Identification and Characterization of a Drosophila Nuclear Proteasome Regulator

A HOMOLOG OF HUMAN 11 S REGgamma (PA28gamma )*

Patrick Masson, Oskar Andersson, Ulla-Maja PetersenDagger, and Patrick Young§

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



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We report the cloning and characterization of a Drosophila proteasome 11 S REGgamma (PA28) homolog. The 28-kDa protein shows 47% identity to the human REGgamma 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 REGgamma proteasome activator has a role independent of the invertebrate immune system. Our results support the idea that gamma  class proteasome activators have an ancient conserved function within metazoans and were present prior to the emergence of the alpha  and beta  REG classes.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha  and beta  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 REGgamma . Currently, it is known that the human REGgamma forms an additional independent complex within mammalian nuclei and can associate reversibly with the 20 S proteasome (18).

A variety of evidence indicates that the mammalian REGalpha beta functions to generate antigens for the major histocompatibility complex class I immune pathway (2, 19-21). Interferon gamma  treatment strongly increases the expression of the alpha  and beta  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 REGalpha beta subunits have reduced ability to generate class I antigen peptides (25). For mammalian REGgamma there is no current evidence that this nuclear protein plays a role in the mammalian immune system. The REGgamma has been reported by several groups not to be induced by interferon gamma , and its location in the nucleus makes it an unlikely candidate to assist antigen presentation by the major histocompatibility complex class I immune system.

The human REGgamma 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 REGgamma 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-kappa B signaling pathways and is also probably related (29-31).

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.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-beta -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.

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 -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-alpha -subunit HC8, purchased from AFFINITI Research Products. The rabbit 20 S proteasome was purchased from AFFINITI Research Products.

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 >= 25% exposure to solvent were grouped as surface residues.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 REGgamma . Comparison of the human REGalpha 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 REGalpha . 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 REGgamma sequences is aligned with the c-Myc NLS. Amino acid residues that form the inner channel in the human REGalpha 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 REGgamma nuclear localization signal. C, mapping sequence conservation to potential REG structural features. Drosophila and human REGgamma channel residues show a much higher similarity than the outside surface residues or the complete sequences.

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 REGalpha ring contains a central channel composed of seven long alpha -helices, with each REG monomer contributing a single helix. The REGalpha beta proteins have different surface channel properties compared with those predicted for REGgamma and the invertebrate REG channel surfaces (Fig. 1, A and C). The REGalpha beta channels contain a repeating number of basic residues on the channel surface, while the proposed corresponding residue positions found within the REGgamma channels are conserved acidic residues.

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 REGalpha , -beta , and -gamma (26). Human REGalpha beta strongly activates hydrophobic peptide degradation in vitro, whereas human REGgamma 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 REGgamma 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 alpha  subunit of the 20 S proteasome.

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 REGgamma , 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.

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 Delta -Tyr dREG may only form a weak or transient complex that is able to inhibit the proteasome but not activate.

Nuclear Localization of dREG-- We wished to determine whether the dREG is a functional homolog of the mammalian REGgamma protein. It has been previously observed that human REGalpha and REGbeta antisera label the cytoplasm and the nucleoli (41). In contrast, mammalian PA28gamma 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.

Identification of a c-Myc-like NLS in Drosophila REG-- While REGgamma 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 REGgamma sequences. Surprisingly, the human REGalpha 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 REGgamma sequences do contain an atypical monopartite NLS first described for the human c-Myc oncogene (43). To investigate whether this region in Drosophila REGgamma 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.

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.



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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.

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).



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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

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 REGgamma (44). The dREG and tick sequences both share high overall sequence similarity to the human REGgamma sequence (Figs. 1 and 7). Our fluorogenic peptide assays demonstrate that the mammalian REGgamma and dREG share similar preferences for activating the proteasome's trypsin-like proteolytic activity. As expected for a REGgamma 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 REGalpha and -beta 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 REGgamma proteasome complex.



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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 alpha -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 REGgamma 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 REGgamma 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 REGgamma sequences but is not present in the REGalpha or REGbeta . 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 REGalpha 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). REGalpha and REGbeta molecules that lack the homolog-specific insert regions are functional in binding and activating the 20 S proteasome (38). It has been proposed for REGalpha and REGbeta 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 REGalpha . The REGalpha beta complex has been proposed to exist both in the cytoplasm and in the nucleus (41, 42). The bipartite NLS may function in transporting the REGalpha beta 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 REGgamma 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 REGgamma complexes share a distinct channel surface compared with the REG alpha  and beta  classes. The preferential activation of certain peptides by the REGgamma 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 REGalpha beta 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 alpha -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 REGgamma sequences. The vertebrate REGgamma 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 REGalpha beta classes and that the modern invertebrate REG sequences group with the mammalian REGgamma class. Recent chromosomal localization of the mouse genes has demonstrated that the REGbeta sequence was probably a gene duplication of an ancestral alpha  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 REGbeta gene is derived from REGalpha (48). It has been proposed that the REGalpha emerged by gene duplication from a gamma -subunit-like precursor system (48, 49). The identification of the Drosophila REG as being a homolog to the REGgamma mammalian proteasome activator is additional evidence that the REGgamma may most closely resemble the ancestral REG sequence and function.

What is the current function of REGgamma in the metazoan cell? The Drosophila model system should be useful for identifying the role for REGgamma 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 REGgamma 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 REGgamma (27). The presented data do not support a role for the REGgamma 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, REGgamma , to the immune related REGalpha beta . 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.


    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.


    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.

Dagger 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.


    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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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