Entamoeba histolytica EhDEAD1 is a conserved DEAD-box RNA helicase with ATPase and ATP-dependent RNA unwinding activities

López-Camarillo, César; García-Hernández, María de la Luz; Marchat, Laurence A.; Luna-Arias, Juan Pedro; Cruz, Olga Hernández de la; Mendoza, Leobardo; Orozco, Esther

doi:10.1016/j.gene.2008.01.024

Cited by 13 publications

(21 citation statements)

References 46 publications

(66 reference statements)

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“…The recent completion of the E. histolytica genome sequence generated a large raw data set that can be used to gain insights into the molecular biology of this organism. The present work and the accompanying paper by López-Camarillo et al (2008) are the first reports about RNA helicases in this protozoan parasite. The EhDEAD and EhDExH-box families of E. histolytica are similar in size to RNA helicases families described in other eukaryotic organisms (de la Cruz et al, 1999;Boudet et al, 2001;Abdelhaleem et al, 2003), including the protozoan parasites L. major (Ivens et al, 2005) and P. falciparum (Tuteja and Pradhan, 2006).…”

Section: Discussionmentioning

confidence: 61%

Putative DEAD and DExH-box RNA helicases families in Entamoeba histolytica

Marchat

Orozco

Guillén

et al. 2008

Gene

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Section: Discussionmentioning

confidence: 61%

Putative DEAD and DExH-box RNA helicases families in Entamoeba histolytica

Marchat

Orozco

Guillén

et al. 2008

Gene

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“…No signal was detected using pre-immune serum (lanes 3 and 4). The use of antibodies against EhPAP that has been immunodetected in both NE and CE [36], confirmed protein integrity (lanes 5 and 6), while the use of antibodies against the nuclear EhPC4 [42] validated cell fractionation of both extracts (lanes 7 and 8). Intriguingly, anti-EhCFIm25 antibodies revealed a 55 kDa band whereas the predicted molecular weight for the polypeptides referred to as C4M2T1, Q7YZC1 and B1N3I5 is 30.4, 28.1 and 15.2 kDa, respectively.…”

Section: Resultsmentioning

confidence: 99%

“…As controls, we used anti-EhPC4 antibodies that specifically identified the putative E. histolytica transcription and polyadenylation factor EhPC4 in the nuclear fraction [42] and antibodies against EhPAP that has been previously located in both CE and NE [36]. In some experiments, proteins were analyzed in the presence of a higher concentration of β-mercaptoethanol (8%) and 8 M urea, in order to improve reducing conditions.…”

Section: Methodsmentioning

confidence: 99%

“…cDNA were synthesized using 1 μg total RNA, 100 ng oligo (dT18), 100 mM DTT, 10 mM dNTPs, 40 U SUPERase-in (Ambion) and 200 U Superscript II reverse transcriptase (Invitrogen) in first-strand buffer, for 1 h at 42°C. Semi-quantitative PCR assays were performed with 1⁄5 volume of the reverse transcription mixture, 10 mM each dNTPs, 5 mM MgCl 2 and 2.5 units of Taq DNA polymerase (Invitrogen) [42], using 5´-GAACAACAGAACCAATGCAAG-3´ (sense) and 5'-CAATTTTTAATCTTCCTCCAACA-3' (antisense) as internal primers for EhCFIm25 transcripts. Amplification was performed as follows: 94 ºC for 5 min; 94 ºC for 1 min, 53 ºC for 1 min and 72 ºC for 1 min, for 25 cycles, and a final extension step at 72 ºC for 7 min.…”

Section: Methodsmentioning

confidence: 99%

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The 25 kDa Subunit of Cleavage Factor Im Is a RNA-Binding Protein That Interacts with the Poly(A) Polymerase in Entamoeba histolytica

Pezet-Valdez¹,

Fernández‐Retana²,

Ospina-Villa³

et al. 2013

PLoS ONE

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In eukaryotes, polyadenylation of pre-mRNA 3´ end is essential for mRNA export, stability and translation. Taking advantage of the knowledge of genomic sequences of Entamoeba histolytica, the protozoan responsible for human amoebiasis, we previously reported the putative polyadenylation machinery of this parasite. Here, we focused on the predicted protein that has the molecular features of the 25 kDa subunit of the Cleavage Factor Im (CFIm25) from other organisms, including the Nudix (nucleoside diphosphate linked to another moiety X) domain, as well as the RNA binding domain and the PAP/PAB interacting region. The recombinant EhCFIm25 protein (rEhCFIm25) was expressed in bacteria and used to generate specific antibodies in rabbit. Subcellular localization assays showed the presence of the endogenous protein in nuclear and cytoplasmic fractions. In RNA electrophoretic mobility shift assays, rEhCFIm25 was able to form specific RNA-protein complexes with the EhPgp5 mRNA 3´ UTR used as probe. In addition, Pull-Down and LC/ESI-MS/MS tandem mass spectrometry assays evidenced that the putative EhCFIm25 was able to interact with the poly(A) polymerase (EhPAP) that is responsible for the synthesis of the poly(A) tail in other eukaryotic cells. By Far-Western experiments, we confirmed the interaction between the putative EhCFIm25 and EhPAP in E. histolytica. Taken altogether, our results showed that the putative EhCFIm25 is a conserved RNA binding protein that interacts with the poly(A) polymerase, another member of the pre-mRNA 3´ end processing machinery in this protozoan parasite.

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“…Several genes from E. histolytica were cloned, sequenced and expressed in an effort to identify new drug targets for this parasite, including the alcohol dehydrogenase gene (Ehadh3) [18], the ferredoxin oxidoreductase gene [19], the EhDEAD1 RNA helicase gene [20], and the Ehvma2 gene (which encodes the B subunit of the vacuolar ATPase) [21]. Mechanisms of drug resistance in E. histolytica were also studied, and it was determined that the multidrug-resistant phenotype is regulated at the transcriptional level by the P-glycoprotein-like genes (EhPgp) 1 and 5 [22].…”

Section: Identification Of Lipopeptidophosphoglycanmentioning

confidence: 99%

The Role of Lipopeptidophosphoglycan in the Immune Response toEntamoeba histolytica

Wong‐Baeza

Alcántara‐Hernández

Mancilla-Herrera

et al. 2010

Journal of Biomedicine and Biotechnology

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The sensing of Pathogen Associated Molecular Patterns (PAMPs) by innate immune receptors, such as Toll-like receptors (TLRs), is the first step in the inflammatory response to pathogens. Entamoeba histolytica, the etiological agent of amebiasis, has a surface molecule with the characteristics of a PAMP. This molecule, which was termed lipopeptidophosphoglycan (LPPG), is recognized through TLR2 and TLR4 and leads to the release of cytokines from human monocytes, macrophages, and dendritic cells; LPPG-activated dendritic cells have increased expression of costimulatory molecules. LPPG activates NKT cells in a CD1d-dependent manner, and this interaction limits amebic liver abscess development. LPPG also induces antibody production, and anti-LPPG antibodies prevent disease development in animal models of amebiasis. Because LPPG is recognized by both the innate and the adaptive immune system (it is a “Pamptigen”), it may be a good candidate to develop a vaccine against E. histolytica infection and an effective adjuvant.

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Entamoeba histolytica EhDEAD1 is a conserved DEAD-box RNA helicase with ATPase and ATP-dependent RNA unwinding activities

Cited by 13 publications

References 46 publications

Putative DEAD and DExH-box RNA helicases families in Entamoeba histolytica

Putative DEAD and DExH-box RNA helicases families in Entamoeba histolytica

The 25 kDa Subunit of Cleavage Factor Im Is a RNA-Binding Protein That Interacts with the Poly(A) Polymerase in Entamoeba histolytica

The Role of Lipopeptidophosphoglycan in the Immune Response toEntamoeba histolytica

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