scholarly journals N6-Adenosine methylation on mRNA is recognized by YTH2 domain protein of human malaria parasite Plasmodium falciparum

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Gayathri Govindaraju ◽  
Rajashekar Varma Kadumuri ◽  
Devadathan Valiyamangalath Sethumadhavan ◽  
C. A. Jabeena ◽  
Sreenivas Chavali ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Transcriptional Control ◽  
Developmental Stages ◽  
Epigenetic Modification ◽  
Site Directed Mutagenesis ◽  
Transcriptional Level ◽  
Major Protein ◽  
Sequence Specificity ◽  
Sequencing Data ◽  
Fluorescence Depolarization

Abstract Background Plasmodium falciparum exhibits high translational plasticity during its development in RBCs, yet the regulation at the post-transcriptional level is not well understood. The N6-methyl adenosine (m6A) is an important epigenetic modification primarily present on mRNA that controls the levels of transcripts and efficiency of translation in eukaryotes. Recently, the dynamics of m6A on mRNAs at all three developmental stages of P. falciparum in RBCs have been profiled; however, the proteins that regulate the m6A containing mRNAs in the parasites are unknown. Results Using sequence analysis, we computationally identified that the P. falciparum genome encodes two putative YTH (YT521-B Homology) domain-containing proteins, which could potentially bind to m6A containing mRNA. We developed a modified methylated RNA immunoprecipitation (MeRIP) assay using PfYTH2 and find that it binds selectively to m6A containing transcripts. The PfYTH2 has a conserved aromatic amino acid cage that forms the methyl-binding pocket. Through site-directed mutagenesis experiments and molecular dynamics simulations, we show that F98 residue is important for m6A binding on mRNA. Fluorescence depolarization assay confirmed that PfYTH2 binds to methylated RNA oligos with high affinity. Further, MeRIP sequencing data revealed that PfYTH2 has more permissive sequence specificity on target m6A containing mRNA than other known eukaryotic YTH proteins. Taken together, here we identify and characterize PfYTH2 as the major protein that could regulate m6A containing transcripts in P. falciparum. Conclusion Plasmodium spp. lost the canonical m6A-specific demethylases in their genomes, however, the YTH domain-containing proteins seem to be retained. This study presents a possibility that the YTH proteins are involved in post-transcriptional control in P. falciparum, and might orchestrate the translation of mRNA in various developmental stages of P. falciparum. This is perhaps the first characterization of the methyl-reading function of YTH protein in any parasites.

scholarly journals Identification and characterization of a novel GGA/C-binding protein, GBP-i, that is rapidly inducible by cytokines.

1994 ◽  
Vol 14 (12) ◽  
pp. 7770-7781 ◽  
Author(s):  
G V Raj ◽  
K Khalili
Keyword(s):  
Phorbol Myristate Acetate ◽  
Transforming Growth Factor Beta ◽  
Transcriptional Control ◽  
Transforming Growth Factor ◽  
Jc Virus ◽  
Interleukin 1 ◽  
Transcriptional Level ◽  
Sequence Specificity ◽  
Myristate Acetate ◽  
Necrosis Factor Alpha

Immunosuppressive states with accompanying alterations in cytokine profiles have been postulated to play a vital role in the reactivation of viruses from latency. Cytokines regulate gene expression by activating transcription factors via well-characterized signal transduction pathways. In this study, we report the identification of a novel inducible protein, GBP-i, that binds to a double-stranded GGA/C-rich region of the transcriptional control region of the human papovavirus JC virus (JCV), specifically within the origin of viral DNA replication. GBP-i is distinct from previously characterized GC-box-binding proteins with respect to both its sequence specificity and its electrophoretic mobility on native and denaturing gels. GBP-i responds within 90 min to phorbol myristate acetate stimulation; however, unlike typical phorbol myristate acetate-inducible factors, this rapid induction is regulated primarily at the transcriptional level. Further, the induction of GBP-i appears to be widespread and mediated by many inflammatory cytokines, including interleukin-1 beta, tumor necrosis factor alpha, gamma interferon, and transforming growth factor beta. Interestingly, the induced protein acts as a transcriptional repressor in its native context in the JCVL promoter. However, when its binding sequence is transposed to a heterologous promoter, GBP-i appears to function as a transcriptional activator. The data presented here suggest a role for GBP-i in cytokine-mediated induction of viral and cellular genes.

Identification and characterization of a novel GGA/C-binding protein, GBP-i, that is rapidly inducible by cytokines

1994 ◽  
Vol 14 (12) ◽  
pp. 7770-7781
Author(s):  
G V Raj ◽  
K Khalili
Keyword(s):  
Phorbol Myristate Acetate ◽  
Transforming Growth Factor Beta ◽  
Transcriptional Control ◽  
Transforming Growth Factor ◽  
Jc Virus ◽  
Interleukin 1 ◽  
Transcriptional Level ◽  
Sequence Specificity ◽  
Myristate Acetate ◽  
Necrosis Factor Alpha

Immunosuppressive states with accompanying alterations in cytokine profiles have been postulated to play a vital role in the reactivation of viruses from latency. Cytokines regulate gene expression by activating transcription factors via well-characterized signal transduction pathways. In this study, we report the identification of a novel inducible protein, GBP-i, that binds to a double-stranded GGA/C-rich region of the transcriptional control region of the human papovavirus JC virus (JCV), specifically within the origin of viral DNA replication. GBP-i is distinct from previously characterized GC-box-binding proteins with respect to both its sequence specificity and its electrophoretic mobility on native and denaturing gels. GBP-i responds within 90 min to phorbol myristate acetate stimulation; however, unlike typical phorbol myristate acetate-inducible factors, this rapid induction is regulated primarily at the transcriptional level. Further, the induction of GBP-i appears to be widespread and mediated by many inflammatory cytokines, including interleukin-1 beta, tumor necrosis factor alpha, gamma interferon, and transforming growth factor beta. Interestingly, the induced protein acts as a transcriptional repressor in its native context in the JCVL promoter. However, when its binding sequence is transposed to a heterologous promoter, GBP-i appears to function as a transcriptional activator. The data presented here suggest a role for GBP-i in cytokine-mediated induction of viral and cellular genes.

scholarly journals The Structure of the Cysteine-Rich Domain of Plasmodium falciparum P113 Identifies the Location of the RH5 Binding Site

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Ivan Campeotto ◽  
Francis Galaway ◽  
Shahid Mehmood ◽  
Lea K. Barfod ◽  
Doris Quinkert ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Binding Site ◽  
Structural Information ◽  
Blood Stage ◽  
Site Directed Mutagenesis ◽  
Effective Vaccine ◽  
Binding Region ◽  
Fab Fragment ◽  
Content Type ◽  
Blood Stage Malaria

ABSTRACT Plasmodium falciparum RH5 is a secreted parasite ligand that is essential for erythrocyte invasion through direct interaction with the host erythrocyte receptor basigin. RH5 forms a tripartite complex with two other secreted parasite proteins, CyRPA and RIPR, and is tethered to the surface of the parasite through membrane-anchored P113. Antibodies against RH5, CyRPA, and RIPR can inhibit parasite invasion, suggesting that vaccines containing these three components have the potential to prevent blood-stage malaria. To further explore the role of the P113-RH5 interaction, we selected monoclonal antibodies against P113 that were either inhibitory or noninhibitory for RH5 binding. Using a Fab fragment as a crystallization chaperone, we determined the crystal structure of the RH5 binding region of P113 and showed that it is composed of two domains with structural similarities to rhamnose-binding lectins. We identified the RH5 binding site on P113 by using a combination of hydrogen-deuterium exchange mass spectrometry and site-directed mutagenesis. We found that a monoclonal antibody to P113 that bound to this interface and inhibited the RH5-P113 interaction did not inhibit parasite blood-stage growth. These findings provide further structural information on the protein interactions of RH5 and will be helpful in guiding the development of blood-stage malaria vaccines that target RH5. IMPORTANCE Malaria is a deadly infectious disease primarily caused by the parasite Plasmodium falciparum. It remains a major global health problem, and there is no highly effective vaccine. A parasite protein called RH5 is centrally involved in the invasion of host red blood cells, making it—and the other parasite proteins it interacts with—promising vaccine targets. We recently identified a protein called P113 that binds RH5, suggesting that it anchors RH5 to the parasite surface. In this paper, we use structural biology to locate and characterize the RH5 binding region on P113. These findings will be important to guide the development of new antimalarial vaccines to ultimately prevent this disease, which affects some of the poorest people on the planet.

scholarly journals Polycombs and microRNA-223 regulate human granulopoiesis by transcriptional control of target gene expression

Blood ◽  
2012 ◽  
Vol 119 (17) ◽  
pp. 4034-4046 ◽  
Author(s):  
Giuseppe Zardo ◽  
Alberto Ciolfi ◽  
Laura Vian ◽  
Linda M. Starnes ◽  
Monia Billi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Sequences ◽  
Transcriptional Control ◽  
Transcriptional Level ◽  
Seed Region ◽  
Mrna Targets ◽  
Epigenetic Events ◽  
Evolutionarily Conserved ◽  
Lineage Decision ◽  
Chromatin Remodeling Complexes

Abstract Epigenetic modifications regulate developmental genes involved in stem cell identity and lineage choice. NFI-A is a posttranscriptional microRNA-223 (miR-223) target directing human hematopoietic progenitor lineage decision: NFI-A induction or silencing boosts erythropoiesis or granulopoiesis, respectively. Here we show that NFI-A promoter silencing, which allows granulopoiesis, is guaranteed by epigenetic events, including the resolution of opposing chromatin “bivalent domains,” hypermethylation, recruitment of polycomb (PcG)–RNAi complexes, and miR-223 promoter targeting activity. During granulopoiesis, miR-223 localizes inside the nucleus and targets the NFI-A promoter region containing PcGs binding sites and miR-223 complementary DNA sequences, evolutionarily conserved in mammalians. Remarkably, both the integrity of the PcGs-RNAi complex and DNA sequences matching the seed region of miR-223 are required to induce NFI-A transcriptional silencing. Moreover, ectopic miR-223 expression in human myeloid progenitors causes heterochromatic repression of NFI-A gene and channels granulopoiesis, whereas its stable knockdown produces the opposite effects. Our findings indicate that, besides the regulation of translation of mRNA targets, endogenous miRs can affect gene expression at the transcriptional level, functioning in a critical interface between chromatin remodeling complexes and the genome to direct fate lineage determination of hematopoietic progenitors.

Biochemical characterization of Plasmodium falciparum CTP:phosphoethanolamine cytidylyltransferase shows that only one of the two cytidylyltransferase domains is active

2013 ◽  
Vol 450 (1) ◽  
pp. 159-167 ◽  
Author(s):  
Sweta Maheshwari ◽  
Marina Lavigne ◽  
Alicia Contet ◽  
Blandine Alberge ◽  
Emilie Pihan ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Cellular Localization ◽  
Biochemical Characterization ◽  
De Novo ◽  
Membrane Lipids ◽  
Polyclonal Antibodies ◽  
Homology Modelling ◽  
Site Directed Mutagenesis ◽  
Recombinant Enzymes ◽  
Parasite Life Cycle

The intra-erythrocytic proliferation of the human malaria parasite Plasmodium falciparum requires massive synthesis of PE (phosphatidylethanolamine) that together with phosphatidylcholine constitute the bulk of the malaria membrane lipids. PE is mainly synthesized de novo by the CDP:ethanolamine-dependent Kennedy pathway. We previously showed that inhibition of PE biosynthesis led to parasite death. In the present study we characterized PfECT [P. falciparum CTP:phosphoethanolamine CT (cytidylyltransferase)], which we identified as the rate-limiting step of the PE metabolic pathway in the parasite. The cellular localization and expression of PfECT along the parasite life cycle were studied using polyclonal antibodies. Biochemical analyses showed that the enzyme activity follows Michaelis–Menten kinetics. PfECT is composed of two CT domains separated by a linker region. Activity assays on recombinant enzymes upon site-directed mutagenesis revealed that the N-terminal CT domain was the only catalytically active domain of PfECT. Concordantly, three-dimensional homology modelling of PfECT showed critical amino acid differences between the substrate-binding sites of the two CT domains. PfECT was predicted to fold as an intramolecular dimer suggesting that the inactive C-terminal domain is important for dimer stabilization. Given the absence of PE synthesis in red blood cells, PfECT represents a potential antimalarial target opening the way for a rational conception of bioactive compounds.

scholarly journals The Most Polymorphic Residue on Plasmodium falciparum Apical Membrane Antigen 1 Determines Binding of an Invasion-Inhibitory Antibody

2006 ◽  
Vol 74 (5) ◽  
pp. 2628-2636 ◽  
Author(s):  
A. M. Coley ◽  
K. Parisi ◽  
R. Masciantonio ◽  
J. Hoeck ◽  
J. L. Casey ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Apical Membrane ◽  
Site Directed Mutagenesis ◽  
Supporting Evidence ◽  
Inhibitory Antibody ◽  
Apical Membrane Antigen 1 ◽  
Apical Membrane Antigen ◽  
Immune Pressure ◽  
Polymorphic Residue

ABSTRACT Apical membrane antigen 1 (AMA1) is currently one of the leading malarial vaccine candidates. Anti-AMA1 antibodies can inhibit the invasion of erythrocytes by Plasmodium merozoites and prevent the multiplication of blood-stage parasites. Here we describe an anti-AMA1 monoclonal antibody (MAb 1F9) that inhibits the invasion of Plasmodium falciparum parasites in vitro. We show that both reactivity of MAb 1F9 with AMA1 and MAb 1F9-mediated invasion inhibition were strain specific. Site-directed mutagenesis of a fragment of AMA1 displayed on M13 bacteriophage identified a single polymorphic residue in domain I of AMA1 that is critical for MAb 1F9 binding. The identities of all other polymorphic residues investigated in this domain had little effect on the binding of the antibody. Examination of the P. falciparum AMA1 crystal structure localized this residue to a surface-exposed α-helix at the apex of the polypeptide. This description of a polymorphic inhibitory epitope on AMA1 adds supporting evidence to the hypothesis that immune pressure is responsible for the polymorphisms seen in this molecule.

scholarly journals Structure-function analysis of human transforming growth factor-α by site-directed mutagenesis

1992 ◽  
Vol 283 (1) ◽  
pp. 91-98 ◽  
Author(s):  
J A Feild ◽  
R H Reid ◽  
D J Rieman ◽  
T P Kline ◽  
G Sathe ◽  
...  
Keyword(s):  
Biological Activity ◽  
Growth Factor ◽  
Receptor Binding ◽  
Transcriptional Control ◽  
Transforming Growth Factor ◽  
Function Analysis ◽  
Protein A ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Transforming Growth Factor Α

Site-directed mutants of transforming growth factor-alpha (TGF-alpha) were expressed in an Escherichia coli outer membrane protein A (ompA) expression/secretion vector under the transcriptional control of the lambda PL promoter. TGF-alpha mutant proteins were isolated from cell pellets using alkaline extraction with 0.1 M-Tris (pH 10.5). The levels of protein expression of 23 TGF-alpha mutants were comparable with those of wild-type TGF-alpha, as determined by immunoblotting and radioimmunoassay. An analysis of biological activity using as assays radioreceptor binding competition and colony formation in soft agar showed that the following mutations destroy the activity of TGF-alpha: Gly-19 to Val, Val-33 to Pro and Gly-40 to Val. Mutations of Arg-42 to Lys, Leu-48 to Ala, Tyr-38 to Trp or Phe-17 to Tyr significantly decrease, but do not destroy, biological activity when compared with the wild-type. Mutations in 14 other residues did not significantly alter receptor binding or colony-forming activity. These studies suggest that two domains localized at the surface of TGF-alpha are important in receptor binding and colony-forming activity. Domain I involves amino acid residues which include Tyr-38 and Leu-48; domain II includes residues Phe-15, Phe-17 and Arg-42.

scholarly journals A Bayesian approach for analysis of whole-genome bisulphite sequencing data identifies disease-associated changes in DNA methylation

2016 ◽  
Author(s):  
Owen J.L. Rackham ◽  
Sarah R. Langley ◽  
Thomas Oates ◽  
Eleni Vradi ◽  
Nathan Harmston ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Epigenetic Modification ◽  
Promoter Hypermethylation ◽  
Whole Genome ◽  
Sequencing Data ◽  
Accurate Identification ◽  
Bisulphite Sequencing ◽  
Consistent Change ◽  
Rat Strain ◽  
Differential Transcription

ABSTRACTDNA methylation is a key epigenetic modification involved in gene regulation whose contribution to disease susceptibility remains to be fully understood. Here, we present a novel Bayesian smoothing approach (called ABBA) to detect differentially methylated regions (DMRs) from whole-genome bisulphite sequencing (WGBS). We also show how this approach can be leveraged to identify disease-associated changes in DNA methylation, suggesting mechanisms through which these alterations might affect disease. From a data modeling perspective, ABBA has the distinctive feature of automatically adapting to different correlation structures in CpG methylation levels across the genome whilst taking into account the distance between CpG sites as a covariate. Our simulation study shows that ABBA has greater power to detect DMRs than existing methods, providing an accurate identification of DMRs in the large majority of simulated cases. To empirically demonstrate the method’s efficacy in generating biological hypotheses, we performed WGBS of primary macrophages derived from an experimental rat system of glomerulonephritis and used ABBA to identify >1,000 disease-associated DMRs. Investigation of these DMRs revealed differential DNA methylation localized to a 600bp region in the promoter of the Ifitm3 gene. This was confirmed by ChIP-seq and RNA-seq analyses, showing differential transcription factor binding at the Ifitm3 promoter by JunD (an established determinant of glomerulonephritis) and a consistent change in Ifitm3 expression. Our ABBA analysis allowed us to propose a new role for Ifitm3 in the pathogenesis of glomerulonephritis via a mechanism involving promoter hypermethylation that is associated with Ifitm3 repression in the rat strain susceptible to glomerulonephritis.

scholarly journals Efficacy of Artemisinin Base Combination Therapy and Genetic Diversity of Plasmodium falciparum from Uncomplicated Ma-laria Falciparum Patient in District of Pesawaran, Province of Lampung, Indonesia

2019 ◽  
Author(s):  
Jhons Fatriyadi SUWANDI ◽  
Widya ASMARA ◽  
Hari KUSNANTO ◽  
Din SYAFRUDDIN ◽  
Supargiyono SUPARGIYONO
Keyword(s):  
Plasmodium Falciparum ◽  
Genetic Variation ◽  
Falciparum Malaria ◽  
Health Centre ◽  
Sequencing Data ◽  
Single Nucleotide ◽  
Pfmdr1 Gene ◽  
Pcr Product ◽  
Nucleotide Mutation ◽  
Reverse Primer

Background: Malaria is an infectious disease caused by Plasmodium sp., that still prevalence in some part of Indonesia. District of Pesawaran is one of malaria endemic area in the Province of Lampung. The purpose of this study was to evaluate the efficacy of the ACT treatment in the District of Pesawaran Province of Lampung, Indonesia from Dec 2012 to Jul 2013 and the genetic variation of the Plasmodium falciparum also studied. Methods: This study was observational analytic study of falciparum malaria patients treated with ACT and primaquine (DHP-PQ and AAQ-PQ) at Hanura Primary Health Centre (Puskesmas). DNA isolation was done with QIAmp DNA Mini Kit. Amplification of PfMDR1, MSP1, and MSP2 genes was done with appropriate forward and reverse primer and procedures optimized first. PCR Product of PfMDR1 gene was prepared for sequencing. Data analysis was done with MEGA 6 software. Results: The results of this research are DHP-PQ effectiveness was still wellness among falciparum malaria patients in District of Pesawaran, Province of Lampung, Indonesia. There is Single-nucleotide mutation of N86Y of PfMDR1 gene. The dominant alleles found are MAD20 and 3D7 alleles with Multiplicity of Infection (MOI) are low. Conclusion: Therapy of DHP-PQ as an antimalarial falciparum in Pesawaran District, Lampung, Indonesia is still good. The genetic variation found was the SNP on the N86Y PfMDR1 gene, with dominant allele MAD20 and 3D7.

scholarly journals Transcriptional Regulation of the Copper Transporter Mfc1 in Meiotic Cells

2013 ◽  
Vol 12 (4) ◽  
pp. 575-590 ◽  
Author(s):  
Jude Beaudoin ◽  
Raphaël Ioannoni ◽  
Stéphane Mailloux ◽  
Samuel Plante ◽  
Simon Labbé
Keyword(s):  
Transcriptional Activation ◽  
Regulatory Element ◽  
Specific Protein ◽  
Copper Transport ◽  
Site Directed Mutagenesis ◽  
Regulatory Sequence ◽  
Transcriptional Level ◽  
Binding Studies ◽  
Copper Transporter ◽  
Content Type

ABSTRACT Mfc1 is a meiosis-specific protein that mediates copper transport during the meiotic program in Schizosaccharomyces pombe . Although the mfc1 + gene is induced at the transcriptional level in response to copper deprivation, the molecular determinants that are required for its copper starvation-dependent induction are unknown. Promoter deletion and site-directed mutagenesis have allowed identification of a new cis -regulatory element in the promoter region of the mfc1 + gene. This cis -acting regulatory sequence containing the sequence TCGGCG is responsible for transcriptional activation of mfc1 + under low-copper conditions. The TCGGCG sequence contains a CGG triplet known to serve as a binding site for members of the Zn (2) Cys (6) binuclear cluster transcriptional regulator family. In agreement with this fact, one member of this group of regulators, denoted Mca1, was found to be required for maximum induction of mfc1 + gene expression. Analysis of Mca1 cellular distribution during meiosis revealed that it colocalizes with both chromosomes and sister chromatids during early, middle, and late phases of the meiotic program. Cells lacking Mca1 exhibited a meiotic arrest at metaphase I under low-copper conditions. Binding studies revealed that the N-terminal 150-residue segment of Mca1 expressed as a fusion protein in Escherichia coli specifically interacts with the TCGGCG sequence of the mfc1 + promoter. Taken together, these results identify the cis -regulatory TCGGCG sequence and the transcription factor Mca1 as critical components for activation of the meiotic copper transport mfc1 + gene in response to copper starvation.

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