1 Departamento de Patología Experimental, Centro de Investigación y de Estudios Avanzados del IPN (CINVESTAV-IPN), AP 14-740, DF 07000, Mexico
2 Departamento de Genética y Biología Molecular, Centro de Investigación y de Estudios Avanzados del IPN (CINVESTAV-IPN), AP 14-740, DF 07000, Mexico
Correspondence
E. Orozco
esther{at}mail.cinvestav.mx
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The GenBank accession number for the sequence reported in this paper is AJ489250.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Few DNA-binding proteins involved in E. histolytica transcription have been identified (Petri et al., 1987; Gómez et al., 1998
; Pérez et al., 1998
; Schaenman et al., 2001
). The E. histolytica TATA-box-binding protein (EhTBP) has 55 % homology with the human TBP (Luna-Arias et al., 1999
) and is located in the nuclei and kinetoplast-like organelles (EhkO), which contain DNA (Orozco et al., 1997
). However, no studies have been done on the genome protection events that occur in E. histolytica, and DNA-binding proteins involved in these processes are unknown. In mammals, the product of the p53 tumour-suppressor gene is a transcriptional factor that has been implicated in safeguarding genomic stability. This protein is located in the cytoplasm of cells during G1-phase, then at the onset of the S-phase it is translocated into the nucleus and accumulated later in the cytoplasm during the G2/M interphase (Shaulsky et al., 1990a
). Genotoxic treatments induce an increase of cellular p53 levels (Cox & Lane, 1995
) which is related to G1-phase arrest (Smith et al., 1994
) and DNA repair, but a high expression of p53 leads to apoptosis (Enoch & Norbury, 1995
). The mechanisms by which p53 specifies its distinct functional options remain unclear. p53-like proteins and their DNA-binding consensus sequences have been found in mammals (Kumaravel & Wafik, 2000
), clams (Van Beneden et al., 1997
), squid (Schmale & Bamberger, 1997
) and Drosophila melanogaster (Ollman et al., 2000
; Brodsky et al., 2000
), among others, but not in lower eukaryotes. D. melanogaster p53 (Dmp53) has 2150 % identity to the human p53 main motifs and shows many, but not all, of the functions discovered for the human p53 (Ollman et al., 2000
; Brodsky et al., 2000
). The study of p53-like proteins in primitive organisms might help to elucidate the primitive functions of this multifunctional protein.
Here, we report the identification and cloning of an E. histolytica protein (Ehp53), which to our knowledge is the first p53-like protozoan protein described so far. Ehp53 binds to oligonucleotides containing the mammalian p53 consensus sequence (oli-p53), is recognized by antibodies against the human p53, accumulates in the cells after UV irradiation, and is mainly located in nuclei and EhkOs. The Ehp53-encoding gene was cloned and the corresponding amino acid sequence was compared with the human p53 and Dmp53. The main domains of the three proteins have significant homology.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Electrophoretic mobility-shift assays.
Aliquots of 15 µg of protein of nuclear extracts (NE) prepared as described by Gómez et al. (1998) were incubated for 10 min at 4 °C with poly dG : dC (1 µg µl-1) in binding buffer containing 12 mM HEPES pH 7·9, 60 mM KCl, 10 % (v/v) glycerol, 1 mM DTT, 1 mM EDTA, 1 mM spermidine and 1 mM MgCl2. Then, [
-32P]dATP-end-labelled (10 000 c.p.m.) oli-p53 (5'-GGACATGCCCGGGCATGTCC-3') (El-Deiry et al., 1992
) or oli-Eh112 (5'-AGAAATTCATGGGCTAGTGG-3') (García-Rivera et al., 2000
) double-stranded oligonucleotides were added to the mixture. Incubation continued for another 10 min at 4 °C. In some experiments, before adding the labelled oligonucleotides, the mixture was incubated for 1 h at 4 °C with 1 µl (10 µg) of Ab-1 or Ab-2 (Banks et al., 1986
) monoclonal antibodies against the C-terminus or the N-terminus of the human p53 protein, respectively, purchased from Oncogene Science. DNAprotein complexes were analysed by 6 % PAGE. Gels were dried and exposed to X-O-MAT film at -70 °C. Oligonucleotides were synthesized by BioSynthesis USA.
Protein extraction, gel electrophoresis and Western blot assays.
Trophozoites (1x107), COS or C33 cells (1x107) were washed with phosphate-buffered saline pH 7·2 (PBS) at 4 °C and disrupted by freezethawing three times in 100 mM Tris/HCl pH 8·0 with 100 mM p-hydroxymercuribenzoate. Protein concentration was measured by the Bradford method (Bradford, 1976) and 15 µg samples of proteins were analysed by one-dimensional electrophoresis in 10 % SDS-PAGE or 30 µg samples by two-dimensional PAGE (10 %), using conventional solutions and Bio-lyte 57 and 3·510 pH range ampholines (Bio-Rad) (O'Farrell et al., 1977
). Duplicate gels were Coomassie blue stained or transferred to nitrocellulose filters for Western blot assays (Towbin et al., 1979
). Filters were blocked with 0·05 % (v/v) Tween 20 and 4 % (w/v) non-fat milk in PBS (PBS-TM) for 23 h at room temperature and incubated for 2 h at room temperature with 10 µg ml-1 of the Ab-1 or Ab-2 monoclonal antibodies. The immunoreactivity was detected by the chemiluminescence method (Amersham) using anti-mouse peroxidase-labelled goat secondary antibodies. In all experiments, the same nitrocellulose filters were probed with anti-actin antibody as an internal control. The membranes were submerged in 100 mM 2-mercaptoethanol, 2 % (w/v) SDS, and 62·5 mM Tris/HCl pH 6·7 and incubated at 60 °C for 30 min. The filters were rinsed twice for 10 min with large volumes of TBS/1 % Tween buffer, and after testing the filters for the absence of the first antibody the immunodetection with the anti-actin antibody was performed as described.
UV irradiation of E. histolytica trophozoites.
Trophozoites (2·5x107) in TYI-S-33 medium were transferred to plastic dishes and incubated for 60 min at 37 °C. The medium and non-adhered trophozoites were then discarded and 10 ml PBS was added to the remaining cells. Trophozoites were irradiated with 254 nm UV light at 80 J m-2 for 4 s. After irradiation, the trophozoites were incubated in TYI-S-33 medium for 0, 30, 60 and 120 min at 37 °C. Trophozoites were lysed, protein concentration was measured and PAGE (10 %) and Western blot assays were done using the Ab-2 antibody.
Confocal laser microscopy experiments.
Trophozoites grown on coverslips were fixed with 4 % (w/v) paraformaldehyde for 1 h at 37 °C and permeabilized with 0·2 % (v/v) Triton X-100 for 30 min at 37 °C. Then, cells were incubated with Ab-2 for 1 h at 37 °C, followed by anti-mouse fluoresceinated second antibody for 1 h at 37 °C. After incubation, cells were washed three times with PBS at room temperature. Before observation, they were counterstained for 5 min with propidium iodide solution (1 µg ml-1). Samples were mounted with antifade and visualized through a confocal scanning system MRC 1024 (Bio-Rad) equipped with a krypton/argon laser and fitted to a Diaphot 200-inverted microscope (Nikon).
Cloning of the Ehp53 gene and expression of the recombinant polypeptide.
The Ehp53 gene was obtained from an E. histolytica ZAP cDNA library (Sánchez et al., 1994
) using the Ab-2 monoclonal antibody against the human p53, according to the methodology described by Sambrook et al. (1989)
. The Ehp53 cDNA clone (1300 bp) was then used as a probe to obtain a DNA clone from an E. histolytica
ZAP genomic DNA library (Descoteaux et al., 1992
). Ehp53 cDNA and DNA clones were subjected to automatic sequencing (Perkin Elmer). Sequence data analysis was performed with the FASTA algorithm (Pearson & Lipman, 1988
) in the EMBL and GenBank databases. The analysis of consensus sequences was performed with the software package of the University of Wisconsin Genetics Computer Group (Devereux et al., 1984
). A DNA fragment (1880 bp) encoding the 293 amino acids at the amino-terminus of Ehp53 cDNA was PCR-amplified and cloned in-frame with the histidine tag of the pRSETA plasmid to be expressed in Escherichia coli. Proteins from IPTG-induced bacteria were separated by 10 % PAGE and submitted to Western blot assays using Ab-2 and a commercial anti-histidine antibody (Invitrogen).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Detection of Ehp53 in total proteins of E. histolytica
To identify the E. histolytica proteins that are recognized by the antibodies against human p53 and that may form complexes with oli-p53, we carried out Western blot assays of total E. histolytica proteins separated by one- and two-dimensional PAGE. In one-dimensional PAGE Ab-1 and Ab-2 recognized a 53 kDa band (Ehp53) in the trophozoites of clone A (strain HMI IMSS) and strain 462, which co-migrated with the p53 protein of COS cells (Fig. 2a, b).
|
The number of proteins recognized by Ab-1 and Ab-2 was investigated by two-dimensional PAGE (Fig. 2d). Ab-1 (not shown) and Ab-2 revealed a single 53 kDa spot in the basic region of the gel, between pI 7·5 and 8·0 (Fig. 2e
).
The results presented in Fig. 2 show that Ehp53 is antigenically related to the amino- and carboxy-terminus of the human p53 and it seems to be a single protein according to the two-dimensional gel results. The electrophoretic migration of Ehp53 predicted a molecular mass similar to those found for members of the p53 family in many species.
UV irradiation increases the amount of Ehp53 in E. histolytica trophozoites
In mammalian cells, UV irradiation produces an accumulation of p53 (Cox & Lane, 1995). The effect of UV irradiation upon live trophozoites on Ehp53 exposed to UV light (80 J m-2) was determined as described in Methods. In Western blot analysis, the Ab-2 antibodies revealed a stronger Ehp53 band in proteins obtained from trophozoites incubated for 30 min after UV irradiation. The band increased in intensity at 60 min and started to diminish in trophozoites incubated for 120 min after UV irradiation (Fig. 3
a). Anti-actin antibodies showed that all lanes were loaded with a similar amount of protein (Fig. 3b
). The increase of Ehp53 may be related to DNA repair, suggesting a putative functional similarity between Ehp53 and the mammalian p53.
|
|
|
A recombinant polypeptide (Ehp53293) is recognized by Ab-2
Western blot assays using a recombinant polypeptide (Ehp53293) obtained by the expression of the first 293 amino acid residues of Ehp53 showed that the anti-histidine and the Ab-2 antibodies recognized Ehp53293 (Fig. 6a), indicating that it is an Ehp53 recombinant protein containing the histidine tag. Given that Ab-2 recognized a single spot in two-dimensional gels, we assume that the 53 kDa band recognized in total proteins and the recombinant polypeptide Ehp53293 correspond to the same protein, except that Ehp53293 lacks the Ehp53 carboxy-terminus.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
As the most evolutionarily distant member of the p53 gene family to be identified, Ehp53 may shed some light on the selective pressures that have maintained p53-like molecules through evolution. It is likely that a protein with important and multiple functions may have arisen very early and may be conserved through evolution. The possibility of horizontal gene transfer of the p53 gene from the human host to the parasite is discarded because of the high divergence in sequence between human p53 and Ehp53. The fact that Ab-2 recognized 53 kDa proteins in E. invadens, a snake parasite, and E. moshkovskii, a free-living Entamoeba species, supports the hypothesis that p53 arose earlier than was thought.
An interesting finding was the presence of higher amounts of Ehp53 in the cytoplasm and EhkOs than in the nuclei of the trophozoites, shown by confocal microscopy. In mammalian cells, mutations in the nuclear localization signal of p53 and p53 proteins complexed with viral or cellular proteins prevent the efficient passage of p53 through the nuclear membrane (Shaulsky et al., 1990b). EhTBP (Luna-Arias et al., 1999
) and the C/EBP-like transcription factor (Marchat et al., 2002
) are also present in nuclei and EhkOs. It would be of interest to investigate whether nuclear and extra-nuclear Ehp53 have similar structure and function.
The results of our experiments strongly support the hypothesis that Ehp53 and the human p53 correspond to homologous proteins. (i) Ehp53 and p53 have been identified by two antibodies directed against different regions of p53, and both proteins showed the same relative mobility in electrophoresis, as was shown by comparison with the COS or C33 cells used throughout this work as controls. (ii) The concentration of both mammalian p53 and Ehp53 protein increases after UV irradiation. In mammals, p53 has a short half-life (1525 min), which increases after UV irradiation (Maltzman & Zyzyk, 1984). Ehp53 may also show an extended half-life after UV-irradiation of the trophozoites. (iii) Ehp53 also binds to specific DNA sequences; it is present in the nucleus and the EhkO organelle, co-localizing with DNA. Altogether, the results presented here suggest that Ehp53 is a part of the DNAprotein complexes detected by gel-shift assays, supporting the idea that this protein is a transcription factor. (iv) oli-Eh112, a 20 bp sequence homologous to the consensus p53-binding sequence present in the Ehadh112 gene promoter, formed complexes with NE of E. histolytica trophozoites and with NE of COS cells. Complexes were competed by non-labelled oli-Eh112 and oli-p53, indicating their specificity and confirming the high homology between oli-Eh112 and oli-p53. (v) The amino acid sequence predicted from the Ehp53 gene showed the presence of conserved domains reported for p53. Additionally, the recombinant Ehp53 protein was recognized by Ab-2.
Questions on the functions of Ehp53 in E. histolytica remain open. In mammalian cells, p53 participates in apoptosis, DNA repair and cell cycle regulation, preventing uncontrolled cellular division, which produces cancer. Ehp53 shows changes in three of the more frequently mutated positions described for p53 in transformed cells and in two of the residues involved in Zn2+ binding (Fig. 5, asterisks). However, at this time, we cannot speculate on the significance of these changes. The functional characterization of Ehp53 is currently under study.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ankri, S., Stolarsky, T., Bracha, R., Padilla-Vaca, F. & Mirelman, D. (1999). Antisense inhibition of expression of cysteine proteinases affects Entamoeba histolytica-induced formation of liver abscess in hamsters. Infect Immun 67, 421422.
Banks, L., Matlashewsky, G. & Crawford, L. (1986). Isolation of human-p53-specific monoclonal antibodies and their use in studies of human p53 expression. Eur J Biochem 159, 529534.[Abstract]
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248254.[CrossRef][Medline]
Brodsky, M. H., Nordstrom, W., Tsang, G., Kwan, E., Rubin, G. M. & Abrams, J. M. (2000). Drosophila p53 binds a damage response element at the reaper locus. Cell 101, 103113.[Medline]
Clark, C. G. & Diamond, L. S. (1991). The Laredo strain and other "Entamoeba histolytica-like" amoebae are E. moshkovskii. Mol Biochem Parasitol 46, 1118.[Medline]
Cox, L. S. & Lane, D. P. (1995). Tumour suppressors, kinases and clamps: how p53 regulates the cell cycle in response to DNA damage. BioAssays 17, 501508.[Medline]
Cox, L. S., Midgley, C. A. & Lane, D. P. (1994). Xenopus p53 is biochemically similar to the human tumor suppressor protein p53 and is induced upon DNA damage in somatic cells. Oncogene 9, 29512959.[Medline]
Descoteaux, S., Ayala, P., Samuelson, J. & Orozco, E. (1992). Primary sequences of two P-glycoprotein genes of Entamoeba histolytica. Mol Biochem Parasitol 54, 200202.
Devereux, J., Haeberli, P. & Smithies, O. (1984). A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12, 387395.[Abstract]
Diamond, L. S., Harlow, D. R. & Cunick, C. C. (1978). A new medium for the axenic cultivation of Entamoeba histolytica and other Entamoeba. Trans R Soc Trop Med Hyg 72, 431432.[Medline]
El-Deiry, W. S., Kern, S. E., Pietenpol, J. A., Kinzler, K. W. & Vogelstein, B. (1992). Definition of a consensus binding site for p53. Nat Genet 1, 4549.[Medline]
Enoch, T. & Norbury, C. (1995). Cellular responses to DNA damage: cell-cycle checkpoints, apoptosis and the roles of p53 and ATM. Trends Biochem Sci 20, 426430.[CrossRef][Medline]
García-Rivera, G., Gómez, C., Pérez, D. G., Flores, E., Rodríguez, M. A. & Orozco, E. (2000). Structural characterization of the 5'-flanking regions of the Entamoeba histolytica Ehcp112 and Ehadh112 genes. Arch Med Res 31, S303S304.[CrossRef][Medline]
Gómez, C., Pérez, D. G., Lopez-Bayghen, E. & Orozco, E. (1998). Transcriptional analysis of the EhPgp1 promoter of Entamoeba histolytica multidrug-resistant mutant. J Biol Chem 273, 72777284.
Guimaraes, S., Urdaneta, H., Silva, E. F. & Tavares, C. A. (1991). Entamoeba histolytica: antigenic characterization of axenic strains from Brazil. Rev Inst Med Trop Sao Paulo 33, 611.[Medline]
Kumaravel, S. & Wafik, E. S. (2000). Tumor suppressor p53: regulation and function. Front Biosci 5, 424437.
Loidl, A. & Loidl, P. (1996). Oncogene and tumor suppressor gene-related proteins in plants and fungi. Crit Rev Oncogenesis 7, 4964.[Medline]
Lu, W., Pochampally, R., Chen, L., Traidej, M., Wang, Y. & Chen, J. (2000). Nuclear exclusion of p53 in a subset of tumors requires MDM2 function. Oncogene 19, 232240.[CrossRef][Medline]
Luna-Arias, J. P., Hernandez-Rivas, R., De Dios-Bravo, G., García, J., Mendoza, L. & Orozco, E. (1999). The TATA-box binding protein of Entamoeba histolytica: cloning of the gene and location of the protein by immunofluorescence and confocal microscopy. Microbiology 145, 3340.[Abstract]
Maltzman, W. & Czyzyk, L. (1984). UV irradiation stimulates levels of p53 cellular tumor antigen in nontransformed mouse cells. Mol Cell Biol 4, 16891694.[Medline]
Marchat, L. A., Gómez, C., Pérez, D. G., Paz, F., Mendoza, L. & Orozco, E. (2002). The CCAAT/enhancer binding protein sites are cis-activator elements of the Entamoeba histolytica Ehpgp1 (mdr-like) gene expresion. Cell Microbiol 4, 725737.[Medline]
Middeler, G., Zerf, K., Jenovai, S., Thulig, A., Tschodrich-Rotter, M., Kubitscheck, U. & Peters, R. (1997). The tumor suppressor p53 is subject to both nuclear import and export, and both are fast, energy-dependent and lectin-inhibited. Oncogene 14, 14071417.[CrossRef][Medline]
O'Farrell, P. Z., Goodman, H. M. & O'Farrell, P. H. (1977). High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell 12, 11331142.[Medline]
Ollman, M., Young, L. M., Di Como, C. J. & 10 other authors (2000). Drosophila p53 is a structural and functional homolog of the tumor suppressor p53. Cell 101, 91101.[Medline]
Orozco, E., Guarneros, G., Martínez-Palomo, A. & Sánchez, T. (1983). Entamoeba histolytica. Phagocytosis as a virulence factor. J Exp Med 158, 15111521.[Abstract]
Orozco, E., Gharaibeh, R., Riverón, A. M., Delgadillo, D. M., Mercado, M. & Sanchez, T. (1997). A novel cytoplasmic structure containing DNA networks in Entamoeba histolytica trophozoites. Mol Gen Genet 254, 250257.[CrossRef][Medline]
Pearson, W. R. & Lipman, D. J. (1988). Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A 85, 24442448.[Abstract]
Pérez, D. G., Gómez, C., Lopez-Bayghen, E., Tannich, E. & Orozco, E. (1998). Transcriptional analysis of the EhPgp5 promoter of the Entamoeba histolytica multidrug-resistant mutant. J Biol Chem 273, 72857292.
Petri, W. A., Smith, R. D., Schlesinger, P. H. & Ravdin, J. I. (1987). Isolation of the galactose binding lectin which mediates the in vitro adherence of Entamoeba histolytica. J Clin Invest 80, 12381244.[Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sánchez, M. A., Peattie, D. A., Wirth, D. & Orozco, E. (1994). Cloning, genomic organization and transcription of the Entamoeba histolytica -tubulin encoding gene. Gene 146, 239244.[CrossRef][Medline]
Schaenman, J. M., Gilchrist, C. A., Mann, B. J. & Petri, W. A., Jr (2001). Identification of two E. histolytica sequence-specific URE4 enhancer binding proteins with homology to the RNA-binding motif RRM. J Biol Chem 276, 16021609.
Scheffner, M., Werness, B. A., Huibregtse, J. M., Levine, A. J. & Howley, P. M. (1990). The E6 oncoprotein encoded by human papillomavirus type 16 and 18 promotes the degradation of p53. Cell 63, 11291136.[Medline]
Schmale, H. & Bamberger, C. (1997). A novel protein with strong homology to the tumor suppressor p53. Oncogene 15, 13631367.[CrossRef][Medline]
Shaulsky, G., Ben-Ze'ev, A. & Rotter, V. (1990a). Subcellular distribution of the p53 protein during the cell cycle of Balb/c 3T3 cells. Oncogene 6, 17071711.
Shaulsky, G., Goldfinger, N., Ben-Ze'ev, A. & Rotter, V. (1990b). Nuclear accumulation of p53 protein is mediated by several nuclear localization signals and plays a role in tumorigenesis. Mol Cell Biol 10, 65656577.[Medline]
Smith, M. L., Chen, I. T., Zhan, Q., Bae, I., Chen, C. Y., Gilmer, T. M., Kastan, M. B., O'Connor, P. M. & Fomace, A. J., Jr (1994). Interaction of the p53-regulated protein GADD45 with proliferating cell nuclear antigen. Science 266, 13761380.[Medline]
Stommel, J. M., Marchenko, N. D., Jimenez, G. S., Moll, U. M., Hope, T. J. & Wahl, G. M. (1999). A leucine-rich nuclear export signal in the p53 tetramerization domain: regulation of subcellular localization and p53 activity by NES masking. EMBO J 18, 16601672.
Towbin, H., Staehelin, T. & Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76, 43504356.[Abstract]
Van Beneden, R. J., Walker, C. W. & Laughner, E. S. (1997). Characterization of gene expression of a p53 homologue in the soft-shell clam (Mya arenaria). Mol Mar Biol Biotechnol 6, 116122.[Medline]
WHO (1997). Amoebiasis. WHO Weekly Epidemiol Record 72, 97100.[Medline]
Received 19 July 2002;
revised 30 October 2002;
accepted 19 January 2003.