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Geraldo G S Oliveira,
Franklin B Magalhães,
Márcia C A Teixeira,
Andrea M Pereira,
Cristiane G M Pinheiro,
Lenita R Santos,
Marília B Nascimento,
Cheila N G Bedor,
Alessandra L Albuquerque,
Washington L C dos-Santos,
Yara M Gomes,
Edson D Moreira,
Maria E F Brito,
Lain C Pontes de Carvalho, Osvaldo P de Melo Neto
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ABSTRACT: To expand the available panel of recombinant proteins that can be useful for identifying Leishmania-infected dogs and for diagnosing human visceral leishmaniasis (VL), we selected recombinant antigens from L. infantum, cDNA, and genomic libraries by using pools of serum samples from infected dogs and humans. The selected DNA fragments encoded homologs of a cytoplasmic heat-shock protein 70, a kinesin, a polyubiquitin, and two novel hypothetical proteins. Histidine-tagged recombinant proteins were produced after subcloning these DNA fragments and evaluated by using an enzyme-linked immunosorbent assays with panels of canine and human serum samples. The enzyme-linked immunosorbent assays with different recombinant proteins had different sensitivities (67.4-93.0% and 36.4-97.2%) and specificities (76.1-100% and 90.4-97.3%) when tested with serum samples from Leishmania-infected dogs and human patients with VL. Overall, no single recombinant antigen was sufficient to serodiagnosis all canine or human VL cases.
The American journal of tropical medicine and hygiene 12/2011; 85(6):1025-34. · 2.59 Impact Factor
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Eden R Freire,
Rafael Dhalia,
Danielle M N Moura,
Tamara D da Costa Lima,
Rodrigo P Lima,
Christian R S Reis,
Katie Hughes,
Regina C B Q Figueiredo,
Nancy Standart,
Mark Carrington, Osvaldo P de Melo Neto
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ABSTRACT: Translation initiation in eukaryotes requires eIF4E, the cap binding protein, which mediates its function through an interaction with the scaffolding protein eIF4G, as part of the eIF4F complex. In trypanosomatids, four eIF4E homologues have been described but the specific function of each is not well characterized. Here, we report a study of these proteins in Trypanosoma brucei (TbEIF4E1 through 4). At the sequence level, they can be assigned to two groups: TbEIF4E1 and 2, similar in size to metazoan eIF4E1; and TbEIF4E3 and 4, with long N-terminal extensions. All are constitutively expressed, but whilst TbEIF4E1 and 2 localize to both the nucleus and cytoplasm, TbEIF4E3 and 4 are strictly cytoplasmic and are also more abundant. After knockdown through RNAi, TbEIF4E3 was the only homologue confirmed to be essential for viability of the insect procyclic form. In contrast, TbEIF4E1, 3 and 4 were all essential for the mammalian bloodstream form. Simultaneous RNAi knockdown of TbEIF4E1 and 2 caused cessation of growth and death in procyclics, but with a delayed impact on translation, whilst knockdown of TbEIF4E3 alone or a combined TbEIF4E1 and 4 knockdown led to substantial translation inhibition which preceded cellular death by several days, at least. Only TbEIF4E3 and 4 were found to interact with T. brucei eIF4G homologues; TbEIF4E3 bound both TbEIF4G3 and 4 whilst TbEIF4E4 bound only to TbEIF4G3. These results are consistent with TbEIF4E3 and 4 having distinct but relevant roles in initiation of protein synthesis.
Molecular and Biochemical Parasitology 03/2011; 176(1):25-36. · 2.55 Impact Factor
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ABSTRACT: Trypanosomatid protozoans are reliant on posttranscriptional processes to control gene expression. Regulation occurs at the levels of mRNA processing, stability, and translation, events that may require the participation of the poly(A) binding protein (PABP). Here, we have undertaken a functional study of the three distinct Leishmania major PABP (LmPABP) homologues: the previously described LmPABP1; LmPABP2, orthologous to the PABP described from Trypanosoma species; and LmPABP3, unique to Leishmania. Sequence identity between the three PABPs is no greater than 40%. In assays measuring binding to A-rich sequences, LmPABP1 binding was poly(A) sensitive but heparin insensitive; LmPABP2 binding was heparin sensitive and less sensitive to poly(A), compatible with unique substitutions observed in residues implicated in poly(A) binding; and LmPABP3 displayed intermediate properties. All three homologues are simultaneously expressed as abundant cytoplasmic proteins in L. major promastigotes, but only LmPABP1 is present as multiple isoforms. Upon transcription inhibition, LmPABP2 and -3 migrated to the nucleus, while LmPABP1 remained predominantly cytoplasmic. Immunoprecipitation assays showed an association between LmPABP2 and -3. Although the three proteins bound to a Leishmania homologue of the translation initiation factor eukaryotic initiation factor 4G (eIF4G) (LmEIF4G3) in vitro, LmPABP1 was the only one to copurify with native LmEIF4G3 from cytoplasmic extracts. Functionality was tested using RNA interference (RNAi) in Trypanosoma brucei, where both orthologues to LmPABP1 and -2 are required for cellular viability. Our results indicate that these homologues have evolved divergent functions, some of which may be unique to the trypanosomatids, and reinforces a role for LmPABP1 in translation through its interaction with the eIF4G homologue.
Eukaryotic Cell 10/2010; 9(10):1484-94. · 3.60 Impact Factor
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ABSTRACT: We report the identification of two distinct homologues of the 70-kDa mitochondrial heat shock protein (mtHSP70) from Leishmania chagasi/Leishmania infantum (Lc2.1 and Lc2.2). In Leishmania species, multiple genes encoding Lc2.2 are present whilst single genes encode Lc2.1. Strikingly, genes encoding Lc2.1-like proteins are absent from Trypanosoma species. Lc2.2 is characterized by a poly-glutamine rich C-terminus, absent from Lc2.1 or mtHSP70 homologues outside the trypanosomatids. Lc2.1 displays unique substitutions within its peptide-binding domain which modify amino acids strictly conserved in cytoplasmic and mitochondrial HSP70 proteins alike. Affinity purified antibodies recognize mainly a single protein in extracts from promastigotes/epimastigotes of various Leishmania/Trypanosoma species. Upon differentiation of Leishmania amazonensis into amastigotes a second protein (presumably Lc2.1) is induced and becomes the predominant mtHSP70 homologue expressed. Subcellular localization of these proteins was investigated and ratified a distribution throughout the mitochondrial matrix. Our results imply novel mtHSP70 functions which evolved within the genus Leishmania.
Molecular and Biochemical Parasitology 09/2008; 160(2):157-62. · 2.55 Impact Factor
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ABSTRACT: Protozoan parasites belonging to the family Trypanosomatidae are characterized by an unusual pathway for the production of mRNAs via polycistronic transcription and trans-splicing of a 5' capped mini-exon which is linked to the 3' cleavage and polyadenylation of the upstream transcript. However, little is known of the mechanism of protein synthesis in these organisms, despite their importance as agents of a number of human diseases. Here we have investigated the role of two Trypanosoma brucei homologues of the translation initiation factor eIF4A (in the light of subsequent experiments these were named as TbEIF4AI and TbEIF4AIII). eIF4A, a DEAD-box RNA helicase, is a subunit of the translation initiation complex eIF4F which binds to the cap structure of eukaryotic mRNA and recruits the small ribosomal subunit. TbEIF4AI is a very abundant predominantly cytoplasmic protein (over 1 x 10(5) molecules/cell) and depletion to approximately 10% of normal levels through RNA interference dramatically reduces protein synthesis one cell cycle following double-stranded RNA induction and stops cell proliferation. In contrast, TbEIF4AIII is a nuclear, moderately expressed protein (approximately 1-2 x 10(4) molecules/cell), and its depletion stops cellular proliferation after approximately four cell cycles. Ectopic expression of a dominant negative mutant of TbEIF4AI, but not of TbEIF4AIII, induced a slow growth phenotype in transfected cells. Overall, our results suggest that only TbEIF4AI is involved in protein synthesis while the properties and sequence of TbEIF4AIII indicate that it may be the orthologue of eIF4AIII, a component of the exon junction complex in mammalian cells.
Nucleic Acids Research 02/2006; 34(9):2495-507. · 8.03 Impact Factor
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ABSTRACT: In eukaryotes protein synthesis initiates with the binding of the multimeric translation initiation complex eIF4F - eIF4E, eIF4A and eIF4G - to the monomethylated cap present on the 5' end of mRNAs. eIF4E interacts directly with the cap nucleotide, while eIF4A is a highly conserved RNA helicase and eIF4G acts as a scaffold for the complex with binding sites for both eIF4E and eIF4A. eIF4F binding to the mRNA recruits the small ribosomal subunit to its 5' end. Little is known in detail of protein synthesis in the protozoan parasites belonging to the family Trypanosomatidae. However, the presence of the highly modified cap structure, cap4, and the spliced leader sequence on the 5' ends of all mRNAs suggests possible differences in mRNA recruitment by ribosomes. We identified several potential eIF4F homologues by searching Leishmania major databases: four eIF4Es (LmEIF4E1-4), two eIF4As (LmEIF4A1-2) and five eIF4Gs (LmEIF4G1-5). We report the initial characterisation of LmEIF4E1-3, LmEIF4A1-2 and LmEIF4G3. First, the expression of these proteins in L. major promastigotes was quantitated by Western blotting using isoform specific antibodies. LmEIF4A1 and LmEIF4E3 are very abundant, LmEIF4G3 is moderately abundant and LmEIF4E1/LmEIF4E2/LmEIF4A2 are rare or not detected. In cap-binding assays, only LmEIF4E1 bound to the 7-methyl-GTP-Sepharose resin. Molecular modelling confirmed that LmEIF4E1 has all the structural features of a cap-binding protein. Finally, pull-down assays were used to investigate the potential interaction between the eIF4A (LmEIF4A1/LmEIF4A2) and eIF4G (LmEIF4G1-3) homologues. Only LmEIF4G3, via the HEAT domain, bound specifically both to LmEIF4A1 as well as to human eIF4A. Therefore for each factor, one of the L. major forms seems to fulfil, in part at least, the expected characteristics of a translational initiation factor.
Molecular and Biochemical Parasitology 04/2005; 140(1):23-41. · 2.55 Impact Factor
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ABSTRACT: The poly(A)-binding protein (PABP), bound to the 3' poly(A) tail of eukaryotic mRNAs, plays critical roles in mRNA translation and stability. PABP autoregulates its synthesis by binding to a conserved A-rich sequence present in the 5'-untranslated region of PABP mRNA and repressing its translation. PABP is composed of two parts: the highly conserved N terminus, containing 4 RNA recognition motifs (RRMs) responsible for poly(A) and eIF4G binding; and the more variable C terminus, which includes the recently described PABC domain, and promotes intermolecular interaction between PABP molecules as well as cooperative binding to poly(A). Here we show that, in vitro, GST-PABP represses the translation of reporter mRNAs containing 20 or more A residues in their 5'-untranslated regions and remains effective as a repressor when an A61 tract is placed at different distances from the cap, up to 126 nucleotides. Deletion of the PABP C terminus, but not the PABC domain alone, significantly reduces its ability to inhibit translation when bound to sequences distal to the cap, but not to proximal ones. Moreover, cooperative binding by multiple PABP molecules to poly(A) requires the C terminus, but not the PABC domain. Further analysis using pull-down assays shows that the interaction between PABP molecules, mediated by the C terminus, does not require the PABC domain and is enhanced by the presence of RRM 4. In vivo, fusion proteins containing parts of the PABP C terminus fused to the viral coat protein MS2 have an enhanced ability to prevent the expression of chloramphenicol acetyltransferase reporter mRNAs containing the MS2 binding site at distal distances from the cap. Altogether, our results identify a proline- and glutamine-rich linker located between the RRMs and the PABC domain as being strictly required for PABP/PABP interaction, cooperative binding to poly(A) and enhanced translational repression of reporter mRNAs in vitro and in vivo.
Journal of Biological Chemistry 12/2003; 278(47):46357-68. · 4.77 Impact Factor
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ABSTRACT: The poly(A)-binding protein (PABP), bound to the 3′ poly(A) tail of eukaryotic mRNAs, plays critical roles in mRNA translation
and stability. PABP autoregulates its synthesis by binding to a conserved A-rich sequence present in the 5′-untranslated region
of PABP mRNA and repressing its translation. PABP is composed of two parts: the highly conserved N terminus, containing 4
RNA recognition motifs (RRMs) responsible for poly(A) and eIF4G binding; and the more variable C terminus, which includes
the recently described PABC domain, and promotes intermolecular interaction between PABP molecules as well as cooperative
binding to poly(A). Here we show that, in vitro, GST-PABP represses the translation of reporter mRNAs containing 20 or more A residues in their 5′-untranslated regions and
remains effective as a repressor when an A61 tract is placed at different distances from the cap, up to 126 nucleotides. Deletion of the PABP C terminus, but not the
PABC domain alone, significantly reduces its ability to inhibit translation when bound to sequences distal to the cap, but
not to proximal ones. Moreover, cooperative binding by multiple PABP molecules to poly(A) requires the C terminus, but not
the PABC domain. Further analysis using pull-down assays shows that the interaction between PABP molecules, mediated by the
C terminus, does not require the PABC domain and is enhanced by the presence of RRM 4. In vivo, fusion proteins containing parts of the PABP C terminus fused to the viral coat protein MS2 have an enhanced ability to
prevent the expression of chloramphenicol acetyltransferase reporter mRNAs containing the MS2 binding site at distal distances
from the cap. Altogether, our results identify a proline- and glutamine-rich linker located between the RRMs and the PABC
domain as being strictly required for PABP/PABP interaction, cooperative binding to poly(A) and enhanced translational repression
of reporter mRNAs in vitro and in vivo.
Journal of Biological Chemistry 11/2003; 278(47):46357-46368. · 4.77 Impact Factor
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ABSTRACT: The poly(A)-binding protein (PABP) is a highly conserved eukaryotic protein whose synthesis is regulated at the post-transcriptional level. The binding of PABP to the poly(A)-rich element found in the 5'-untranslated region (5'UTR) of PABP mRNA specifically inhibits its own translation. In this report, we show that similar adenosine-rich elements in the 5'UTR of the chloramphenicol acetyl-transferase (CAT) gene can significantly reduce the reporter mRNA abundance and translation in human 293 cells. The reduction in mRNA level, but not CAT expression, is dependent on the size of the 5'UTR poly(A) element. Furthermore, one 5'UTR-tethered PABP molecule is enough to inhibit CAT expression without affecting its mRNA level. We propose that the control of PABP synthesis may involve mRNA decay and the repression of translation.
FEBS Letters 08/2003; 546(2-3):329-34. · 3.54 Impact Factor
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ABSTRACT: In eukaryotes protein synthesis initiates with the binding of the multimeric translation initiation complex eIF4F – eIF4E, eIF4A and eIF4G – to the monomethylated cap present on the 5′ end of mRNAs. eIF4E interacts directly with the cap nucleotide, while eIF4A is a highly conserved RNA helicase and eIF4G acts as a scaffold for the complex with binding sites for both eIF4E and eIF4A. eIF4F binding to the mRNA recruits the small ribosomal subunit to its 5′ end. Little is known in detail of protein synthesis in the protozoan parasites belonging to the family Trypanosomatidae. However, the presence of the highly modified cap structure, cap4, and the spliced leader sequence on the 5′ ends of all mRNAs suggests possible differences in mRNA recruitment by ribosomes. We identified several potential eIF4F homologues by searching Leishmania major databases: four eIF4Es (LmEIF4E1-4), two eIF4As (LmEIF4A1-2) and five eIF4Gs (LmEIF4G1-5). We report the initial characterisation of LmEIF4E1-3, LmEIF4A1-2 and LmEIF4G3. First, the expression of these proteins in L. major promastigotes was quantitated by Western blotting using isoform specific antibodies. LmEIF4A1 and LmEIF4E3 are very abundant, LmEIF4G3 is moderately abundant and LmEIF4E1/LmEIF4E2/LmEIF4A2 are rare or not detected. In cap-binding assays, only LmEIF4E1 bound to the 7-methyl-GTP-Sepharose resin. Molecular modelling confirmed that LmEIF4E1 has all the structural features of a cap-binding protein. Finally, pull-down assays were used to investigate the potential interaction between the eIF4A (LmEIF4A1/LmEIF4A2) and eIF4G (LmEIF4G1-3) homologues. Only LmEIF4G3, via the HEAT domain, bound specifically both to LmEIF4A1 as well as to human eIF4A. Therefore for each factor, one of the L. major forms seems to fulfil, in part at least, the expected characteristics of a translational initiation factor.
Molecular and Biochemical Parasitology.
