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Eukaryotic Cell, June 2009, p. 844-851, Vol. 8, No. 6
1535-9778/09/$08.00+0     doi:10.1128/EC.00165-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Characterization of a REG/PA28 Proteasome Activator Homolog in Dictyostelium discoideum Indicates that the Ubiquitin- and ATP-Independent REG Proteasome Is an Ancient Nuclear Protease ^,

Patrick Masson,¹ Daniel Lundin,² Fredrik Söderbom,³ and Patrick Young^4*

Swiss Institute of Bioinformatics, Swiss-Prot Group CMU, 1, rue Michel Servet, CH-1211 Geneva 4, Switzerland,¹ Department of Molecular Biology and Functional Genomics, Stockholm University, S-10691 Stockholm, Sweden,² Department of Molecular Biology, Swedish University of Agricultural Sciences (SLU), Uppsala Biomedical Center (BMC), Box 590, S-75124 Uppsala, Sweden,³ Department of Genetics Microbiology and Toxicology, Stockholm University, S-10691 Stockholm, Sweden⁴

Received 12 May 2008/ Accepted 10 April 2009

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The nuclear proteasome activator REG/PA28 is an ATP- and ubiquitin-independent activator of the 20S proteasome and has been proposed to degrade and thereby regulate both a key human oncogene, encoding the coactivator SRC-3/AIB1, and the cyclin-dependent kinase inhibitor p21 (Waf/Cip1). We report the identification and characterization of a PA28/REG homolog in Dictyostelium. Association of a recombinant Dictyostelium REG with the purified Dictyostelium 20S proteasome led to the preferential stimulation of the trypsin-like proteasome peptidase activity. Immunolocalization studies demonstrated that the proteasome activator is localized to the nucleus and is present in growing as well as starving Dictyostelium cells. Our results indicate that the Dictyostelium PA28/REG activator can stimulate both the trypsin-like and chymotrypsin-like activities of the 20S proteasome and supports the idea that the REG-20S proteasome represents an early unique nuclear degradation pathway for eukaryotic cells.

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The proteasome is a large multicatalytic enzyme involved in non-lysosome-regulated protein degradation (7) and has been shown to be an essential factor in controlling various cellular processes, such as cell cycle progression, transcriptional regulation, and metabolic pathways, through regulated proteolysis (3). The 20S proteasome is a barrel-shaped cylinder made up of four stacked rings of seven subunits each. In isolation, the 20S proteasome active sites are sequestered behind closed gates that are formed by the outer subunit rings of the 20S proteasome (9). It is currently thought that the vast majority of proteins are degraded by the proteasome system only when the 20S proteasome is associated with an activating subcomplex, such as the 19S regulator complex, to form the 26S proteasome (22, 27). Several additional activating complexes have also been found to associate with the 20S proteasome, such as PA200 (26) and the REG/PA28 family, which is the focus of this work (8, 10, 23).

While the composition and structure vary considerably between the proteasome activator complexes, a common feature of the divergent complexes is the ability to associate and change the conformational position of the rings to open the closed gate on either end of the 20S proteasome (28).

The human REG family constitutes a distinct class of proteasome regulatory complexes. Three subunits, , β, and , are able to assemble two distinct heptameric rings: REGβ can associate as a three--subunit, four-β-subunit heptameric ring (31), while the REG complex is proposed to be a homopolymer of seven identical subunits. Unlike the 19S proteasome, the REG activator has been previously characterized only in metazoans and is apparently absent in plants and yeasts. An activator similar or distantly related to the REG has been characterized in Trypanosoma brucei and termed PA26 but demonstrates little sequence identity or similarity with the three isoforms of mammalian proteasome REG, , β, and (29). Surprisingly considering the lack of sequence homology, the PA26 is capable of forming a heptamer ring structure like REG and activates the 20S proteasome in a similar manner (5).

While the REG activators have been well characterized in terms of their ability to promote the degradation of small peptides, evidence for their role in promoting degradation of full-length proteins has only recently been obtained. The first proposed protein target for the nuclear REG proteasome complex has been identified and corresponds to the steroid receptor SRC-3/AIB1 coactivator, encoded by an important oncogene that is commonly present at high concentrations in human breast cancers (14). The SRC-3/AIB1 coactivator is proposed to be degraded in a ubiquitin- and ATP-independent manner by the REG proteasome. Recently, two groups reported that the key central cyclin-dependent kinase inhibitor, p21(Waf/Cip1) is another endogenous target. RNA interference knockdown, gain-of-function analysis, and pulse-chase experiments substantiate the idea that REG promotes degradation of unbound p21 (2, 13). In vitro assays using purified REG, p21, and the 20S proteasome confirm that REG directly mediates degradation of free p21 in an ATP- and ubiquitin-independent manner. These two recent examples suggest that further studies using various model systems and assays will likely identify additional protein substrates that are degraded by the REG proteasome complex.

In this study, we cloned a Dictyostelium gene that has clear sequence similarity to the human REG gene. Expression and purification of the Dictyostelium gene product in Escherichia coli generated a PA28/REG complex that can associate and activate the 20S proteasome and allowed us to identify conserved and divergent properties between the human and Dictyostelium forms of this proteasome activator.

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Chemical reagents and antibodies. Rabbit polyclonal antibodies against a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-purified recombinant Dictyostelium REG were raised in rabbits by Agrisera. Initial Western immunoblot tests confirmed the production of an anti-REG antibody that yielded a single band with the expected molecular weight migration value that matched the Dictyostelium REG migration value. The specificity of the anti-REG antibody was confirmed by immunoblotting against purified REG protein and by depleting the anti-REG antibody against polyvinylidene difluoride-bound purified REG protein. For specificity testing, an excess of purified REG protein was incubated for 1 h in Tris-buffered saline (TBS) with 1 cm² of polyvinylidene difluoride transfer membrane, washed three times, and then blocked with 5% milk in TBS. A total of 10 µl of anti-REG antibody was then incubated for 1 h at 4°C in 1 ml TBS buffer with the immobilized REG protein. The 1-ml solution was then used to probe an immunoblot of purified REG protein and crude Dictyostelium extract, and results were compared to the results for anti-REG serum that was not preabsorbed against REG protein. A TRITC (tetramethyl rhodamine isothiocyanate)-conjugated secondary antibody was purchased from Sigma. t-Butoxycarbonyl (Boc)-Leu-Arg-Arg-7-amino-4-methyl-coumarin (MCA), Suc-Leu-Leu-Val-Tyr-MCA, and Boc-Gly-Gly-Leu-MCA were purchased from Affiniti Research Products. The peptide acetyl-Asp-Glu-Val-Asp-MCA was purchased from Peninsula Laboratories Europe.

Purification of Dictyostelium 20S proteasome. Dictyostelium was grown in HL5 medium according to Sussman (24). The Dictyostelium cells were collected by centrifugation followed by freezing at –80°C. A total of 20 g (wet weight) was French pressed three times and resuspended in 3 volumes (60 ml) of MVB buffer (20 mM MOPS [pH 7.5], 20 mM sodium acetate, 20 mM KCl, 10 mM NaCl, 2 mM MgCl₂, 1 mM dithiothreitol [DTT] with 10% glycerol). The crude lysate was centrifuged for 1 h at 20,000 x g at 4°C. The crude supernatant was incubated for 1 h at 4°C with 10% streptomycin sulfate, centrifuged at 20,000 x g for 15 min, and then dialyzed overnight against 2 liters of MVB buffer with 10% glycerol at 4°C. The dialyzed extract was centrifuged an additional time for 5 min at 10,000 x g. An extract of 90 ml, 6 mg/ml, was passed over a DEAE cellulose column that had been equilibrated in TBS buffer and 10% glycerol. The 20S proteasome was eluted with 1 liter KCl gradient, 0 to 1 M, and the fractions were assayed for 20S proteasome LLVY-MCA activity in TS buffer (10 mM Tris-HCl [pH 8.8], 25 mM KCl, 10 mM NaCl, 1.1 mM MgCl₂, and 0.1 mM EDTA) containing 0.035% SDS. Fractions containing proteasome activity were concentrated using Amicon Centricons. The Dictyostelium 20S proteasome extract was passed over a Superose 6 gel filtration column equilibrated in MVB buffer and 10% glycerol. The fractions were assayed for 20S proteasome activity and stored at –80°C. The purity of the 20S proteasome was analyzed by electrophoresis on a 10 to 15% SDS-polyacrylamide gel.

Purification of the Dictyostelium proteasome activator. The Dictyostelium REG cDNA, clone series SSH1-D, clone SSH185 (19), was PCR amplified and inserted into PET26b between NdeI and EcoRI restriction sites. Escherichia coli BL21(DE3) transformed with pET26b containing the Dictyostelium REG 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 µM isopropyl-1-thio-D-galactopyranoside and harvested after a 2-h induction. The soluble protein fraction was obtained from induced recombinant cells using a French press and resuspending 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 x g for 30 min at 4°C. The soluble protein extract was treated with 10% streptomycin sulfate and centrifuged at 20,000 x g, 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-to-1 M KCl gradient in TS buffer, pH 8.8. Fractions containing Dictyostelium REG were identified by SDS-PAGE and Coomassie staining. Fractions with the recombinant Dictyostelium REG were further purified by gel filtration using a Superdex 200 column equilibrated with TS buffer and 1 mM dithiothreitol. The Dictyostelium REG eluted as a complex of the expected size.

Gel filtration of Dictyostelium proteasome and REG. To identify a physical interaction between the REG complex and the proteasome, the two purified complexes, 1 µg of purified Dictyostelium proteasome, and 7.5 µg of purified recombinant Dictyostelium REG were preincubated for 15 min and passed over a Superose 6 fast protein liquid chromatography column in 20 mM MOPS (pH 7.5), 20 mM sodium acetate, 20 mM KCl, 100 mM NaCl, 2 mM MgCl₂, 1 mM dithiothreitol at room temperature. Individual runs of 20S proteasomes or REG at the same protein concentrations were also performed under identical conditions.

Fluorometric assays of proteasome activities. Spectrofluorometric assays were performed in the presence of fluorogenic peptides, 1 µg of purified Dictyostelium proteasome, and 7.5 µg of purified recombinant Dictyostelium REG, in 20 mM MOPS pH 7.5, 20 mM sodium acetate, 20 mM KCl, 10 mM NaCl, 2 mM MgCl₂, 1 mM DTT. Proteasome and Dictyostelium REG were incubated together for 15 min at room temperature to allow association, prior to the addition of the fluorogenic peptide substrates. Reactions were performed at room temperature for 30 min 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.

Immunostaining of Dictyostelium cells. Dictyostelium cells were either cultured in growth medium (HL-5) or subjected to starving conditions in PDF buffer (20 mM KCl, 5 mM MgCl₂, 20 mM KPO₄ [pH 6.4]). Cells were placed on plastic coverslips, and allowed to attach for 30 min. Cells were fixed with a solution of 3.7% formaldehyde in 15 mM NaPO₄-KPO₄ buffer, pH 6.5, for 10 min and permeabilized for 5 min in –20°C methanol containing 1% formaldehyde. Cells were incubated with Dictyostelium REG polyclonal antibody (1/500) for 1 h at room temperature in 0.1% BSA in PBS, washed three times with PBS containing 0.05% Tween 20, and then incubated with TRITC-conjugated secondary antibody to a dilution of 1/100 at room temperature in 0.1% BSA, 0.05% Tween 20 in PBS. Finally, three additional washes of 5 min each were carried out in PBS Tween, and Hoechst dye was added during the final wash to a 5 µg/ml concentration. Finally, coverslips were mounted in a 50% glycerol-50% PBS solution. The immunostaining protocol was repeated with only the secondary antibody in order to rule out its hybridization to the nucleus.

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Sequence similarities of the REG gene family. Initial BLAST searches revealed a number of partial sequences from the Dictyostelium development cDNA database (19) that showed high similarity to the REG proteasome activator present in metazoans. The cDNA clone SSH185 appeared to contain the longest cDNA sequence of the matching sequences but was originally sequenced to contain only a partial open reading frame that matched part of the REG proteasome activator sequence. However, sequencing of this clone revealed instead a full-length open reading frame that contained high similarity to the REG family throughout the sequence. The open reading frame translated into a corresponding protein of 26 kDa and the gene to psmE3, designated DDB0191141 in dictyBase (http://dictybase.org/). Alignment of metazoan REG sequences with the Dictyostelium protein reveals high conservation of the activation domain (Fig. 1 and Table 1). For metazoan REG subunits, the classes, , β, and , can be identified directly from their primary sequences within two specific regions, the conserved region that contains the single long alpha helix that forms the seven-member channel and the homolog-specific region. The homolog-specific region codes for a flexible loop region that is present on top of the REG-proteasome and has been shown to contain a conserved nuclear localization signal (NLS) (17). For the homolog-specific region, the Dictyostelium REG sequence shows by far the shortest loop sequence of all identified REG sequences: the loop region is almost absent except for a small region that shows strong similarity to the proposed REG NLS. For the alpha helix channel, the Dictyostelium REG sequence shows only limited similarity to any of the metazoan classes. A limited number of residues are conserved both in the class and the Dictyostelium REG. For the channel alpha helix region, the Dictyostelium sequence shows similarity of 34% with the human sequence but only 23% and 21% with the human and β sequences, respectively.

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FIG. 1. Alignment of the deduced amino acid sequence of Dictyostelium REG with various homologues, including Drosophila REG, C. elegans REG, zebrafish REG, -β, and -, and human REG, -β, and -. The sequence alignment was obtained with the MegAlign program from DNASTAR. Specifically, the alignment of REG protein sequences was carried out using ClustalW (25) and a PAM 250 scoring matrix. Identical residues are shaded in black. The nuclear localization signal region is boxed. The proposed residues for the -helix forming the inner channel are marked with a thin line. Another thin black line indicates the activation region that interacts with the 20S proteasome. The asterisk marks the residue corresponding to the human REG Lys188 that converts the activation pattern from REG to REG in human when mutated to an Asp or Glu residue (12). The percentages of sequence similarity are shown in Table 1.

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TABLE 1. Comparison of sequence similarities in percent scores for the different members of the REG family

The genomic sequence of the Dictyostelium REG gene confirms the sequence of our cDNA clone and reveals the position of the introns within the Dictyostelium REG gene on chromosome 4. The gene contains three small introns, which is well above average for such a small gene. The average number of introns in Dictyostelium is 1.9, with 30% of genes containing no introns (4). Genomic searches yield a single copy of the REG gene without any apparent duplications or related sequences with significant similarity scores. In order to address whether the Dictyostelium REG gene was a recent acquisition, we compared the GC content of the REG sequence against that of other Dictyostelium genes. The AT content is 74% for the REG open reading frame. This extremely high AT content matches the overall ratio found in Dictyostelium open reading frames, which is 72% (4), and does not indicate that the REG gene was recently acquired by horizontal transfer from a metazoan.

Proteasome activation by Dictyostelium REG. The open reading frame for Dictyostelium REG was cloned into an expression vector, and the corresponding Dictyostelium protein was overexpressed in E. coli. The purified recombinant Dictyostelium REG protein was analyzed for its ability to stimulate peptide degradation by the 20S proteasome. Initially, purified Dictyostelium 20S proteasome was obtained using standard DEAE and gel filtration chromatography. The purification was analyzed by SDS-PAGE (Fig. 2A) as well as by a simple protease fluorogenic peptide assay. To confirm that that the purified 20S proteasome was functional and in a closed gated state, LLVY-MCA assays were carried out with and without the presence of low concentrations of SDS (data not shown). As expected, the purified Dictyostelium 20S proteasome was active, as shown by its activation by a low concentration (0.035%) of SDS.

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FIG. 2. Purification of the Dictyostelium 20S proteasome and Dictyostelium REG. (A) Coomassie stained SDS-PAGE after the various purification steps for the 20S proteasome and for purification of the recombinant Dictyostelium REG expressed in E. coli. Equivalent amounts of total proteins were loaded on a 10 to 15% SDS-PAGE gel. The line represents the expected migration area for various 20S proteasome subunits. (B) To identify a physical interaction between the REG complex and the proteasome, the two purified complexes (1 µg of purified Dictyostelium 20S proteasome and 7.5 µg of purified recombinant Dictyostelium REG) were preincubated for 15 min and passed over a Superose 6 fast protein liquid chromatography column, and the results were compared to chromatographic runs of the individual complexes.

The recombinant Dictyostelium REG was also purified using DEAE ion-exchange and gel filtration chromatography. The location of the Dictyostelium REG from the DEAE-eluted fractions was determined by SDS-PAGE (Fig. 2A). The presence of Dictyostelium proteasome activator was also detected by a simple proteasome fluorogenic peptide assay. For the assay, partially purified fractions from the Dictyostelium REG purification were mixed with 20S proteasome and LLVY-MCA fluorogenic peptide solution. Fractions that stimulated LLVY-MCA proteolytic activity were found to colocalize with a band that matched the Dictyostelium REG molecular weight when run on denaturing SDS-polyacrylamide gels. Control E. coli extracts that lacked the Dictyostelium REG plasmid did not show this stimulatory activity or the presence of the band on gels (data not shown). Finally, gel filtration of the REG-20S proteasome complex was carried out. An excess of REG complex was preincubated with 20S proteasome and passed over a Superose 6 column at room temperature (Fig. 2B). Comparisons of chromatographic runs of the individual complexes versus preincubated REG and 20S reveal the formation of a high-molecular-weight cocomplex. Immunoblots of specific fractions using a REG antibody confirmed the presence of REG in the higher-molecular-weight complex (data not shown).

To determine relative increases in peptidase activation, purified proteasomes from Dictyostelium cells were mixed with purified Dictyostelium REG and the C-terminal cleavage of these fluorogenic tri- or tetrapeptides were monitored for increased production of cleaved fluorogenic MCA. The assays were performed with the fluorogenic peptides LLR-MCA, LLVY-MCA, and either DEVD-MCA or LLE-MCA to measure the trypsin, chymotrypsin, and acidic residue activities, respectively. As shown in Fig. 3, the Dictyostelium REG was able to enhance peptide cleavage in a broad spectrum for different peptide substrates. However, the relative stimulation showed significant differences. The chymotrypsin-like activity monitored by the cleavage of LLVY-MCA peptide was enhanced 6-fold in the presence of Dictyostelium REG, whereas the trypsin-like activity evaluated by LRR-MCA was increased 14-fold when the 20S proteasome was incubated and allowed to associate with the Dictyostelium REG. As expected, the proteasome activation did not require the addition of ATP (data not shown).

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FIG. 3. Activation of the purified Dictyostelium 20S proteasome by Dictyostelium recombinant REG. (A) Fluorescence intensity representing the fluorogenic peptide cleavage by the 20S proteasome purified from Dictyostelium cell extract without (black bars) or with (gray bars) Dictyostelium REG. Dictyostelium REG and proteasomes were preincubated 10 min at room temperature and then incubated with a specific fluorogenic peptide. (B) Comparison of the ability of Dictyostelium REG to stimulate fluorogenic peptide degradation by the 20S proteasome.

Dictyostelium REG is a nuclear protein in growing and starving cells. In order to investigate the localization of the REG complex in Dictyostelium cells, we overexpressed the recombinant protein in E. coli, extracted the corresponding band from polyacrylamide gels, and generated several polyclonal antibodies. For cellular staining studies, a high-titer antibody that had minimum cross-reactivity with E. coli or other Dictyostelium proteins was selected (Fig. 4A).

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FIG. 4. The Dictyostelium REG is predominantly nuclear, in both growing and starving cells. (A) An immunoblot was prepared and transferred with E. coli-expressed recombinant REG or crude protein extract from Dictyostelium cells in duplicate and divided into two (left and middle). The two halves of the immunoblot were incubated with equal volumes of anti-Dictyostelium REG polyclonal sera. Sera were preadsorbed with recombinant REG, before incubation with the membrane (middle). (Right) Coomassie staining of the transferred proteins. (B to E) Dictyostelium cells were stained with polyclonal anti-Dictyostelium REG polyclonal antibody followed by rhodamine secondary antibody (B and D). Cells show nuclear localization of the proteasome regulator compared to DNA staining with Hoechst 33258 (C and E). (B and C) Individual Dictyostelium cells; (D and E) Dictyostelium cells under starvation.

This polyclonal antibody was used for immunofluorescence studies on fixed Dictyostelium cells (Fig. 4B to E). Dictyostelium cells were harvested both before and after medium starvation. To visualize the localization of the nucleus, Hoechst fluorochrome staining of the DNA was carried out after immunostaining of the fixed cells with Dictyostelium REG antibody and fluorescent secondary antibody (Fig. 4C and E). The commercial secondary antibody does not itself stain the nuclei of fixed Dictyostelium cells (data not shown). The Hoechst stain did not affect REG immunostaining, and cells stained only with anti-REG antibody showed the same pattern as the dually stained cells (data not shown). For Dictyostelium REG, a strong nuclear staining in the nucleus was observed. The cytoplasm contained very little or no staining, suggesting that the Dictyostelium REG complex is a nuclear protein, as has been seen for REG class of metazoan proteasome activators. Initial comparisons between growing and starving Dictyostelium cells showed no differences in the amount of Dictyostelium REG present within the nuclei of the two stages, with both showing strong NLSs.

Gene-disrupted REG Dictyostelium strains were not isolated. To determine the role of the PA28/REG proteasome-activating complex, homologous recombination was attempted to knock out the PA28/REG gene by transformation of a plasmid containing the Dictyostelium REG genomic sequence disrupted with selectable markers. Multiple attempts to generate a REG knockout Dictyostelium were carried out. However, Western blot analysis revealed that all isolated colonies still expressed the Dictyostelium REG proteasome activator at wild-type levels (data not shown).

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It is currently unclear why a small subset of nuclear proteins bypass the requirement for ubiquitination and degradation by the 26S proteasome and instead are apparently degraded by the REG 20S proteasome. Is this type of degradation a recent adaptation in animal cell nuclei or is it an ancient pathway that precedes the ubiquitin proteasome system? A key feature for Dictyostelium is their ability to grow as single cells and then differentiate into multicellular stalked fruiting bodies that contain haploid spores. The proteasome is required for Dictyostelium developing from the proliferative to the differentiated state. Using differential display, a Dictyostelium 20S proteasome subunit gene was isolated as one preferentially expressed during the transition from growth to differentiation (1). The UbpA deubiquitinating enzyme has also been identified as being essential for development but not growth for Dictyostelium (15).

In general, the metazoan REG activators are present both in the cytoplasm and the nucleus. REGβ is found in the cytoplasm of animals possessing an adaptive immune system, and the presence of this complex in zebrafish suggests that the origin of these genes preceded the divergence of bony fishes and tetrapods (20). The REG gene appears to have a more ancient origin, since it has been characterized in invertebrates such as Drosophila, Rhipicephalus appendiculatus, and Caenorhabditis elegans. The NLS for REG has been identified in Drosophila and resembles the c-myc monopartite NLS (16, 17). The REG NLS is present in the middle of a flexible loop region that is disordered in the crystal structure for REG and was termed the homology insert region (30) (Fig. 5A). Both Dictyostelium REG and metazoan activators are present in the nucleus but not obviously attached to structural elements of the nuclei.

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FIG. 5. Nuclear REG proteasomes are an ancient eukaryotic pathway. (A) Illustration of the proposed REG complex based on the known crystal structures (10, 11). The main difference between the Dictyostelium and human REG complexes is the length of the homolog insert regions. However, both contain monopartite NLS sequences in this unstructured region. (B) Alignment of monopartite NLSs found within the homolog insert regions from various organisms. The overall identities and expect values of predicted proteins to the entire length of human REG are as follows: Coprinopsis cinerea (XP_001829111), 35%, 2e-34; Ustilago maydis (XP_756858), 27%, 7e-24; Nematostella vectensis (XP_001638241), 48%, 8e-63; E. huxleyi (DQ658283 [GenBank] ), 19%, 1e-07. The c-myc NLS sequence motif is from humans (16). (C) Comparison of the insert position to the known human REG (11) crystal structure reveals that the insert aligns with the turn of the adjacent long alpha-helical pair of the REG monomer. (D) The position of Dictyostelium in eukaryotic phylogeny and mapping the absence or presence of the REG activators on the eukaryotic tree. Red lines represent groups that have REG proteasome activator sequences in their genomes. Whole-proteome comparisons of Dictyostelium and representatives of key groups, rooted on archaeal species, were used to generate the phylogenetic tree, which was developed from original data in reference 4.

The activity characterization of Dictyostelium REG suggests that the Dictyostelium REG protein is similar in nuclear localization to the class of REG proteasome activators in metazoans with some differences in activity toward peptide cleavage activation. It has been observed that treatment of metazoan REG activators with ammonium sulfate results in changes in activation. Previous studies have shown that ammonium sulfate broadens the substrate specificity for mammalian REG (6). During the purification of the Dictyostelium REG complex, ammonium sulfate precipitation was not carried out on the Dictyostelium activator. For Dictyostelium REG, the trypsin-like proteasome activity was enhanced over other tested protease activities, and this preference to stimulate cleavage after basic amino acid residues is similar to a characteristic of the class. However, the proposed inner channel residues of the Dictyostelium REG are significantly different from those of any of the three classes found in metazoans. Furthermore, Dictyostelium REG showed a broader range of proteasome activation than the metazoan nuclear activators. The Dictyostelium protein was able to stimulate the proteolysis of a chymotrypsin model substrate, while metazoan REG complexes inhibit this proteasome activity (17).

A number of close relationships between Dictyostelium and metazoans have been identified at the protein level, and the recent ability to compare complete proteomes has greatly helped in the understanding of the lineage relationships (4). It is currently accepted that Dictyostelium diverged from the animal line shortly after the plants and shortly before fungi and yeasts. A search for genomes that contain sequences with high similarity to REG proteasome activators reveals a number of new candidates. Recently finished fungus genomes show the presence of genes with high similarity to REG and conservation in both location and sequence of a potential monopartite NLS signal for a number of sequences (Fig. 5B). Alignment searches of newly sequenced genomes indicate that a wide range of eukaryotes contain REG proteasome activators and that the complex has been lost both in yeasts and in modern plants. For the phytoplankton Emiliania huxleyi, an initial low overall similarity was found between a candidate gene and the human REG protein sequence. The low initial score was due to the lack of a homolog insert-specific domain in the phytoplankton sequence; instead, a large insertion is present near the C terminus of the activation domain region (see Fig. S1 in the supplemental material). Interestingly, mapping the predicted E. huxleyi REG onto the structure of the human REG revealed that the extra sequence is inserted into the neighboring conserved turn domain atop the REG structure (Fig. 5C). This suggests that the observed large differences in the primary sequences between the human and phytoplankton proteins may reflect only small structural tertiary differences. The mapping evidence indicates that the homolog-specific inserts are evolutionarily conserved at the opening of the REG complex and that they function as localization domains and possibly contribute to acquisitions of protein substrates. Mapping the presence of REG on the tree of eukaryotes indicates that the nuclear REG proteasome degradation pathway is ancient and was likely lost or replaced in plants and yeast (Fig. 5D). Cluster analysis based on sequence similarity of the newly obtained REG sequences supports the idea that three classes with the nonmetazoan REG sequences are within the REG class (our unpublished data). The apparent conservation of an NLS in the majority of REG sequences suggests that a REG proteasome was a nuclear complex in ancestral eukaryotes.

A role for REG in cell cycle progression was suggested after it was reported that REG is abnormally overexpressed in thyroid cancer cells, especially in cells at the peripheral region of the cancer (21). In Drosophila, study of the complex promoter region revealed that transcription of REG is under the control of DREF, a transcription factor typically found to activate Drosophila genes involved in cell cycle progression and DNA replication (18). Currently, the oncogene SRC-3/AIB1 and cyclin-dependent kinase inhibitor p21 (Waf/Cip1) are the only protein substrates known to be degraded by the REG-proteasome complex in mammalian cells (2, 13, 14). From the studies cited above, the nuclear REG complex appears to function in processes that promote or repress cell cycle progression, suggesting that this conserved degradation complex may degrade a number of nuclear targets that have not yet been identified. The Dictyostelium system should serve as a useful model to discover additional proteins that are degraded in a ubiquitin- and ATP-independent manner by the proteasome. The properties and cellular location of the Dictyostelium REG suggest an ancient lineage for the REG class of proteasome activators.

 
We thank Anthony Poole for discussions and comments. Takahiro Morio from the University Tsukuba, Tsukuba, Ibaraki Japan, provided the original cDNA clone from the Dictyostelium cDNA project that was supported by the Japan Society for the Promotion of Science (RFTF96L00105) and Ministry of Education, Science, Sports and Culture of Japan (08283107).

This work was supported by grants from the Swedish Cancer Society and the Swedish Research Council.

 
* Corresponding author. Mailing address: Department of Genetics Microbiology and Toxicology, Stockholm University, S-10691 Stockholm, Sweden. Phone: 46-8-163107. Fax: 46-8-164315. E-mail: patrick.young{at}gmt.su.se

Published ahead of print on 1 May 2009.

Supplemental material for this article may be found at http://ec.asm.org/.

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Eukaryotic Cell, June 2009, p. 844-851, Vol. 8, No. 6
1535-9778/09/$08.00+0     doi:10.1128/EC.00165-08
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