Cloning of Schistosoma mansoni Seven in Absentia (SmSINA)+ homologue cDNA, a gene involved in ubiquitination of SmRXR1 and SmRXR2

Molecular & Biochemical Parasitology 131 (2003) 45–54

Cloning of Schistosoma mansoni Seven in Absentia (SmSINA)+ homologue cDNA, a gene involved in ubiquitination of SmRXR1 and SmRXR2夽 Marcelo R. Fantappié1,2 , Ahmed Osman1 , Christer Ericsson3 , Edward G. Niles, Philip T. LoVerde∗ Department of Microbiology and Immunology, Witebsky Center for Microbial Pathogenesis and Immunology, School of Medicine and Biomedical Sciences, 138 Farber Hall, State University of New York, Buffalo, NY 14214, USA Received 10 April 2003; accepted 1 July 2003

Abstract Drosophila (SINA) and human Seven in Absentia (SIAH-1 and SIAH-2) have been implicated in ubiquitin-mediated proteolysis of different target proteins. Using the Schistosoma mansoni nuclear receptor SmRXR2 as bait in a yeast two-hybrid system, we identified a DNA fragment that encodes part of the schistosome homologue of the Seven in Absentia protein (SmSINA). Screening of S. mansoni cDNA expression library resulted in the isolation of a cDNA containing the full-length coding region of SmSINA. SmSINA contains the characteristic structural features of other SINA proteins including a conserved N-terminal RING finger domain and a cysteine-rich C-terminus. We demonstrate that SmSINA associates with SmRXR2 and SmRXR1 both in vivo and in vitro, and define the binding domains in SmRXR2 and SmRXR1 that mediate their interaction. Schistosome SINA co-localizes with SmRXR2 and SmRXR1 in vitelline cells. In addition, we show that SmSINA stimulates the ubiquination of both SmRXR2 and SmRXR1 in vitro. Our findings suggest that SmSINA regulates ubiquitination and ubiquitin-induced degradation of schistosome nuclear receptors (RXR1 and RXR2) via the ubiquitin–proteasome pathway. © 2003 Elsevier B.V. All rights reserved. Keywords: Seven in Absentia; Retinoid X receptor; Ubiquitination; Proteasomal degradation; Schistosoma mansoni

1. Introduction Schistosomiasis is a chronic debilitating disease caused by a parasitic helminth, Schistosoma mansoni [1]. These helminth parasites are dioecious, that is, they have separate sexes. The pathology of the disease results from the immune Abbreviations: aa, amino acid; 3-AT, 3-amino-1,2,4-triazole; bp, base pairs; GST, glutathione-S-transferase; nt, nucleotide; RT–PCR, reverse transcriptase–polymerase chain reaction; IPTG, isopropyl-1-thio-␤-dgalactospyranoside; RXR, retinoid X receptor; SmRXR, Schistosoma mansoni RXR; SmSINA, Schistosoma mansoni Seven in Absentia; GAL4-DBD, GAL4-DNA binding domain; GAL4-AD, GAL4 transcription activation domain 夽 Note: The nucleotide sequence reported in this paper has been submitted to the GenBankTM with the accession number AY227022. ∗ Corresponding author. Tel.: +1-716-8292459; fax: +1-716-8292169. E-mail address: [email protected] (P.T. LoVerde). 1 These authors contributed equally to the work. 2 Present address: Departamento de Bioqu´ımica M´ edica ICB/CCS, Universidade Federal do Rio de Janeiro, Ilha do Fundão 21941-590, Brazil. 3 Present address: Department of Oncology and Pathology, Cancer Center Karolinska, Karolinska Institute, R8:04, 171 76 Stockholm, Sweden. 0166-6851/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0166-6851(03)00188-9

response of the host against eggs laid by the female worms. Interestingly, reproductive development and egg production by the female schistosome is regulated by a stimulus from the male parasite (see [2] for review). The male stimulus regulates female-specific gene expression. The p14 gene, encoding a major eggshell precursor, as well as other developmentally regulated genes, reviewed by LoVerde and Chen [3], were shown to be transcriptionally active only in mature females of S. mansoni in response to worm pairing. Previous results suggest that nuclear receptors SmRXR1 and SmRXR2 may be involved in the regulation of p14 gene expression [4–6]. Retinoid X receptors (RXR) contain six defined regions [7,8]. The N-terminal A/B region contains the ligand-independent transactivation domain (AF-1). The C-region, contains two zinc fingers responsible for DNA binding. The D domain is a hinge region that confers flexibility to the molecule. The E/F region functions in dimerization and ligand binding, and is also involved in mediating ligand-induced interactions with transcriptional coactivators. Modulation of the assembly of transcription pre-initiation complexes by nuclear receptors involves indirect actions on

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components of the transcriptional machinery. Coactivators and corepressors bind to the AF-2 site in the C-terminal region of nuclear receptors and mediate communication between nuclear receptors, transcriptional machinery and the chromatin environment (reviewed in [8]). In this context, active components covalently modify chromosomal proteins resulting in activation or repression of transcription. However, other coregulators for the nuclear receptor superfamily, such as SUG-1 [9] and E6-AP [10] are also involved in the ubiquitin–proteasome pathway and mediate their action through degradation of transcription factors. The Seven in Absentia (SINA) protein was discovered in Drosophila as a factor required for R7 photoreceptor development [11]. In R7, SINA acts together with Phyllopod, induced by the sevenless-Ras-Raf pathway [12–14] to target the repressor of cell fate determination Tramtrack for degradation through the proteasome pathway [14]. The mammalian homologues of Drosophila SINA (Siah-1a, Siah-1b, and Siah-2 in mouse, and SIAH-1 and SIAH-2 in human) were implicated in regulating proteasomal degradation of certain proteins to which they bind. For example, SIAH-1 mediates degradation of DCC (Deleted in Colorectal Cancer) and of the nuclear receptor corepressor N-CoR [15,16]. Mutational analysis revealed that the RING finger containing N-terminal domain of SIAH proteins is required for proteolysis, while the C-terminus is involved in binding DCC and N-CoR [15,16]. In this report, we demonstrate that schistosome SINA stimulates the ubiquitination of SmRXR1 and SmRXR2 in vitro. These results indicate that SmSINA may play an important role in regulating the turnover of schistosome nuclear receptors.

2. Materials and methods 2.1. Identification of SmSINA by a yeast two-hybrid system Construction of S. mansoni yeast two-hybrid cDNA library in GAL4-AD vector (pAD-GAL4-2.1; Stratagene) and SmRXR1 and SmRXR2 bait vectors was previously described [4,6]. Unlike SmRXR1, SmRXR2-GAL4-BD (SmRXR2-DBD) fusion protein did not activate transcription of the reporter genes of the yeast host strains [6]. The bait vector RXR2-DBD was co-transformed into PJ69-4a [17] competent cells, along with S. mansoni adult worm pair yeast two-hybrid cDNA library using the LiOAc/PEG method [18]. Transformed cells were plated onto synthetic dextrose medium (SD) supplemented with amino acid dropout solutions lacking tryptophan (Trp) and leucine (Leu) (to determine transformation efficiency, a total of 200,000 clones were screened) or lacking Trp, Leu, adenine (Ade), and histidine (His), and containing 3 mM 3-amino-1,2,4triazole (3-AT), a metabolic inhibitor for histidine biosynthesis. Colonies that were able to grow on SD medium lacking histidine (-His) in the presence of 3 mM 3-AT were selected.

Potential positive colonies were retransformed into Y190 yeast competent cells, using the Frozen-EZ Transformation II kit (Zymo Research) and the transformed cells were plated onto SD medium lacking Trp, Leu, and His in the presence of 25 mM 3-AT. The LacZ filter lift assay was performed according to Stratagene two-hybrid instruction manual (http://www.stratagene.com/displayProduct.asp?productId= 256). Control plasmids, p53 and pSV40 (Stratagene) were co-transformed along with each transformation experiment to provide positive control for interacting proteins. As a negative control, RXR2-DBD vector was co-transformed with an irrelevant gene, coding for S. mansoni dynein protein, cloned into pAD-GAL4-2.1 (Stratagene). 2.2. Cloning of SmSINA cDNA In order to isolate a cDNA clone containing the entire coding region of SmSINA, 400,000 plaques of a S. mansoni adult worm pair cDNA ␭-Zap II library were screened using the partial sequence originally identified in the yeast two-hybrid screen as a probe. SmSINA DNA fragment was radiolabeled with [␣-32 P]dCTP by random priming. The bacteriophage harboring full-length SmSINA was excised in vivo into the recombinant phagemid pSmSINABlueScript-SK(+) and both strands of the cloned DNA fragment were sequenced. The predicted amino acid sequence of SmSINA was compared to other SINA proteins using the Pile-Up program (Wisconsin GCG Package version 10.1). 2.3. Reverse transcriptase–polymerase chain reaction To evaluate the levels of SmSINA mRNA in different stages of the parasite life cycle, semi-quantitative RT–PCR was performed. Oligonucleotides corresponding to nucleotide 666–685 and the complementary sequence of nucleotide 997–1020 were used as forward and reverse primers, respectively, to amplify a 355 bp PCR product. Alpha-tubulin-specific primers [19] were used in each reaction as a representative constitutively expressed gene control. Due to the relative abundance of ␣-tubulin cDNA compared to that of SmSINA and to ensure the intensity of PCR products comparison are determined in the linear range, ␣-tubulin PCR reactions were cycled 26 times while SmSINA cDNA was amplified for 28 cycles. Equal amounts were used as templates and to load the agarose gels. Band intensities were determined using BioRad gel documentation system (Gel Doc 1000 system and Quantity one software; version 4.2.3). 2.4. SmSINA interaction with SmRXR1 and SmRXR2 in vitro by GST pull-down assay GST pull-down experiments were carried out using several constructs containing SmSINA or SmRXR1 and SmRXR2 fused to GST. SmRXR1 and SmRXR2 were divided into three domains. SmRXR1 cDNA, encoding the

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AF-1 ligand-independent transactivation domain (aa 1–270; GST-RXR1-AF-1), the DNA binding domain (aa 271–340; GST-RXR1-DBD) and the E–F region (aa 399–743; GSTRXR1-AF-2), encoding the AF-2 ligand-dependent transactivation domain and ligand-binding domain [4]. SmRXR2 cDNA, encoding the AF-1 ligand-independent transactivation domain (aa 1–197; GST-RXR2-AF-1), the DNA binding domain (aa 198–263; GST-RXR2-DBD) and the E–F region (aa 327–784; GST-RXR2-AF-2), encoding the AF-2 ligand-dependent transactivation domain and ligand-binding domain [5]. PCR was performed using gene-specific primers containing EcoRI restriction site in the 5 primer and a XhoI restriction site in the 3 primer. The PCR products were cloned into TOPO-TA vector (pCR2.1-TOPO; Invitrogen), excised and then recloned into pGEX-4T-1 (Amersham Pharmacia Biotech). The entire coding regions of SmSINA and SmRXR1 were also inserted into pCITE-4a (Novagen) expression vector. Recombinant prokaryotic expression vectors carrying all constructs were overexpressed in bacteria and the GST-fusion proteins were affixed to glutathione-Sepharose beads. A SmSINA recombinant pCITE-4a vector was in vitro transcribed and translated using STP3 transcription/translation system (Novagen) in the presence of 35 S-methionine. Binding reactions between SmSINA and SmRXR1 and SmRXR2 were performed by adding 2 ␮l of the 35 S-labeled-translation reactions to GST-fusion coupled beads equivalent to about 2 ␮g of one of the fusion proteins. The final volume was adjusted to 300 ␮l by adding binding buffer (50 mM Tris–HCl, pH 7.5, 100 mM NaCl, 10% glycerol, 0.15% Nonidet P-40). The reactions were rocked overnight at 4 ◦ C. Glutathione-Sepharose beads were collected by centrifugation, washed five times, and boiled in 1× SDS loading buffer. Bound proteins were size-separated in 12% SDS–PAGE. The gels were stained, destained, treated with Amplify (Amersham Pharmacia Biotech), dried and exposed to X-ray film. An equivalent of 2 ␮g of GST-coupled beads was included in the binding reactions to serve as negative control in the pull-down experiment.

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RXR2-AF-1) (10 ␮g/ml, each) were used to detect SmSINA and SmRXR2 native proteins in paraformaldehyde-fixed adult worm cryo-sections. Biotinylated goat anti-rabbit and goat anti-mouse IgG (10 ␮g/ml; Molecular Probes, Inc.) and Alexa Fluor-647 streptavidin conjugates (10 ␮g/ml; Molecular Probes, Inc.) were used to localize the primary IgG antibodies. Probed sections were evaluated using a Bio-Rad MRC1024 confocal microscope equipped with a krypton-argon laser and a 680 and a 522 nm filters. The 680 nm filter was used to visualize the specific immunoreactivity at a wavelength beyond the range at which the auto-fluorescence observed in the female parasites could be visualized (seen by using the 522 nm filter). 2.7. In vitro ubiquitination assay SmSINA cDNA was divided into three main domains, encoding its N-terminal (aa 1–89; GST-SINA-N), its RING finger (aa 90–127; GST-SINA-R) and its cysteine-rich C-terminus (aa 128–331; GST-SINA-C). GST-fusion proteins GST-RXR1-AF-2 and GST-RXR2-AF-2 (5 ␮g) were incubated with either GST-SmSINA-
-C (full-length protein lacking aa 345–371; deletion of the last 27 amino acids of GST-SmSINA significantly enhanced its expression levels), GST-SmSINA, GST-SINA-N, GST-SINA-R or GST-SmSINA-C. Ubiquitination reactions were assembled in a 40 ␮l reaction volume, containing ubiquitination buffer (40 mM Tris–HCl, pH 7.6, 5 mM KCl, and 5 mM MgCl2 ), 2 mM ATP, 5 ␮g ubiquitin (Sigma), 100 ␮g of HeLa cell extract (Fraction II) (Affinity Research) and the proteasome inhibitors clasto-lactacystin-␤-lactone and ubiquitin aldehyde (Affinity Research). Reactions were incubated for 1 h 30 min at 30 ◦ C, then for an additional 1 h at room temperature after addition of 20 ml of glutathione-Sepharose beads. The reactive beads were washed four times and reactions were resolved by SDS–PAGE, transferred to a PVDF membrane (Immobilon-P; Millipore) and analyzed by immunoblotting using anti-ubiquitin antibody (Sigma).

2.5. Production of specific antisera 3. Results DNA fragments encoding the RING finger of SmSINA (GST-SINA-R) and the ligand-independent activation domain of SmRXR2 (GST-RXR2-AF-1) were used to immunize New Zealand rabbits and BALB/c mice with complete Freund’s adjuvant for the primary injection and incomplete Freund’s adjuvant for two additional booster doses. The sera were then passed over a protein A-Sepharose column (Amersham Biosciences) and the IgG fractions were eluted and used in immunolocalization studies. 2.6. Immunolocalization of SmSINA The purified IgG fractions of mice antisera (pre-immune and anti-SmSINA-RING) and rabbit serum (anti-GST-

3.1. Identification of SmSINA as SmRXR2-interacting protein To identify proteins that interact with S. mansoni nuclear receptors, we used SmRXR2 as bait to screen a yeast two-hybrid S. mansoni adult worm pair cDNA library. Out of 200,000 transformants screened, using the PJ69-4a yeast strain [17], we isolated 32 potential positive clones. Plasmid DNA prepared from these 32 clones was transformed into Y190 yeast competent cells, and the LacZ filter lift assay was performed. One clone turned blue in the LacZ assay indicating a positive interaction with SmRXR2 (Fig. 1, streaks 2 and 3). The recovered plasmid was sequenced and the insert was

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Fig. 1. Yeast two-hybrid interaction of SmSINA with SmRXR1 and SmRXR2. Panel A: Growth on SD -Trp/-Leu media. Yeast strain Y190 was co-transformed using the two-hybrid plasmid DNA clone obtained in the screening and dynein DNA (streak 1), with two independent transformants of SmRXR2 (streaks 2 and 3), and with two independent colonies of SmRXR1 (streaks 4 and 5). Co-transformation of p53 and pSV40 was performed as positive control (streak 6). Colonies were grown on -Trp/-Leu plates to demonstrate growth and a LacZ filter lift assay performed. Panel B: A mirror image of the plate in panel A showing positive two-hybrid interactions by the induction of LacZ expression (blue color). A cartoon above panel B shows the components and interaction in the yeast two-hybrid system to give a positive result.

found to share high homology to a portion of the Drosophila protein, SINA [11]. Schistosome SINA (SmSINA) was also found to interact, in vivo, with SmRXR1, since Y190 yeast cells co-transformed with SmSINA-GAL4-DBD (the isolated SmSINA sequence cloned in pAS2.1; Clontech) and SmRXR1-AD, could grow on selective medium lacking histidine that contained 30 mM 3-AT and turned blue in a LacZ assay (Fig. 1, streaks 4 and 5). Co-transformation of p53 and pSV40 control plasmids was used as a positive control (Fig. 1, streak 6). As a negative control, we co-transformed SmRXR2-DBD with a S. mansoni dynein-AD (an irrelevant gene), which was not able to drive the ␤-galactosidase reporter gene (Fig. 1, streak 1). 3.2. Isolation and structural analysis of SmSINA cDNA A 641-bp DNA fragment (pAD-GAL4-2.1-SmSINA) that showed homology to Drosophila SINA was labeled and used to screen an adult worm ␭-Zap II cDNA library. A cDNA containing the entire coding region of schistosome SINA was identified and shown to encode a protein of 371 amino acids. Fig. 2 shows a comparison of the deduced amino acid sequence of SmSINA and SINA proteins from different species. SINA was originally identified as a protein required for R7 photoreceptor development in Drosophila [11]. Database

searches revealed the presence of SINA homologues in a number of organisms, including, human, mouse, C. elegans and Arabidopsis. The amino acid sequence of SINA proteins are well-conserved [11,21,22]. All SINA proteins contain an N-terminal RING finger motif (C3 HC4 ) followed by a conserved cysteine/histidine region (C2 HC3 H2 ) which may represent a novel class of Zn2+ -binding motif (Fig. 2). RING finger motifs are cysteine/histidine-rich, Zn2+ -binding domains that are thought to mediate protein–protein interactions [20,21]. Recent studies indicate that RING finger motifs may function in ubiquitination as ubiquitin ligases via interaction with ubiquitin-conjugating enzymes [20,21,23]. Deletion analysis reveals that, while the N-terminal RING finger is required for proteolysis function, the C-terminal region of SINA proteins may be involved in the interaction with target proteins [23]. Consistent with prior observation, the positive pAD-GAL4-2.1-SmSINA clone contains amino acids 157–371 which includes the conserved C-terminal region of the SINA protein (Fig. 2), suggesting that the C-terminal region of SmSINA is the target protein-binding domain. Schistosome SINA RING finger motif and its cysteine-rich C-terminal region are highly conserved when compared to Drosophila and mammalian SINA proteins. SmSINA also shares an identical nuclear localization signal with SINA from other species (Fig. 2).

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Fig. 2. Sequence analysis of schistosome SmSINA. (A) Domain structure of SmSINA: RING finger motif; cysteine-rich region; NLS, nuclear localization signal. (B) Amino acid sequence alignment of SmSINA protein with SINA from the Caenorhabditis elegans (accession no. AAB94380) mouse Siahb (accession no. Z19580), human SIAH-1 (accession no. U76247), human SIAH-2 (accession no. U76248), and Drosophila (accession no. M38384) homologues. The conserved RING finger motif is boxed and in blue. The conserved cystienes and histidines are in color in the RING finger and C-rich domains. The conserved nuclear localization signal is shown in red. %I and %S are identity and similarity scores.

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Fig. 3. Expression of SmSINA. RT–PCR analysis of SmSINA and ␣-tubulin from different developmental stages. Top panel shows intensity of SmSINA RT–PCR products (middle panel) relative to Sm ␣-tubulin RT–PCR products (bottom panel). The lanes are numbered in the top panel. Middle panel shows SmSINA RT–PCR products using RNA isolated from various S. mansoni life cycle stages and controls. Lanes 1, 3, and 4, mRNA was prepared from uninfected Biomphalaria glabrata, S. mansoni in vitro transformed primary sporocysts, and 30-day infected B. glabrata, respectively. Lane 2 represents the egg stage. Lane 5, 3-day schistosomule; lane 6, 7-day lung stage; lane 7, 15-day schistosomule; lane 8, 21-day immature worm; lane 9, 28-day worms; lane 10, 30-day worm pairs; lane 11, 32-day worm pairs; lane 12, 35-day worm pairs; lane 13, 45-day worm pairs; lane 14, mature 45-day female worm; lane 15, mature 45-day male worm; lane 16, 45-day single sex female worm; lane 17, 45-day single sex male worm; lane 18, 25-day single sex female worm; and lane 19, no template negative control. Bottom panel shows results from ␣-tubulin RT–PCR using RNA isolated from the same stages and controls as for the middle panel.

3.3. RT–PCR analysis of SmSINA RT–PCR analysis was performed on total RNA isolated from various developmental stages of the parasite life cycle to evaluate the expression of SmSINA throughout development. ␣-Tubulin specific primers were included in each reaction as an internal control representing a constitutivelyexpressed gene (Fig. 3). SmSINA was expressed more or less constituitively in the different life stages tested. 3.4. In vitro interaction of SmSINA with SmRXR1 and SmRXR2 To determine if the SmSINA interaction with SmRXR1 and SmRXR2 detected in the yeast two-hybrid occurs in vitro, and also to identify the domains involved in this interaction, GST-SmRXR1/SmRXR2 fusion proteins immobilized on glutathione beads were used to affinity-purify (“pull-down”) radiolabeled SmSINA produced in a rabbit reticulocyte system. Fig. 4A shows GST-fusion proteins of different domains of SmRXR1 and SmRXR2. SmSINA was able to interact with the C-terminus (the AF-2 domain, E–F region) of either SmRXR1 or SmRXR2 (Fig. 4B, lanes 5 and 8). SmSINA was also able to interact with sequences present in the AF-1 domain (AB region) of SmRXR2 (Fig. 4B, lane 6). However, no binding was observed in the internal DNA binding domain of either SmRXR1 or SmRXR2 (Fig. 4B, lanes 4 and 7).

3.5. SmSINA-dependent ubiquitination of SmRXR1 and SmRXR2 To evaluate a possible role for SmSINA in SmRXR1 and SmRXR2 degradation, we tested the effect of SmSINA on ubiquitination of SmRXR1 and SmRXR2 in an in vitro ubiquitination reaction, containing SmSINA, ubiquitin and a HeLa cell fraction containing the enzymes for ubiquitin conjugation. In order to determine which regions of schistosome RXRs serve as substrates in ubiquitination reactions, we used the three regions of SmRXR1 and SmRXR2 (AF-1, DBD, and AF-2 domains) in the reactions. The RING finger domain present in the N-terminal region of Drosophila SINA and mammalian SIAH/Siah has been implicated in the degradation of interacting proteins [14,15,23–25]. The C-termini of these proteins were shown to be involved in mediating protein–protein interactions [15]. In order to investigate the role of these domains in the function of schistosome SINA, we analyzed the effects of GST-SINA-N, GST-SINA-R, GST-SINA-C, as well as GST-SmSINA-
-C in the polyubiquitination of SmRXR1 and SmRXR2 AF-2 domains. As shown in Fig. 5A, GST-SmSINA-
-C was able to promote a clear polyubiquitination in the AF-2 regions of SmRXR1 (lane 9) and SmRXR2 (lane 4). However, no polyubiquitination was observed when SmRXR1-AF-2 (Fig. 5A, lanes 6, 7, and 8), or SmRXR2-AF-2 (Fig. 5A, lanes 1, 2, and 3) were incubated with the three independent domains of SmSINA. Fig. 5B shows the negative controls, highlighting the specificity of

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Fig. 4. Biochemical characterization of the association between SmSINA and SmRXR proteins. (A) Schematic representation of SmRXR1 and SmRXR2 proteins and deletion mutants encoded by GST-fusion cDNA constructs used in the pull-down experiments. These fusion proteins were immobilized on glutathione-Sepharose beads and incubated with 35 S-SmSINA prepared by coupled in vitro transcription and translation system. After five washes, bound proteins were analyzed by SDS–PAGE and exposed to X-ray film. (B) Identification of SmSINA-binding to domains of SmRXR1 and SmRXR2.

Fig. 5. SmSINA dependent in vitro ubiquitination of SmRXR1 and SmRXR2. Equivalent amounts of GST-fusion-SmRXR1 and GST-fusion-SmRXR2 E–F regions were incubated with HeLa extract (fraction II) and ubiquitin in the absence (−) or presence (+) of purified recombinant SmSINA
-C (lacking the last 27 amino acids of the protein), SmSINA-N (N-terminal only), SmSINA-R (RING finger only) or SmSINA-C (C-terminus only). GST-fusion proteins were isolated, separated by gel electrophoresis and ubiquitinated SmRXR1 and SmRXR2 were observed by Western blot analysis using anti-ubiquitin antibody. (A) Experimental groups; (B) controls.

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the polyubiquitination reactions of schistosome RXRs due to the function of schistosome SINA protein. 3.6. Co-localization of endogenous SmSINA with SmRXR2 Affinity-purified anti-SmSINA and anti-SmRXR2 antibodies were used to localize the corresponding native proteins in adult schistosome cryo-sections. The results revealed a substantial immunoreactivity against SmSINA (Fig. 6, pan-

els H and J) and SmRXR2 (Fig. 6, panel F) in the vitelline cells and the tegument layer. In sections probed with the pre-immune mouse antibodies (Fig. 6, panels A–C) and pre-immune rabbit IgG (data not shown) only female worm auto-fluorescence could be observed with the 522 nm filter (Fig. 6, panel B). No specific reactivity was observed with a 680 nm filter (Fig. 6, panel C). Similarly, in sections probed with RXR2 antibodies, the sections visualized with 522 nm filter showed auto-fluorescence of female vitellaria (Fig. 6,

Fig. 6. Immunolocalization of SmSINA and SmRXR2. S. mansoni adult worm cryo-sections were probed with pre-immune mouse IgG (panels A–C), anti-SmRXR2 IgG (panels D–F) or anti-SmSINA (panels G–J). Panels A, D, G, and I are phase contrast fields, panels B and E were visualized using the 522 nm filter (representing auto-fluorescence), and panels C, F, H, and J were visualized using the 680 nm filter (representing specific reactivity). In panels B and E, auto-fluorescence of female vitelline cells (V) can be seen. Specific reactivity was only observed in sections probed with anti-SmRXR2 and anti-SmSINA antibodies and visualized with the 680 nm filter (panels C, F, H, and J) in the tegument (T) and vitelline cells (V). I, intestine. Original magnification: 600×.

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panel E) which could easily be separated from the specific reactivity observed using the Alexa Fluor-647 conjugate visualized at 680 nm (panel F).

4. Discussion The ubiquitin–proteasome pathway plays a crucial role in the regulation of intracellular levels of a wide range of proteins, including transcriptional regulators [26]. Ubiquitination is implicated in the control of key cellular functions such as cell cycle progression, signal transduction, cell differentiation and cell death [26–31]. Although members of the nuclear receptor superfamily are known to be degraded in a hormone-dependent manner through the ubiquitin–proteasome pathway [28], the exact mechanism underlying the degradation and turnover of these receptors remains unsolved. In the current study, we demonstrate that S. mansoni SINA protein interacts with SmRXR1 and SmRXR2 and stimulates their ubiquitination in vitro. It has been proposed that SINA proteins promote ubiquitin– proteasome dependent degradation of several nuclear proteins [26–28] as well as DCC, a neuronal plasma membrane protein [15]. The evidence presented here suggests that SmSINA may also regulate the degradation of SmRXR1 and SmRXR2, two nuclear proteins with putative roles in the regulation of the p14-female specific gene expression [4,6]. SINA proteins are widely expressed in embryo and adult tissues. Subcellular fractionation studies showed that SINA proteins are present in the nucleus, the cytosol and in membrane-associated pools [12,13,22,27,31,32]. Our immunofluorescence and RT–PCR studies revealed that SmSINA localizes to a number of tissues and developmental stages. Importantly, SmSINA and both SmRXR1 and SmRXR2 are localized in the vitelline cells of mature female worms. The co-localization patterns suggest that SmSINA may play an important role in the regulation of the expression levels of SmRXR1 and SmRXR2, as well as other proteins. The ubiquitin–proteasome pathway consists of two major steps: the conjugation of ubiquitin to the substrate and subsequent degradation of the ubiquitinated protein by the 26S proteasome [33]. Ubiquitin conjugation involves sequential reactions in which ubiquitin is first activated by an ubiquitin-activating enzyme (E1), then transferred to an ubiquitin-conjugating enzyme (Ubc or E2) and finally ligated to the substrate by an ubiquitin–protein ligase (E3) [33]. The specificity of ubiquitin conjugation is conferred by the E3, which binds the substrate protein and cooperates with the E2 enzyme to catalyze the covalent attachment of ubiquitin to the substrate. The AF-2 domain of nuclear receptors is essential for ligand-activated transcription, involved in coupling the receptor to transcriptional coregulator protein complexes. This domain also contacts components of the proteasome ma-

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chinery, such as the SUG-1, a component of the 26S proteasome [9,34] and the E6-AP coactivator, a member of the E3 ubiquitin–protein ligases [10,35,36]. Our data support a model in which SmSINA stimulates the polyubiquitination of SmRXR1 and SmRXR2 by acting as bridge between the nuclear receptors and the E2/E3 enzymes. Alternatively, SmSINA may bind to the receptors and act as an E3 ligase per se. In this regard, the E3 ligase activity has been demonstrated for the human SIAH-1 protein [23]. The structural requirements underlying the recognition of substrates by E3 ligases are largely unknown. E3 can recognize several substrates [23,25], which is clearly the case for SIAH/Siah proteins. Previous studies showed that while the N-terminal RING finger domain of SINA proteins is required for interacting with E2/E3, the C-terminal region is involved in binding substrates, such as DCC and N-CoR, although the exact binding site has not been determined [15,16,25]. We tested the capacity of SmSINA to promote ubiquitin conjugation as well as the requirement of its putative functional domains to ubiquitinate SmRXR1 and SmRXR2. Our in vitro ubiquitination data, showed clearly that the polyubiquitination of SmRXR1 and SmRXR2 required the entire coding region of SmSINA, including both functional domains, the RING finger and the cysteine-rich C-terminal domains. As expected, each functional domain alone did not promote ubiquitination of SmRXR1 and SmRXR2. This result supports the hypothesis that schistosome SINA protein retains the evolutionary function identified for other SINA homologues. In order to map the interaction sites between SmSINA and SmRXR1 and SmRXR2 in vitro, we carried out pull-down assays. Our protein interaction data indicate that the SmRXR1- and SmRXR2-binding sites for SmSINA reside in the E–F region of the proteins. This finding agrees well with the fact that the AF-2 domains of SmRXR1 and SmRXR2 contain putative ubiquitination sites. A common feature of short-lived regulatory proteins is the presence of regions that are rich in proline (P), glutamate (E), serine (S), and threonine (T) residues [37,38]. These PEST sequences confer enhanced susceptibility to proteolysis. The manner in which this process occurs is not entirely clear, but likely involves recognition of PEST sequences by the ubiquitinating enzymes or by a component of the proteasome complex. Recognition of PEST sequences may be direct or may require phosphorylation [37,38]. Interestingly, analysis of SmRXR1 and SmRXR2 E–F regions, identified putative PEST motifs. By the same token, the AB region of SmRXR2 contains a putative phosphorylation site, which may function as a potential ubiquitination motif. Indeed, SmSINA was able to interact with the AB region of SmRXR2. These findings suggest that SmRXR1 and SmRXR2 are candidates to be rapidly turned over. Whether this involves a SmSINA-dependent mechanism, awaits further investigation.

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M.R. Fantappi´e et al. / Molecular & Biochemical Parasitology 131 (2003) 45–54

Acknowledgements This work was supported by National Institutes of Health Grant AI46762. We thank Dr. Wade Sigurdson, Head of the Confocal Microscopy and 3-Dimensional Imaging Laboratory for help with microscopy.

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