in the degradation of intact proteins when
associated in a hybrid proteasome.
The original identification of the 11 S REG proteasome activator was
from bovine and human red blood cells using the fluorogenic peptide
Suc-LLVY-MCA,1
7-amino-4-methylcoumarin. A novel protein complex was characterized that was capable of enhancing proteasomal activity without the addition
of ATP (13, 15). This activator was found to be composed of two
subunits termed A variety of evidence indicates that the mammalian REG The human REG Data base searches of cDNA libraries and the completed
Drosophila genome sequence indicate that only a single REG
gene is present. The following results demonstrate the presence of a
REG activator in Drosophila that is localized to the nucleus
by a monopartite NLS and activates the trypsin-like activity of the proteasome while potentially inhibiting chymotrypsin-like activities.
Cloning and Site-directed Mutagenesis of the Drosophila
REG--
Cloning of the dREG gene was carried out by PCR amplification
of a Drosophila cDNA adult library. The mRNA was
poly T-primed, and the resulting cDNA was directionally cloned
using XhoI and EcoRI restriction sites into a
pBK-CMV phagemid vector using Lambda Zap Express from Stratagene. The
Drosophila library was not reamplified and was diluted
4-fold with water and heated for 15 min at 65 °C. The denatured
sample was cleared by centrifugation, and a 5-µl aliquot was used in
the individual PCRs. The initial primer used was an upstream
primer (5'-GCTAACGAAATGCCTAACGACACTGTGG-3') that was specific for the
Drosophila REG 5' leader sequence. This primer sequence was
obtained from EST sequences available from a data base search, Berkeley
Drosophila Genome Project/Howard Hughes Medical
Institute EST Project. The downstream sequence of the
Drosophila REG was not present in any EST sequence at the time, and a primer (5'-GCGTTAAGATACATTGATGAGTTTGG-3') that corresponded to the pBK-CMV phagemid vector was used for the initial PCR
amplification. PCR products were directly cloned into a Promega EZ-TA
cloning vector and finally sequenced. A clone was isolated that matched a full-length cDNA sequence of the Drosophila REG
cDNA. Subsequent PCRs used an upstream primer of
5'-AGCTAACCATATGCCTAACGACACTGTGG-3' that introduced a NdeI
restriction site at the initial Met residue of the REG sequence. The
downstream primer, 5'-GTTGAGGCTCGGGCTTCGGAAATACC-3', matched the 3'-end
of the REG open reading frame sequence. The resulting PCR products were
initially cloned into a TA Promega plasmid and then subsequently cloned
into a pET26b expression vector. From the 14 previous cDNAs
available on the data base and independent PCR products of the library,
a consensus sequence for the dREG was obtained. This sequence was later
confirmed by the genomic sequence of dREG.
Site-directed mutagenesis was carried out to identify a nuclear
localization signal in the Drosophila REG. The mutagenesis was performed using the Stratagene Quick-Change site-directed mutagenesis kit on a Drosophila REG pACT expression vector,
and the corresponding mutant was then subcloned into a pET26b bacterial expression vector. The two primers used for mutagenesis were 5'- CGTGACAGTCTGCCCACCAGTAGCCAGAGCGTGGATGTCATCG-3' and
5'-CGATGACATCCACGCTCTGGCTACTGGTGGGCAGACTGTCACG-3'. The resulting mutant
constructs were confirmed by sequencing.
Expression and Purification of Drosophila REG--
BL21(DE3)
Escherichia coli transformed with pET26b-dREG was grown at
30 °C in LB medium until an A600 of
0.3 was reached. The bacteria were induced with a final concentration
of 300 mM isopropyl-1-thio- Purification of Drosophila Proteasome--
Staged dechorionated
embryos were harvested from Oregon-R wild type Drosophila.
The embryos were extensively washed with water and frozen at
Fluorometric Assays of Proteasome
Activities--
Spectrofluorometric assays were performed in the
presence of fluorogenic peptides, 1 µg of fly proteasome, or 0.2 µg
of rabbit proteasome and various amounts of dREG in a final volume of
100 µl of 10 mM Tris, pH 7.45. Proteasome and REG were
incubated together for 10 min at room temperature to allow association,
prior to the addition of the fluorogenic peptide substrates. Reactions were performed at room temperature and terminated by the addition of
200 µl of ice-cold ethanol. Fluorescence was measured with a Bio-Rad
fluorometer using an excitation wavelength of 380 nm and an emission
wavelength of 440 nm. All substrate peptides contained the MCA
fluorogenic reporter group. Fluorogenic peptides, Boc-Leu-Arg-Arg-MCA, Suc-Leu-Leu-Val-Tyr-MCA, and benzyloxycarbonyl-Gly-Gly-Leu-MCA, were
purchased from AFFINITI Research Products. The peptide
Ac-Asp-Glu-Val-Asp-MCA was purchased from Peninsula Laboratories Europe.
Immune Induction of mbn-2 Cells and RT-PCR--
Extracts were
made of 107 mbn-2 cells after activation of an immune
response by the addition of 10 µg/ml lipopolysaccharide to the
cells. Cells were determined to be immune induced by monitoring increased transcription of the CecA1 gene using semiquantitative RT-PCR. The mbn-2 Drosophila cells were grown at 25 °C in
Schneider's medium supplemented with 10% fetal calf serum, 1.8 g/liter stable L-glutamine, 50 units/ml penicillin, 50 µg/ml streptomycin, and 50 µg/ml gentamycin. To confirm immune
inductions, RT-PCR was carried out by RNA isolation of mbn-2 cells
after exposure to lipopolysaccharide. The RNA was isolated using
a Qiagen QIAshredder homogenizer and RNeasy mini kits. The RT-PCRs were
carried out using the PerkinElmer Life Sciences GeneAmp EZ rTth RNA PCR
kit. Each RNA sample was run in a series of semiquantitative RT-PCRs, which were withdrawn after an increasing number of cycles. As an
internal control, oligonucleotides specific for the RP49 gene 5'-GACCATCCGCCCAGCATACAGGC-3' and 5'-GAGAACGCAG GCGACCGTTGG-3 were
present in the reactions with oligonucleotides for the gene cecropin
A1, an antibacterial protein, 5'-GTCGCTCAGACCTCACTGCAATATC-3' and 5'-CGAGGTCAACCTCGGGCAGTTGC-3', and finally the
Drosophila REG gene, 5'-CGCCAGCGCGTGGATGTCATCG-3' and
5'-CCACATCTTAAGTAGATTGGAATCC-3'.
Antibodies and 20 S Proteasome--
A dREG antibody was
generated against purified recombinant protein in rabbit. The
polyclonal antibody was generated by AGRI SERA AB using standard
injection protocols. The initial injection used Freund's complete
adjuvant followed with booster injections with Freund's incomplete
adjuvant. After two secondary injections, Western blots were carried
out on the initial prebleeds and compared with the preimmune sera. A
total of three injections of recombinant REG yielded a high titer
rabbit polyclonal antibody.
Western Blot Analysis--
After immune induction, equivalent
amounts of mbn-2 cell protein extracts were separated by
electrophoresis on 11% SDS-polyacryamide gels. Drosophila
cells were plated on 15-cm Petri dishes and allowed to grow for 24 h until they reached a cell density of 2.0 × 106
cells/ml, a total of 1 × 107 cells. Cells were
treated with lipopolysaccharide to induce the innate immune
system, and the inductions were carried out in triplicate plates. At
selected times postinduction, the medium was removed, and the cells
were directly lysed in SDS-PAGE sample buffer. The proteins were
transferred to a Hybond-P membrane (Amersham Pharmacia Biotech). The
membrane was incubated in TBS buffer (25 mM Tris base, pH 7.5, 100 mM NaCl, 50 mM KCl) with 10%
dried milk for 1 h, followed by primary antibody for 1 h in
TBS with 5% dried milk, and finally with 125I-labeled
anti-rabbit secondary antibody (Amersham Pharmacia Biotech) for 1 h in TBS with 5% dried milk. The Western blots were quantitated using
a Molecular Dynamics PhosphorImager.
Immunomicroscopy of Drosophila REG with mbn-2 Cells--
The
immunochemistry was done essentially as described in Ref. 32. In brief,
mbn-2 cells were grown as monolayers on glass coverslips at a density
of 0.5 × 106 cells/ml. The cells were fixed in 3.7%
(w/v) freshly prepared paraformaldehyde in PBS, pH 7.3, for 30 min at
room temperature, rinsed in PBS three times for 20 min each, and
permeabilized with 0.2% Triton X-100 in PBS for 2 min followed by
several changes of PBS. The cells were preincubated with either 2%
normal swine serum for 30 min at room temperature or 10% dried milk
protein in PBS, followed by incubation with polyclonal rabbit anti-REG antibody (1:300) at 4 °C. Cells were rinsed and incubated with either secondary fluorescein isothiocyanate-labeled swine anti-rabbit antibody (1:250) (DAKO, Denmark) or anti-rabbit antibody, whole molecule, tetramethylrhodamine isothiocyanate conjugate for 2 h at
room temperature. The cells were also costained with Hoechst 33258 dye
for 40 s and rinsed with PBS. The slides were observed by
fluorescence microscopy or with a confocal laser-scanning microscope.
Sequence Analysis of the REG Sequences--
Both the multiple
sequence alignment and phylogenetic tree were obtained using the
MegAlign program from DNASTAR. Specifically, the alignment of REG
protein sequences was carried out using ClustalV (33). The phylogenetic
tree was derived by using the application of the UPGMA algorithm (34),
which guides the alignment of ancestral sequences, and applying the
neighbor joining method (35) to the distance and alignment data.
Surface residues were determined using a surface modeling program
within the Swiss-PdbViewer package (36). Amino acid residues with
Cloning and Sequence Determination of Drosophila REG--
To
obtain a complete sequence for the Drosophila REG, PCR
amplification was carried out on a previously generated
Drosophila cDNA library using a primer specific for the
upstream 5' region of the REG open reading frame and a second primer
that corresponded to a downstream vector site. The isolated PCR clones
contained a single complete open reading frame that matched the
consensus identified from the EST data base. The total length of the
isolated gene and flanking sequences consisted of a cDNA sequence
with a total of 1359 nucleotides containing an open reading frame of 735 nucleotides and 450 nucleotides within the 3' downstream region. A
search of the Drosophila genomic sequence identified the
genomic location of the REG gene on the X chromosome at a genetic map position of 1-41, the gene contains a single intron of 196 base pairs.
A sequence alignment of the putative dREG with other REG proteins is
shown in Fig. 1A. The
Drosophila REG sequence shares the highest overall
similarity with the tick sequence and human REG
An additional difference between the REG homologs that may be
functionally significant involves the predicted inner channel surfaces
of the REG sequences (Fig. 1C). The human REG Activation of the 20 S Proteasome with Drosophila REG--
The
PA28 or REG was originally found by the use of fluorogenic peptides.
The ability of the proteasome to degrade small substrates has been
tested in vitro with human REG
Fluorogenic peptide assays demonstrated that the recombinant dREG was
able to strongly stimulate the trypsin-like activity of the
Drosophila 20 S proteasome (Fig.
3A). The activation of the
Drosophila proteasome by the REG complex was
ATP-independent. To confirm that activation is not due to the presence
of contaminating E. coli proteases in the purified
recombinant REG sample, assays were carried out in the absence of
Drosophila 20 S proteasome. The purified REG sample had no
independent protease activity present and required the addition of 20 S
proteasome for peptide proteolysis (Fig. 3A). The dREG, as
previously found with the human REG Inhibiting the Neutral Chymotrypsin-like Activity of the
Proteasome--
To further examine the apparent decrease of the
neutral chymotrypsin-like proteasome activity, assays were carried out
to titrate the inhibition of the 20 S proteasome by dREG. As shown in
Fig. 3C, inhibition of Suc-LLVY-MCA cleavage was titratable, requiring an excess ratio of REG versus the
Drosophila proteasome. To address whether this inhibition
depended upon unique interactions between the dREG and the
Drosophila 20 S proteasome or instead involved interactions
with conserved elements, we tested the Suc-LLVY-MCA hydrolysis activity
of the mammalian 20 S proteasome with dREG at varying molar
concentrations. The results (Fig. 3C) demonstrate that the
dREG can act as an inhibitor for both the invertebrate and mammalian
proteasomes. The dREG was found to inhibit ~50% of the Suc-LLVY-MCA
cleavage activity of the rabbit 20 S proteasome. These results suggest
that the dREG inhibitory activity probably functions upon conserved
features of the 20 S proteasome.
To better understand the inhibition of LLVY-MCA cleavage, we have
recently compared the ability of site-directed mutants of dREG to
function as activators or inhibitors (data not shown). Surprisingly, a
mutant form of dREG can function as an inhibitor of the
chymotrypsin-like activity under conditions when activation is
abolished. A mutant dREG was generated that lacks the conserved C-terminal Tyr residue. For all REG classes, the C terminus has been
demonstrated to be important for tight binding to the 20 S proteasome
(40). The Drosophila REG lacking the C-terminal was found to
activate the proteasome only at low ionic salts concentrations, less
than 10 mM. Surprisingly, this mutant was as active as wild type REG in its ability to inhibit the 20 S proteasome
chymotrypsin-like activity under moderate ionic salt conditions. These
preliminary results suggest that the activation and inhibition
activities are functionally distinct. Our current hypothesis is that
the two forms of dREG have distinctly different interactions with the
20 S proteasome under moderate salt conditions. The wild type dREG
forms a tightly coupled complex with the proteasome that can promote
activation, while the Nuclear Localization of dREG--
We wished to determine whether
the dREG is a functional homolog of the mammalian REG Identification of a c-Myc-like NLS in Drosophila
REG--
While REG
Transfection of the NLS mutant dREG into mbn-2 cells localizes the
proteasome activator to the cytosol (Fig.
5E), while wild type REG
transfection, using an identical vector, efficiently localizes to the
nucleus even when overexpressed (Fig. 5B). A majority of
cells that contained cytoplasmic dREG had dramatic cell morphological
changes with large increases in cell volumes.
Drosophila REG Is Not Induced after Innate Immune
Induction--
The expression pattern for Drosophila REG
RNA during embryogenesis had been previously mapped in a large EST
expression screen.2
Drosophila REG mRNA is present throughout embryogenesis
but is preferentially expressed in the midgut region. The REG protein is also present throughout embryogenesis and matches the broad expression pattern of the 20 S proteasome. Using semiquantitative RT-PCR, we examined the mRNA levels of the dREG gene after heat shock induction and innate immune induction. Under both conditions mRNA levels for dREG did not dramatically change (data not shown). Induction of the innate immune system was confirmed by the strong induction of the cecropin A1 gene monitored by semiquantitative RT-PCR.
Western blot analysis of the REG protein demonstrated that the dREG did
not significantly increase compared with control cells after immune
induction (Fig. 6). Comparison of immune
induced cells stained with dREG antibody revealed no significant
changes in either the apparent levels or changes in localization. Heat shock induction also did not change the staining pattern for dREG within mbn-2 cells (data not shown).
Our results are the first demonstration that nonmammalian REG
sequences do in fact code for functionally active REG complexes. Previously, a gene sequence with similarity to the mammalian 11 S REG
sequence was identified from a tick salivary gland cDNA library and
proposed to be related to the mammalian REG (PA28) homolog.
The 28-kDa protein shows 47% identity to the human REG
and strongly
enhances the trypsin-like activities of both Drosophila and
mammalian 20 S proteasomes. Surprisingly, the Drosophila
REG was found to inhibit the proteasome's chymotrypsin-like activity
against the fluorogenic peptide
succinyl-LLVY-7-amino-4-methylcoumarin. Immunocytological
analysis reveals that the Drosophila REG is localized to
the nucleus but is distributed throughout the cell when nuclear
envelope breakdown occurs during mitosis. Through site-directed
mutagenesis studies, we have identified a functional nuclear
localization signal present in the homolog-specific insert region. The
Drosophila PA28 NLS is similar to the oncogene c-Myc nuclear localization motif. Comparison between uninduced and innate immune induced Drosophila cells suggests that the REG
proteasome activator has a role independent of the invertebrate immune
system. Our results support the idea that
class proteasome
activators have an ancient conserved function within metazoans and were
present prior to the emergence of the
and
REG classes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and
and was shown to have high similarity to a
previously described nuclear protein: Ki autoantigen. This antigen
target was originally identified in patients with the autoimmune
disease systemic lupus erythematosus (16, 17) and named REG
.
Currently, it is known that the human REG
forms an additional
independent complex within mammalian nuclei and can associate
reversibly with the 20 S proteasome (18).
functions
to generate antigens for the major histocompatibility complex class I
immune pathway (2, 19-21). Interferon
treatment strongly increases
the expression of the
and
subunits and the formation of
cytoplasmic REG-immunoproteasomal complexes (18, 22-24). Expression of
PA28 in a fibroblast line increases the presentation of viral epitopes
from influenza nucleoprotein and a cytomegalovirus protein (19).
Finally, transgenic mice that lack the REG
subunits have reduced
ability to generate class I antigen peptides (25). For mammalian REG
there is no current evidence that this nuclear protein plays a role in
the mammalian immune system. The REG
has been reported by several
groups not to be induced by interferon
, and its location in the
nucleus makes it an unlikely candidate to assist antigen presentation
by the major histocompatibility complex class I immune system.
is able to markedly stimulate the trypsin-like
activity of the 20 S proteasome in vitro, preferentially
hydrolyzing peptides with basic residues in the P1 position but only
modestly activating the cleavage of other peptides (26). Recently, it has been observed that mice deficient in REG
have modestly reduced growth rates, and derived cell lines were shown to have altered cell
division with impeded entry into S phase (27). We wished to examine if
the Drosophila 11 S REG has a role in the insect's immune
system. Drosophila lacks an acquired immune system but depends upon an innate immune system that generates antimicrobial peptides for defense against pathogens. This system is well conserved among mammals and insects and has been proposed to share a common evolutionary origin (28). In addition, the vertebrate acquired immune
system shares similar signal transduction pathways with the
interleukin-1 receptor-NF-
B signaling pathways and is also probably related (29-31).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside and harvested
after a 2-h induction. The soluble protein fraction was obtained from
induced recombinant cells using a French press and resuspended in two pellet volumes of TS buffer (10 mM Tris-HCl, pH 8.8, 25 mM KCl, 10 mM NaCl, 1.1 mM
MgCl2, and 0.1 mM EDTA), followed by
centrifugation at 39,000 × g for 30 min at 4 °C.
The soluble protein extract was treated with 10% streptomycin sulfate
and centrifuged, and the supernatant was dialyzed overnight in TS
buffer at 4 °C. The extract was initially passed over a 50-ml DEAE
cellulose column followed by elution with a 1-liter 0-1 M
KCl gradient in TS buffer, pH 8.8. Fractions containing dREG were
identified by SDS-PAGE and Coomassie staining. Fractions with the
recombinant dREG were further purified by gel filtration using a
Superdex 200 column equilibrated with TS buffer and 1 mM
dithiothreitol. The dREG eluted as a complex, and no monomeric form was
apparent during the purification.
80 °C. For a 20 S proteasome purification, 10 g of frozen
embryos were resuspended in TBS buffer (25 mM
Tris-HCl, pH 7.5, 50 mM KCl, 100 mM NaCl) with
10% glycerol and 1 mM dithiothreitol. The lysate was
homogenized with a Dounce homogenizer and centrifuged at 20,000 × g for 60 min at 4 °C. The extract was incubated for 1 h at 4 °C with 10% streptomycin sulfate, centrifuged at
20,000 × g for 10 min at 4 °C, and then dialyzed
overnight in TBS with 10% glycerol. The extract was passed over a
DEAE-cellulose column that had been equilibrated in TBS buffer and 10%
glycerol. The 20 S proteasome was eluted with a 1-liter KCl gradient,
0-1 M, and the fractions were assayed for 20 S proteasome
LLVY-MCA activity in TS buffer containing 0.035% SDS. Fractions
containing proteasome activity were concentrated using Amicon Centricon
concentrators. The Drosophila 20 S proteasome extract was
passed over a Superdex 200 gel filtration column equilibrated in 20 mM MOPS, pH 7.5, 20 mM sodium acetate, 20 mM KCl, 1 mM dithiothreitol, and 10% glycerol.
The fractions were assayed for 20 S proteasome activity and stored
frozen at
80 °C. The purity of the 20 S proteasome was analyzed by
electrophoresis on a 10-15% SDS-polyacrylamide gel and Western blot
analysis using a proteasome monoclonal antibody, anti-
-subunit HC8,
purchased from AFFINITI Research Products. The rabbit 20 S proteasome
was purchased from AFFINITI Research Products.
25% exposure to solvent were grouped as surface residues.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
. Comparison of the
human REG
crystal structure (37) with the location of conserved
residues suggests that the invertebrate REG proteins probably share the
same overall structure as seen for human REG
. Important secondary
structural features, such as proline residues in predicted turns, are
highly conserved among the three classes of REG proteins. However, a
nonessential flexible loop region termed the homolog-specific insert
region present N-terminal to the activation domain shows little
sequence conservation between the REG classes (38). This region does
show conservation within the classes and the alignment of the
vertebrate and invertebrate REG sequences revealed a conserved domain
that resembled a c-Myc nuclear localization domain present in the human
oncogene. Comparison of the c-Myc nuclear localization domain and the
REG sequences are shown within the homolog-specific insert region (Fig.
1A).
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Fig. 1.
Similarities between three classes of
proteasome human activators and invertebrate REG sequences.
A, amino acid sequence alignment using a PAM 250 matrix for
the Drosophila, tick (44), and the three human REG subunits
(26). Residues that are identical to the Drosophila REG are
shaded in black. The homolog-specific insert
region for the REG sequences is boxed. The proposed
monopartite NLS region for the REG sequences is aligned with the
c-Myc NLS. Amino acid residues that form the inner channel in the human
REG
crystal structure (37) are underlined with a
thick line. Asterisks mark the
residues that are predicted to have exposed side chains on the inner
channel. The thin line corresponds to the region
used to generate a protein sequence phylogenetic tree in Fig. 2.
B, schematic diagram of the heptameric ring of REG with a
central channel and an activation domain on the lower face. The
homolog-specific inserts present on the top face are diagrammed as a
single loop containing the REG
nuclear
localization signal. C, mapping sequence conservation to
potential REG structural features. Drosophila and human
REG
channel residues show a much higher similarity than the outside
surface residues or the complete sequences.
ring
contains a central channel composed of seven long
-helices, with
each REG monomer contributing a single helix. The REG
proteins
have different surface channel properties compared with those predicted for REG
and the invertebrate REG channel surfaces (Fig. 1,
A and C). The REG
channels contain a
repeating number of basic residues on the channel surface, while the
proposed corresponding residue positions found within the REG
channels are conserved acidic residues.
, -
, and -
(26).
Human REG
strongly activates hydrophobic peptide degradation
in vitro, whereas human REG
preferentially activates the
basic trypsin-like peptide hydrolysis. To determine whether the
isolated Drosophila gene encodes an active proteasome
activator, fluorogenic peptide assays were carried out using purified
Drosophila 20 S proteasomes and recombinant REG (Fig.
2). As expected, the dREG protein was found as a large complex during size exclusion purification and not as
a monomer. Purification of Drosophila 20 S proteasomes was
also carried out from wild type Oregon-R Drosophila embryos (Fig. 2B). The homogenization protocol used for
Drosophila embryos has previously been shown not to
efficiently rupture the nuclei, and the isolated proteasomes are
therefore preferentially from the cytoplasm. The specific activity of
Drosophila 20 S proteasome for the cleavage of Suc-LLVY-MCA
peptide, 5 pmol/min/µg, agreed with the published activities of
isolated Drosophila 20 S proteasome (39) and for the
mammalian form (13). Western blot analysis of the
Drosophila proteasome showed no detectable REG
protein present in the purification fractions (data not shown).
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Fig. 2.
Purification of the Drosophila
REG and 20 S proteasome. A,
Coomassie-stained SDS-PAGE of the purification steps of dREG expressed
in E. coli. B, purification profile of the 20 S
proteasome. Equivalent amounts of total protein from each step of the
proteasome purification were loaded on an 11% SDS-PAGE gel, and a
Western blot was carried out using a monoclonal antibody specific for
the HC8 subunit of the 20 S proteasome.
, is a strong activator of the
trypsin-like activity of the proteasome but a poor activator of other
proteolytic activities (Fig. 3B). Initial kinetic analysis
indicates that doubling the Vmax for hydrolysis
of Boc-Leu-Arg-Arg-MCA peptide led to no observable change in the
Km (80 µM). Incubation of dREG with
rabbit 20 S proteasome moderately activated the trypsin-like activity 5-fold. Surprisingly, during the initial survey of peptides, the chymotrypsin-like activity for cleavage of Suc-LLVY-MCA was apparently inhibited by dREG.
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Fig. 3.
Activation of the 20 S proteasome by
dREG. A, increasing amounts of recombinant dREG were
preincubated with 1 µg of 20 S Drosophila
(filled circles) or 0.2 µg of rabbit 20 S
proteasome (filled squares). The reaction was
started by the addition of 170 µM boc-LRR-MCA. After an
additional 10-min incubation, the reaction was stopped with cold
ethanol. For the activation profiles, the REG was in molar excess of
proteasome from 2- to 40-fold. The purified 20 S proteasome had an
initial activity of 3.6 pmol/min/µg prior to activation. To confirm
that the increase in cleavage was not due to the presence of
contaminating proteases from the REG sample, a control was carried out
with increasing amounts of the dREG incubated with boc-LRR-MCA peptide
in the absence of proteasome (open circles).
B, comparison of the ability of the dREG to activate various
fluorogenic peptides. 1 µg of Drosophila 20 S proteasome
was assayed by itself (open squares) or with 7.5 µg of recombinant dREG (filled squares). REG
and proteasome were preincubated for 10 min at 20 °C and then
incubated with a specific fluorogenic peptide (final concentration 170 µM). The results are an average of triplicate assays.
C, inhibition of chymotrypsin activity. Increasing amounts
of dREG were incubated with 1 µg of Drosophila or 0.2 µg
of mammalian 20 S proteasome for 10 min, and then the chymotrypsin-like
activity was measured by the addition of the peptide Suc-LLVY-MCA. The
Drosophila proteasome alone had a specific activity of 5 pmol/min/µg for the hydrolysis of Suc-LLVY-MCA.
-Tyr dREG may only form a weak or transient
complex that is able to inhibit the proteasome but not activate.
protein. It
has been previously observed that human REG
and REG
antisera
label the cytoplasm and the nucleoli (41). In contrast, mammalian
PA28
antiserum labels the nucleus but leaves nucleoli as unstained
hollows (41, 42). To determine the cellular localization of the dREG, a
Drosophila leukemic blood cell line, mbn-2, was examined by
immunofluorescence. The mbn-2 cells were immunolabeled with a dREG
polyclonal antibody and a fluorochrome Hoechst stain for DNA. As shown
in Fig. 4B, the dREG is
localized to the nucleus, while the nucleoli are excluded from
immunostaining. To determine whether REG was associated with the
outside of the nuclear membrane or present within the nucleus, confocal
imaging of the samples was carried out (Fig. 4D). The dREG
protein is localized within the nucleus and is not found present within
the nucleoli or on the outside of the nuclear membrane. A second
Drosophila cell line, S2, was also examined and had the identical localization for dREG (data not shown). A small number of
cells from both cell lines did show staining throughout the cell
volume. Staining with 4',6-diamidino-2-phenylindole and
phase-contrast imaging revealed that this small population corresponded
to cells with condensed chromosomes that were in mitosis when nuclear
envelope breakdown was occurring.
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Fig. 4.
Drosophila REG is present in the
nucleus but not nucleoli of mbn-2 cells. A,
phase-contrast image of mbn-2 Drosophila cells.
B, immunofluorescence image probed with polyclonal dREG
antibody and fluorescein secondary antibody. C,
identical cell image of A with Hoechst 33258 staining of DNA
in the nucleus. D, laser confocal image of mbn-2 cells
immunoprobed with polyclonal REG antibody. The image represents the
central image section of mbn-2 nuclei.
activators are clearly localized to the nucleus,
the location of the NLS or other localization signals have not been
identified within any class of REG activators. An initial motif search
of the different REG proteins did not identify a bipartite SV40-like
NLS within the REG
sequences. Surprisingly, the human REG
sequence contains a match to this most common type of NLS with two
clusters of basic residues within its homolog-specific insert region.
At the same aligned position within the homolog-specific insert region,
the REG
sequences do contain an atypical monopartite NLS first
described for the human c-Myc oncogene (43). To investigate whether
this region in Drosophila REG
functions as an NLS, we carried out site-directed mutagenesis on the three basic residues within the proposed domain. Residues Lys79,
Arg80, and Arg82 were converted to Ser
residues, and the resulting clone was expressed both in E. coli and Drosophila mbn-2 cells. The mutant dREG
was expressed and purified as described previously for the wild type dREG complex. The NLS mutant dREG expressed from E. coli was
assayed for its ability to stimulate the trypsin-like activity of the Drosophila 20 S proteasome. Using similar concentrations of
proteasome and activators, wild type REG stimulated the trypsin-like
activity 10.8-fold, while the addition of NLS mutant gave a 9.6-fold
increase in proteasome activity. We conclude that the mutant is active and forms a complex that can interact with the proteasome.
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Fig. 5.
Drosophila REG contains a
NLS within the homolog-specific insert region.
Drosophila mbn-2 cells were cotransfected with dREG and
green fluorescent protein pACT expression plasmids. A-C,
immunofluorescence images of cells transiently overexpressing
wild type dREG. D-F, cells transfected with mutant dREG
that replace residues Lys79, Arg80, and
Arg82 with Ser residues. A and C,
images identifying transfected cells by green fluorescent protein
fluorescence. Cells were stained with anti-dREG antibody followed by
rhodamine secondary antibody and then mounted for viewing
(B and E). Cells transfected with
dREG-Ser79-Ser80-Ser82 show little
nuclear localization of the proteasome activator and increased overall
cell volumes. C and F, images of Hoechst 33258 staining of DNA in the nucleus.
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Fig. 6.
Immune induction does not increase the
synthesis of Drosophila REG. Western blot
analysis of dREG from mbn-2 cells after innate immune induction. Total
cellular protein was harvested at the indicated times by the addition
of SDS-PAGE sample buffer to cell pellets. Equivalent volumes of cell
extracts were analyzed by Western blotting using the polyclonal REG
antibody and an 125I-labeled secondary antibody and
quantitation using a PhosphorImager. The immune induced values
represent triplicate samples for each time point and did not have
increased levels of REG protein compared with uninduced control. The
bottom panel represents one set of time points
from Western blot analysis of the immune induced mbn-2 cells.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(44). The dREG and tick
sequences both share high overall sequence similarity to the human
REG
sequence (Figs. 1 and 7). Our
fluorogenic peptide assays demonstrate that the mammalian REG
and
dREG share similar preferences for activating the proteasome's
trypsin-like proteolytic activity. As expected for a REG
activator,
the dREG functions as a strong activator of the trypsin-like activity
of the 20 S proteasome and could activate Drosophila and
mammalian 20 S proteasome. Surprisingly, the dREG also acts as an
inhibitor to the cleavage of the Suc-LLVY-MCA substrate. The 11 S
REG
and -
subunits were initially discovered in mammalian
extracts by their ability to stimulate the proteasomal activity against
the Suc-LLVY-MCA peptide. The neutral chymotrypsin-like inhibition with
dREG is the first example where an 11 S REG can function as a positive
and negative regulator of proteasomal activity. Examining the cleavage
of unmodified peptides will help in addressing the true substrate
preferences for the REG
proteasome complex.
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[in a new window]
Fig. 7.
Phylogenetic tree of REG sequences. A
61-amino acid block that required no insertion of gaps was selected for
the phylogenetic analysis and is shown as a thin
line in Fig. 1A. The region corresponds to the
conserved activation domain and the proposed -helix that forms the
central channel. The alignment of REG protein sequences was carried out
using a ClustalV method (33). The phylogenetic tree was derived by
using the application of the UPGMA algorithm (34). The percentage of
identical residues shared between the human REG
and the other
sequences is shown in the right column, and the
tree is drawn to scale. The percentages represent identity for the
aligned region and not the complete REG sequences. The zebrafish
sequences were grouped into specific classes as described previously
(48).
Our results also show that the dREG complex is localized to the nucleus
and indicate that the REG class can be expected to have a conserved
nuclear function that predates the division of vertebrates and
invertebrates. We have identified through site-directed mutagenesis on
dREG, a c-Myc like NLS in the homolog-specific insert region that is
conserved in all REG
sequences but is not present in the REG
or
REG
. The homolog-specific insert region was previously identified as
a mutation-free zone in a screen for mutations that would affect
REG activity (45). The human REG
crystal structure does not resolve
the homolog-specific insert region, and the region is probably a
flexible loop on the top face of the REG complex in solution (37).
REG
and REG
molecules that lack the homolog-specific insert
regions are functional in binding and activating the 20 S proteasome
(38). It has been proposed for REG
and REG
that the
homolog-specific insert regions could function in targeting the
proteasome activator to chaperonins or the calnexin·TAP
complex (38). Surprisingly, a bipartite NLS may be present in
the homolog-specific insert region of REG
. The REG
complex has
been proposed to exist both in the cytoplasm and in the nucleus (41,
42). The bipartite NLS may function in transporting the REG
to
the nuclear compartment. Our results support the hypothesis that the
homolog-specific insert regions contain targeting domains with the
first demonstration of a NLS target sequence centered within the REG
domain.
How does a REG complex modulate the activity of the proteasome? The
PA28, 11 S REG, has been proposed to function by opening the ends of
the 20 S proteasome and by the potential allosteric activation of the
catalytic sites (46). The REG complexes show a strong bias in
stimulating the cleavage of specific peptides. Our sequence comparisons
of vertebrate and invertebrate REG sequences suggest that all REG
complexes share a distinct channel surface compared with the REG
and
classes. The preferential activation of certain peptides by the
REG
may be due to the interaction of the channel surface with the
peptides. We favor a model where the dREG channel functions as a gate
or filter that promotes or excludes specific peptides. Site-directed
mutagenesis studies on the surface channel residues should provide a
means to determine whether the channel surface is important in
proteasomal activation.
The REG class proteasome activators are not essential components of
eukaryotic proteasomes. Yeast and plant species apparently lack any
form of REG activators. A common feature of organisms that possess REG
activators is the presence of adaptive or innate immune systems. From
data base searches, it is apparent that only organisms that possess an
advanced acquired immune system encode REG genes within their
genomes. To further examine the relationship between
Drosophila REG and the mammalian REG sequence, a
phylogenetic tree was generated (Fig. 7). The analysis focused on a
conserved region corresponding to the activation domain and the
proposed
-helical central cavity. The three classes of REG are
present as separate branches on the phylogenetic tree and the
invertebrate REG sequences group with the vertebrate REG
sequences.
The vertebrate REG
sequences show extreme conservation within their
activation domains and central channel sequences. The
Dictostelium sequence rooted the phylogenetic tree and does
not have distinct affinity for any of the three classes, either when
compared by its activation domain region or by the overall sequence.
Overall, the diagram supports the idea that gene duplication and
divergence may have generated the REG
classes and that the modern
invertebrate REG sequences group with the mammalian REG
class.
Recent chromosomal localization of the mouse genes has demonstrated
that the REG
sequence was probably a gene duplication of an
ancestral
subunit that occurred relatively recently during the
evolution of the adaptive immune system (47). Recent work on the
zebrafish PA28 genes also supports the idea that the REG
gene is
derived from REG
(48). It has been proposed that the REG
emerged
by gene duplication from a
-subunit-like precursor system (48, 49). The identification of the Drosophila REG as being a homolog
to the REG
mammalian proteasome activator is additional evidence that the REG
may most closely resemble the ancestral REG sequence and function.
What is the current function of REG in the metazoan cell? The
Drosophila model system should be useful for identifying the role for REG
due to its powerful genetics and the presence of a
single REG gene. We are currently sequencing the dREG genes from mutant
fly strains that have had their phenotypes mapped near or within the
REG genomic region. We have recently sequenced the REG
gene from a
wing deformation mutant Drosophila strain, wavy,
and determined that the strain does not contain a mutated REG open
reading frame. A second candidate phenotype we are investigating is
puny (50). For the puny phenotype, adults are
small, with slightly shorter wings than wild type. This mutant may be
similar to the phenotype observed in knockout mice lacking REG
(27). The presented data do not support a role for the REG
class in innate
immunity. It is therefore likely that the ancestral REG subunit also
had a nonimmune function within early metazoans. Modifications and
renewal of preexisting nonimmune genes has been proposed to be an
important mechanism in the emergence of adaptive immunity (2). The REG
complexes may be another example of modification of a nonimmune gene,
REG
, to the immune related REG
. Further examination of the
role of the REG activator in Drosophila and primitive
eukaryotes, such as Dictostelium, should increase our understanding of how the proteasome-REG complex functions and how it
has evolved to play a role in the mammalian immune system.
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ACKNOWLEDGEMENTS |
---|
We thank Martin Rechsteiner and Gregory Pratt for recognizing an 11 S REG-like cDNA sequence within Drosophila using a data base search; Josefin Lundgren for site-directed mutagenesis; Sophia Ekengren for supplying the Drosophila cDNA library; Petra Björk and Gunnel Björklund for assistance with immunocytological analysis and transfections; and Ylva Engström, Anthony Poole, Britt-Marie Sjöberg, Uli Theopold, Lars Wieslander, and Marie Öhman for helpful comments on the manuscript.
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FOOTNOTES |
---|
* This work was supported by grants from the Swedish Natural Science Research Council and the Lars Hiertas Trust.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Dana-Farber Cancer Institute Pediatric Oncology,
M64844 Binney Street Boston, MA 02115.
§ To whom correspondence should be addressed. Tel.: 46-8-164135; Fax: 46-8-152350; E-mail: patrick.young@molbio.su.se.
Published, JBC Papers in Press, October 10, 2000, DOI 10.1074/jbc.M007379200
2 Berkeley Drosophila Genome Project/Howard Hughes Medical Institute EST Project, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are: Suc, succinyl; MCA, 7-amino-4-methylcoumarin; Boc, t-butoxycarbonyl; dREG, Drosophila REG; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase-PCR; EST, expressed sequence tag; TBS, Tris-buffered saline; MOPS, 4- morpholinepropanesulfonic acid; NLS, nuclear localization signal.
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