Nucleic acid binding properties of SmZF1, a zinc finger protein of Schistosoma mansoni

Calzavara-Silva, Carlos Eduardo; Prosdocimi, Francisco; Abath, Frederico G. C.; Pena, Sérgio D.J.; Franco, Glória Regina

doi:10.1016/j.ijpara.2004.07.006

Cited by 5 publications

(9 citation statements)

References 36 publications

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“…Furthermore, unrelated oligonucleotides were not able to abolish this interaction. Taken together, these initial results suggested that SmZF1 may act as a putative transcription factor in S. mansoni [36].…”

Section: Introductionmentioning

confidence: 60%

“…Although it has also been detected in cDNA libraries of other developmental stages of this parasite (i.e., egg, 3 h schistosomulum and cercaria), the biological function of the protein coded by this gene remains to be elucidated. The SmZF1 protein contains three C 2 H 2 -type zinc finger motifs and binds specific DNA oligonucleotides, as do similar nuclear proteins involved in gene transcriptional regulation [35],[36]. Therefore, to investigate whether SmFZ1 is present in the nucleus, where it could act as a transcription factor, we decided to verify its subcellular localization at diverse S. mansoni life stages.…”

Section: Resultsmentioning

confidence: 99%

“…In order to obtain anti-SmZF1 antibodies, the MBP (Maltose Binding Protein) portion of a MBP-SmZF1 recombinant protein [36] was cleaved using Factor Xa protease (New England Biolabs, Ipswitch, MA, USA). The cleavage reaction was carried out for 48 h at 4°C in a 1∶25 enzyme: protein proportion.…”

Section: Methodsmentioning

confidence: 99%

“…In addition, the viral thymidine kinase (tk) promoter region was inserted ( Nhe I/ Bgl II) into the commercial vector pGL3-basic (Promega), generating the vector pGL3-tk-luc, with the luciferase (luc) reporter gene under control of the thymidine kinase promoter. Subsequently, an oligonucleotide containing four repetitions of the putative SmZF1 DNA binding site, D1-3DNA [36], was inserted ( Kpn I/ Nhe I) upstream of the minimal tk promoter, producing the vector pGL3-zf-tk-luc. The oligonucleotide sequence was as follows: 5′-CAGGAAACAGCTATGACCGG CGAGGGAGT GATCGG CGAGGGAGT GATCGG CGAGGGAGT GATCGG CGAGGGAGT GTCGTGACTGGGAAAACCCTGGCG-3′ (specific binding sites D1-3DNA are indicated in bold).…”

Section: Methodsmentioning

confidence: 99%

“…Samples were maintained on ice for 30 min, centrifuged (15000 g, 20 min, 4°C) and then the supernatant was collected. Protein concentrations were measured and normalized as previously described [36]. The extract/DNA binding reactions were carried out in a final volume of 15 µL of binding solution (4 mM Tris-HCl pH 8.0, 40 mM NaCl, 1 mM ZnSO 4 , 4 mM MgCl 2 , 5% glycerol) for 15 min at 4°C.…”

Section: Methodsmentioning

confidence: 99%

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Molecular Characterization of the Schistosoma mansoni Zinc Finger Protein SmZF1 as a Transcription Factor

et al. 2009

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BackgroundDuring its development, the parasite Schistosoma mansoni is exposed to different environments and undergoes many morphological and physiological transformations as a result of profound changes in gene expression. Characterization of proteins involved in the regulation of these processes is of importance for the understanding of schistosome biology. Proteins containing zinc finger motifs usually participate in regulatory processes and are considered the major class of transcription factors in eukaryotes. It has already been shown, by EMSA (Eletrophoretic Mobility Shift Assay), that SmZF1, a S. mansoni zinc finger (ZF) protein, specifically binds both DNA and RNA oligonucleotides. This suggests that this protein might act as a transcription factor in the parasite.Methodology/Principal FindingsIn this study we extended the characterization of SmZF1 by determining its subcellular localization and by verifying its ability to regulate gene transcription. We performed immunohistochemistry assays using adult male and female worms, cercariae and schistosomula to analyze the distribution pattern of SmZF1 and verified that the protein is mainly detected in the cells nuclei of all tested life cycle stages except for adult female worms. Also, SmZF1 was heterologously expressed in mammalian COS-7 cells to produce the recombinant protein YFP-SmZF1, which was mainly detected in the nucleus of the cells by confocal microscopy and Western blot assays. To evaluate the ability of this protein to regulate gene transcription, cells expressing YFP-SmZF1 were tested in a luciferase reporter system. In this system, the luciferase gene is downstream of a minimal promoter, upstream of which a DNA region containing four copies of the SmZF1 putative best binding site (D1-3DNA) was inserted. SmZF1 increased the reporter gene transcription by two fold (p≤0.003) only when its specific binding site was present.ConclusionTaken together, these results strongly support the hypothesis that SmZF1 acts as a transcription factor in S. mansoni.

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Section: Introductionmentioning

confidence: 60%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Molecular Characterization of the Schistosoma mansoni Zinc Finger Protein SmZF1 as a Transcription Factor

et al. 2009

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The molecular characterization and RNAi silencing of SjZFP1 in Schistosoma japonicum

et al. 2014

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During development, Schistosoma japonicum undergoes many morphological and physiological transformations as a result of profound changes in gene expression. Proteins containing zinc finger motifs usually play an important role in DNA recognition, RNA packaging, and transcriptional activation. In our current study, we cloned the open reading frame (ORF) of SjZFP1 of S. japonicum, which encodes a zinc finger protein. We analyzed the complementary DNA (cDNA) sequence of SjZFP1 and examined the expression of SjZFP1 messenger RNA (mRNA) at various developmental stages. We also tested the effects of RNA interference (RNAi) silencing on worm burden, spawning, and egg hatching. The ORF in the SjZFP1 cDNA was 1017 bp in length and was predicted to encode a 338-aa protein with a molecular mass of approximately 38.5 kDa and theoretical isoelectric point (pI) of 7.08. Several conserved regions, including a B-box-type zinc-binding domain, two bipartite nuclear localization signal domains, a paired amphipathic helix repeat, and overlapping RING and PHD finger domains, were identified in the predicted amino acid sequence of SjZFP1. Using real-time PCR, we showed that the SjZFP1 mRNA was expressed across all of the developmental stages of the parasite and that the level of transcription was highest in the cercariae, eggs, schistosomula, and mature adult worms. The level of SjZFP1 mRNA expression in cultured schistosomula treated with one of two SjZFP1-specific small interfering RNAs (siRNAs; AY770 and AY546) was reduced by over 80 %, compared with that in the controls. In RNAi experiments in BALB/c mice, the level of SjZFP1 mRNA increased significantly when the mice were treated with the same SjZFP1-specific siRNAs during the early stages of infection. By contrast, the level of SjZFP1 mRNA decreased significantly when the mice were treated with the SjZFP1-specific siRNAs during the middle to late stages of infection. In four independent experiments, fewer worms were recovered from mice treated with the SjZFP1-specific siRNAs, compared with the number of worms recovered from the control mice. Both the average number and hatching rates of liver eggs recovered from mice treated with the SjZFP1-specific siRNAs during the middle to late stages of infection were significantly lower than those of the liver eggs recovered from the control mice. Our results suggest that the SjZFP1 gene might be important for parasite development, spawning in the vertebrate host, and egg hatching.

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Identification and characterization of the zinc finger protein SjZF in Schistosoma japonicum

Zhang

Jian

Xiang

et al. 2018

Biochemical and Biophysical Research Communications

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Nucleic acid binding properties of SmZF1, a zinc finger protein of Schistosoma mansoni

Cited by 5 publications

References 36 publications

Molecular Characterization of the Schistosoma mansoni Zinc Finger Protein SmZF1 as a Transcription Factor

Molecular Characterization of the Schistosoma mansoni Zinc Finger Protein SmZF1 as a Transcription Factor

The molecular characterization and RNAi silencing of SjZFP1 in Schistosoma japonicum

Identification and characterization of the zinc finger protein SjZF in Schistosoma japonicum

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