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1. A bioinformatic approach for the prediction and functional classification of Toxoplasma gondii long non-coding RNAs

Author

Laura Vanagas, Constanza Cristaldi, Gino La Bella, Agustina Ganuza, Sergio O. Angel, and Andrés M. Alonso

Subjects
Toxoplasma gondii, Non-coding RNAs, Stress response, Differentiation, Medicine, Science
Abstract

Abstract Long non-coding RNAs (lncRNAs) have emerged as significant players in diverse cellular processes, including cell differentiation. Advancements in computational methodologies have facilitated the prediction of lncRNA functions, enabling insights even in non-model organisms like pathogenic parasites, in roles such as parasite development, antigenic variation, and epigenetics. In this work, we focus on the apicomplexan Toxoplasma gondii differentiation process, where the infective stage, tachyzoite, can develop into the cysted stage, bradyzoite, under stress conditions. Using a publicly available transcriptome dataset, we predicted putative lncRNA sequences associated with this differentiation process. Notably, a substantial proportion of these putative lncRNAs exhibited stage-specific expression, particularly at the bradyzoite stage. Furthermore, co-expression patterns between coding transcripts and putative TglncRNAs suggest their involvement in shared processes, such as bradyzoite development. Putative TglncRNA loci analysis revealed their potential influence on the expression of nearby coding genes, including subtelomeric genes unique to the T. gondii genome. Finally we propose a k-mer analysis approach to predict putative functional relationships between characterized lncRNAs from model organisms like Homo sapiens and the putative T. gondii lncRNAs. Our perspective led to predict putative T. gondii lncRNA that potentially could act mediating DNA damage repair pathways, opening a new study field to validate this kind of adaptive mechanisms of T. gondii in response to stress conditions.

Published
2024
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2. Evaluation of topotecan and 10-hydroxycamptothecin on Toxoplasma gondii: Implications on baseline DNA damage and repair efficiency

Author

Constanza Cristaldi, Ana M. Saldarriaga Cartagena, Agustina Ganuza, William J. Sullivan, Jr., Sergio O. Angel, and Laura Vanagas

Subjects
Toxoplasma gondii, Topotecan, 10-Hydroxycamptothecin, DNA damage, Stress replication, DNA damage response, Infectious and parasitic diseases, RC109-216
Abstract

Toxoplasma gondii is an obligate intracellular parasite in the phylum Apicomplexa that causes toxoplasmosis in humans and animals worldwide. Despite its prevalence, there is currently no effective vaccine or treatment for chronic infection. Although there are therapies against the acute stage, prolonged use is toxic and poorly tolerated. This study aims to explore the potential of repurposing topotecan and 10-hydroxycamptothecin (HCPT) as drugs producing double strand breaks (DSBs) in T. gondii. DSBs are mainly repaired by Homologous Recombination Repair (HRR) and Non-Homologous End Joining (NHEJ). Two T. gondii strains, RHΔHXGPRT and RHΔKU80, were used to compare the drug's effects on parasites. RHΔHXGPRT parasites may use both HRR and NHEJ pathways but RHΔKU80 lacks the KU80 protein needed for NHEJ, leaving only the HRR pathway. Here we demonstrate that topotecan and HCPT, both topoisomerase I venoms, affected parasite replication in a concentration-dependent manner. Moreover, variations in fluorescence intensity measurements for the H2A.X phosphorylation mark (γH2A.X), an indicator of DNA damage, were observed in intracellular parasites under drug treatment conditions. Interestingly, intracellular replicative parasites without drug treatment show a strong positive staining for γH2A.X, suggesting inherent DNA damage. Extracellular (non-replicating) parasites did not exhibit γH2A.X staining, indicating that the basal level of DNA damage is likely to be associated with replicative stress. A high rate of DNA replication stress possibly prompted the evolution of an efficient repair machinery in the parasite, making it an attractive target. Our findings show that topoisomerase 1 venoms are effective antiparasitics blocking T. gondii replication.

Published
2023
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3. Proteomics Applications in Toxoplasma gondii: Unveiling the Host–Parasite Interactions and Therapeutic Target Discovery

Author

Bin Deng, Laura Vanagas, Andres M. Alonso, and Sergio O. Angel

Subjects
Toxoplasma gondii, proteomics, host–parasite interactions, PTMs, drug discovery, Medicine
Abstract

Toxoplasma gondii, a protozoan parasite with the ability to infect various warm-blooded vertebrates, including humans, is the causative agent of toxoplasmosis. This infection poses significant risks, leading to severe complications in immunocompromised individuals and potentially affecting the fetus through congenital transmission. A comprehensive understanding of the intricate molecular interactions between T. gondii and its host is pivotal for the development of effective therapeutic strategies. This review emphasizes the crucial role of proteomics in T. gondii research, with a specific focus on host–parasite interactions, post-translational modifications (PTMs), PTM crosstalk, and ongoing efforts in drug discovery. Additionally, we provide an overview of recent advancements in proteomics techniques, encompassing interactome sample preparation methods such as BioID (BirA*-mediated proximity-dependent biotin identification), APEX (ascorbate peroxidase-mediated proximity labeling), and Y2H (yeast two hybrid), as well as various proteomics approaches, including single-cell analysis, DIA (data-independent acquisition), targeted, top-down, and plasma proteomics. Furthermore, we discuss bioinformatics and the integration of proteomics with other omics technologies, highlighting its potential in unraveling the intricate mechanisms of T. gondii pathogenesis and identifying novel therapeutic targets.

Published
2023
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4. Genome-wide localization of histone variants in Toxoplasma gondii implicates variant exchange in stage-specific gene expression

Author

Sheila C. Nardelli, Natalie C. Silmon de Monerri, Laura Vanagas, Xiaonan Wang, Zoi Tampaki, William J. Sullivan, Sergio O. Angel, and Kami Kim

Subjects
Histones, Parasites, Toxoplasma, ChIP-seq, Apicomplexa, Epigenetic, Biotechnology, TP248.13-248.65, Genetics, QH426-470
Abstract

Abstract Background Toxoplasma gondii is a protozoan parasite that differentiates from acute tachyzoite stages to latent bradyzoite forms in response to environmental cues that modify the epigenome. We studied the distribution of the histone variants CenH3, H3.3, H2A.X, H2A.Z and H2B.Z, by genome-wide chromatin immunoprecipitation to understand the role of variant histones in developmental transitions of T. gondii parasites. Results H3.3 and H2A.X were detected in telomere and telomere associated sequences, whereas H3.3, H2A.X and CenH3 were enriched in centromeres. Histones H2A.Z and H2B.Z colocalize with the transcriptional activation mark H3K4me3 in promoter regions surrounding the nucleosome-free region upstream of the transcription start site. The H2B.Z/H2A.Z histone pair also localizes to the gene bodies of genes that are silent but poised for activation, including bradyzoite stage-specific genes. The majority of H2A.X and H2A.Z/H2B.Z loci do not overlap, consistent with variant histones demarcating specific functional regions of chromatin. The extent of enrichment of H2A.Z/H2B.Z (and H3.3 and H2A.X) within the entire gene (5’UTR and gene body) reflects the timing of gene expression during the cell cycle, suggesting that dynamic turnover of H2B.Z/H2A.Z occurs during the tachyzoite cell cycle. Thus, the distribution of the variant histone H2A.Z/H2B.Z dimer defines active and developmentally silenced regions of the T. gondii epigenome including genes that are poised for expression. Conclusions Histone variants mark functional regions of parasite genomes with the dynamic placement of the H2A.Z/H2B.Z dimer implicated as an evolutionarily conserved regulator of parasite and eukaryotic differentiation.

Published
2022
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5. Analysis of the Interactome of the Toxoplasma gondii Tgj1 HSP40 Chaperone

Author

Jonathan Munera López, Andrés Mariano Alonso, Maria Julia Figueras, Ana María Saldarriaga Cartagena, Miryam A. Hortua Triana, Luis Diambra, Laura Vanagas, Bin Deng, Silvia N. J. Moreno, and Sergio Oscar Angel

Subjects
Toxoplasma, Hsp40, protein–protein interaction, Hsp70/Hsp90 cycle, Microbiology, QR1-502
Abstract

Toxoplasma gondii is an obligate intracellular apicomplexan that causes toxoplasmosis in humans and animals. Central to its dissemination and pathogenicity is the ability to rapidly divide in the tachyzoite stage and infect any type of nucleated cell. Adaptation to different cell contexts requires high plasticity in which heat shock proteins (Hsps) could play a fundamental role. Tgj1 is a type I Hsp40 of T. gondii, an ortholog of the DNAJA1 group, which is essential during the tachyzoite lytic cycle. Tgj1 consists of a J-domain, ZFD, and DNAJ_C domains with a CRQQ C-terminal motif, which is usually prone to lipidation. Tgj1 presented a mostly cytosolic subcellular localization overlapping partially with endoplasmic reticulum. Protein–protein Interaction (PPI) analysis showed that Tgj1 could be implicated in various biological pathways, mainly translation, protein folding, energy metabolism, membrane transport and protein translocation, invasion/pathogenesis, cell signaling, chromatin and transcription regulation, and cell redox homeostasis among others. The combination of Tgj1 and Hsp90 PPIs retrieved only 70 interactors linked to the Tgj1-Hsp90 axis, suggesting that Tgj1 would present specific functions in addition to those of the Hsp70/Hsp90 cycle, standing out invasion/pathogenesis, cell shape motility, and energy pathway. Within the Hsp70/Hsp90 cycle, translation-associated pathways, cell redox homeostasis, and protein folding were highly enriched in the Tgj1-Hsp90 axis. In conclusion, Tgj1 would interact with a wide range of proteins from different biological pathways, which could suggest a relevant role in them.

Published
2023
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6. Architecture, Chromatin and Gene Organization of Toxoplasma gondii Subtelomeres

Author

Susana M. Contreras, Romina T. Zambrano Siri, Elías M. Rivera, Constanza Cristaldi, Laura Kamenetzky, Kami Kim, Marina Clemente, Josefina Ocampo, Laura Vanagas, and Sergio O. Angel

Subjects
Toxoplasma, telomere, subtelomere, chromatin, multigene family, environmental adaptation, Genetics, QH426-470, Biotechnology, TP248.13-248.65
Abstract

Subtelomeres (ST) are chromosome regions that separate telomeres from euchromatin and play relevant roles in various biological processes of the cell. While their functions are conserved, ST structure and genetic compositions are unique to each species. This study aims to identify and characterize the subtelomeric regions of the 13 Toxoplasma gondii chromosomes of the Me49 strain. Here, STs were defined at chromosome ends based on poor gene density. The length of STs ranges from 8.1 to 232.4 kbp, with a gene density of 0.049 genes/kbp, lower than the Me49 genome (0.15 kbp). Chromatin organization showed that H3K9me3, H2A.X, and H3.3 are highly enriched near telomeres and the 5′ end of silenced genes, decaying in intensity towards euchromatin. H3K4me3 and H2A.Z/H2B.Z are shown to be enriched in the 5′ end of the ST genes. Satellite DNA was detected in almost all STs, mainly the sat350 family and a novel satellite named sat240. Beyond the STs, only short dispersed fragments of sat240 and sat350 were found. Within STs, there were 12 functional annotated genes, 59 with unknown functions (Hypothetical proteins), 15 from multigene FamB, and 13 from multigene family FamC. Some genes presented low interstrain synteny associated with the presence of satellite DNA. Orthologues of FamB and FamC were also detected in Neospora caninum and Hammondia hammondi. A re-analysis of previous transcriptomic data indicated that ST gene expression is strongly linked to the adaptation to different situations such as extracellular passage (evolve and resequencing study) and changes in metabolism (lack of acetyl-CoA cofactor). In conclusion, the ST region of the T. gondii chromosomes was defined, the STs genes were determined, and it was possible to associate them with high interstrain plasticity and a role in the adaptability of T. gondii to environmental changes.

Published
2022
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7. Emerging Therapeutic Targets Against Toxoplasma gondii: Update on DNA Repair Response Inhibitors and Genotoxic Drugs

Author

Sergio O. Angel, Laura Vanagas, Diego M. Ruiz, Constanza Cristaldi, Ana M. Saldarriaga Cartagena, and William J. Sullivan

Subjects
Toxoplasma gondii, DNA repair, DNA damage, drug, therapy, Microbiology, QR1-502
Abstract

Toxoplasma gondii is the causative agent of toxoplasmosis in animals and humans. This infection is transmitted to humans through oocysts released in the feces of the felines into the environment or by ingestion of undercooked meat. This implies that toxoplasmosis is a zoonotic disease and T. gondii is a foodborne pathogen. In addition, chronic toxoplasmosis in goats and sheep is the cause of recurrent abortions with economic losses in the sector. It is also a health problem in pets such as cats and dogs. Although there are therapies against this infection in its acute stage, they are not able to permanently eliminate the parasite and sometimes they are not well tolerated. To develop better, safer drugs, we need to elucidate key aspects of the biology of T. gondii. In this review, we will discuss the importance of the homologous recombination repair (HRR) pathway in the parasite's lytic cycle and how components of these processes can be potential molecular targets for new drug development programs. In that sense, the effect of different DNA damage agents or HHR inhibitors on the growth and replication of T. gondii will be described. Multitarget drugs that were either associated with other targets or were part of general screenings are included in the list, providing a thorough revision of the drugs that can be tested in other scenarios.

Published
2020
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8. Evaluation of ATM Kinase Inhibitor KU-55933 as Potential Anti-Toxoplasma gondii Agent

Author

Jonathan Munera López, Agustina Ganuza, Silvina S. Bogado, Daniela Muñoz, Diego M. Ruiz, William J. Sullivan, Laura Vanagas, and Sergio O. Angel

Subjects
Toxoplasma gondii, DNA repair, cell cycle, fork collapse, antiparasitic drugs, Microbiology, QR1-502
Abstract

Toxoplasma gondii is an apicomplexan protozoan parasite with a complex life cycle composed of multiple stages that infect mammals and birds. Tachyzoites rapidly replicate within host cells to produce acute infection during which the parasite disseminates to tissues and organs. Highly replicative cells are subject to Double Strand Breaks (DSBs) by replication fork collapse and ATM, a member of the phosphatidylinositol 3-kinase (PI3K) family, is a key factor that initiates DNA repair and activates cell cycle checkpoints. Here we demonstrate that the treatment of intracellular tachyzoites with the PI3K inhibitor caffeine or ATM kinase-inhibitor KU-55933 affects parasite replication rate in a dose-dependent manner. KU-55933 affects intracellular tachyzoite growth and induces G1-phase arrest. Addition of KU-55933 to extracellular tachyzoites also leads to a significant reduction of tachyzoite replication upon infection of host cells. ATM kinase phosphorylates H2A.X (γH2AX) to promote DSB damage repair. The level of γH2AX increases in tachyzoites treated with camptothecin (CPT), a drug that generates fork collapse, but this increase was not observed when co-administered with KU-55933. These findings support that KU-55933 is affecting the Toxoplasma ATM-like kinase (TgATM). The combination of KU-55933 and other DNA damaging agents such as methyl methane sulfonate (MMS) and CPT produce a synergic effect, suggesting that TgATM kinase inhibition sensitizes the parasite to damaged DNA. By contrast, hydroxyurea (HU) did not further inhibit tachyzoite replication when combined with KU-55933.

Published
2019
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9. Apicomplexan and histone variants: What's new?

Author

Sergio O. Angel, Susana Marisol Contreras, Laura Vanagas, Logie, Colin, and Knoch, Tobias Aurelius

Subjects
purl.org/becyt/ford/1 [https], media_common.quotation_subject, TOXOPLASMA, PLASMODIUM, Art, DAÑO DE ADN, purl.org/becyt/ford/1.6 [https], Humanities, EPIGENETICA, Histone variants, media_common
Abstract

Plasmodium spp. and Toxoplasma gondii present a conserved nucleosome composition based on canonical H3 and variants, H4, canonical H2A and variants, and H2B. One-off, the phylum has also a variant H2B, named H2B.Z, which was shown to form a double variant nucleosome H2A.Z/H2B.Z. These histones also present conserved and unique post-translational modifications (PTMs). Histone variants have shown particular genomic localization and PTMs along euchromatin and heterochromatin, including telomere-associated sequences (TAS), suggesting fine-grained chromatin structure modulation. Several other nonhistone proteins present remarkable participation in controlling chromatin state, especially at TAS. Based on that, we discuss the role of epigenetics (PTMs and histone variants) in Plasmodium and Toxoplasma gene expression, replication, and DNA repair. We also discuss TAS structures and chromatin composition and its impact on antigenic variant expression in Plasmodium. Fil: Vanagas, Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Contreras, Susana Marisol. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Ángel, Sergio Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina

Published
2019

10. Evaluation of ATM Kinase Inhibitor KU-55933 as Potential Anti

Author

Jonathan, Munera López, Agustina, Ganuza, Silvina S, Bogado, Daniela, Muñoz, Diego M, Ruiz, William J, Sullivan, Laura, Vanagas, and Sergio O, Angel

Subjects
Cellular and Infection Microbiology, Pyrones, Morpholines, fork collapse, Antiprotozoal Agents, Toxoplasma gondii, DNA repair, Drug Synergism, cell cycle, Protein Kinase Inhibitors, Toxoplasma, Original Research, antiparasitic drugs
Abstract

Toxoplasma gondii is an apicomplexan protozoan parasite with a complex life cycle composed of multiple stages that infect mammals and birds. Tachyzoites rapidly replicate within host cells to produce acute infection during which the parasite disseminates to tissues and organs. Highly replicative cells are subject to Double Strand Breaks (DSBs) by replication fork collapse and ATM, a member of the phosphatidylinositol 3-kinase (PI3K) family, is a key factor that initiates DNA repair and activates cell cycle checkpoints. Here we demonstrate that the treatment of intracellular tachyzoites with the PI3K inhibitor caffeine or ATM kinase-inhibitor KU-55933 affects parasite replication rate in a dose-dependent manner. KU-55933 affects intracellular tachyzoite growth and induces G1-phase arrest. Addition of KU-55933 to extracellular tachyzoites also leads to a significant reduction of tachyzoite replication upon infection of host cells. ATM kinase phosphorylates H2A.X (γH2AX) to promote DSB damage repair. The level of γH2AX increases in tachyzoites treated with camptothecin (CPT), a drug that generates fork collapse, but this increase was not observed when co-administered with KU-55933. These findings support that KU-55933 is affecting the Toxoplasma ATM-like kinase (TgATM). The combination of KU-55933 and other DNA damaging agents such as methyl methane sulfonate (MMS) and CPT produce a synergic effect, suggesting that TgATM kinase inhibition sensitizes the parasite to damaged DNA. By contrast, hydroxyurea (HU) did not further inhibit tachyzoite replication when combined with KU-55933.

Published
2018

11. Canonical histone H2Ba and H2A.X dimerize in an opposite genomic localization to H2A.Z/H2B.Z dimers in Toxoplasma gondii

Author

Maria Carolina Dalmasso, Sergio O. Angel, Kami Kim, Silvina S. Bogado, Agustina Ganuza, William J. Sullivan, and Laura Vanagas

Subjects
CHROMATIN, Chromatin Immunoprecipitation, animal structures, TOXOPLASMA, NUCLEOSOME, Models, Biological, Article, law.invention, purl.org/becyt/ford/1 [https], Ciencias Biológicas, Histones, Biología Celular, Microbiología, law, Nucleosome, purl.org/becyt/ford/1.6 [https], Molecular Biology, biology, CHIP, Antibodies, Monoclonal, Toxoplasma gondii, Promoter, CANONICAL HISTONES, ChIP-on-chip, biology.organism_classification, Molecular biology, Recombinant Proteins, EPIGENETICS, Nucleosomes, Chromatin, Protein Transport, Histone, embryonic structures, biology.protein, Recombinant DNA, Parasitology, Protein Multimerization, Genome, Protozoan, Toxoplasma, Chromatin immunoprecipitation, CIENCIAS NATURALES Y EXACTAS
Abstract

Histone H2Ba of Toxoplasma gondii was expressed as recombinant protein (rH2Ba) and used to generate antibody in mouse that is highly specific. Antibody recognizing rH2Ba detects a single band in tachyzoite lysate of the expected molecular weight (12 kDa). By indirect immunofluorescence (IFA) in in vitro grown tachyzoites and bradyzoites, the signal was detected only in the parasite nucleus. The nucleosome composition of H2Ba was determined through co-immunoprecipitation assays. H2Ba was detected in the same immunocomplex as H2A.X, but not with H2A.Z. Through chromatin immunoprecipitation (ChIP) assays and qPCR, it was observed that H2Ba is preferentially located at promoters of inactive genes and silent regions, accompanying H2A.X and opposed to H2A.Z/H2B.Z dimers. Fil: Bogado, Silvina Solange. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina. Universidad Nacional de San Martín; Argentina Fil: Dalmasso, Maria Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina. Universidad Nacional de San Martín; Argentina Fil: Ganuza, Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; Argentina. Universidad Nacional de San Martín; Argentina Fil: Kim, Kami. Albert Einstein College of Medicine; Estados Unidos Fil: Sullivan, William J.. Indiana University School of Medicine; Estados Unidos Fil: Ángel, Sergio Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina. Universidad Nacional de San Martín; Argentina Fil: Vanagas, Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina. Universidad Nacional de San Martín; Argentina

Published
2014
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12. Toxoplasmahistone acetylation remodelers as novel drug targets

Author

William J. Sullivan, Laura Vanagas, Silvina S. Bogado, Victoria Jeffers, Sergio O. Angel, and Maria Carolina Dalmasso

Subjects
Microbiology (medical), Molecular Sequence Data, Biology, Microbiology, Article, Histone Deacetylases, Histones, Virology, parasitic diseases, Humans, Nucleosome, Amino Acid Sequence, Histone Acetyltransferases, Genetics, Histone deacetylase 5, Antiparasitic Agents, HDAC11, Histone deacetylase 2, HDAC10, Acetylation, Chromatin, Nucleosomes, Cell biology, Histone Deacetylase Inhibitors, Infectious Diseases, Histone, biology.protein, Histone deacetylase, Protein Processing, Post-Translational, Sequence Alignment, Toxoplasma, Toxoplasmosis
Abstract

Toxoplasma gondii is a leading cause of neurological birth defects and a serious opportunistic pathogen. The authors and others have found that Toxoplasma uses a unique nucleosome composition supporting a fine gene regulation together with other factors. Post-translational modifications in histones facilitate the establishment of a global chromatin environment and orchestrate DNA-related biological processes. Histone acetylation is one of the most prominent post-translational modifications influencing gene expression. Histone acetyltransferases and histone deacetylases have been intensively studied as potential drug targets. In particular, histone deacetylase inhibitors have activity against apicomplexan parasites, underscoring their potential as a new class of antiparasitic compounds. In this review, we summarize what is known about Toxoplasma histone acetyltransferases and histone deacetylases, and discuss the inhibitors studied to date. Finally, the authors discuss the distinct possibility that the unique nucleosome composition of Toxoplasma, which harbors a nonconserved H2Bv variant histone, might be targeted in novel therapeutics directed against this parasite.

Published
2012
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13. Conversion of High and Low Pollen Protein Diets Into Protein in Worker Honey Bees (Hymenoptera: Apidae)

Author

Marina Basualdo, H. Solana, C. García, E. Bedascarrasbure, Santiago Barragán, Edgardo Rodríguez, and Laura Vanagas

Subjects
animal structures, FAT BODY, Fat Body, PROTEIN, Hymenoptera, medicine.disease_cause, complex mixtures, Ciencias Biológicas, Honey Bees, Vitellogenin, Vitellogenins, Animal science, Pollen, Hemolymph, Botany, medicine, Diet, Protein-Restricted, Animals, Ecology, Apidae, biology, fungi, food and beverages, Proteins, General Medicine, Honey bee, POLLEN DIETS, Bees, NUTRITIONAL STATUS, Bioquímica y Biología Molecular, biology.organism_classification, Worker bee, HONEY BEES, Insect Science, behavior and behavior mechanisms, biology.protein, CIENCIAS NATURALES Y EXACTAS
Abstract

Adequate protein levels are necessary to maintain strong honey bee [Apis mellifera (L.)] colonies. The aim of this study was to quantify how pollens with different crude protein contents inßuence protein stores within individual honey bees. Caged bees were fed one of three diets, consisting of highÐprotein-content pollen, lowÐprotein-content pollen, or protein-free diet as control; measurements were made based on protein content in hemolymph and fat body, fat body weight, and body weight. Vitellogenin in hemolymph was also measured. Bees fed with high crude protein diet had signiÞcantly higher levels of protein in hemolymph and fat bodies. Caged bees did not increase pollen consumption to compensate for the lower protein in the diet, and ingesting 4 mg of protein per bee could achieve levels of 20 g/ l protein in hemolymph. Worker bees fed with low crude protein diet took more time in reaching similar protein content of the bees that were fed with high crude protein diet. The data showed that fat bodies and body weight were not efÞcient methods of measuring the protein status of bees. The determination of total protein or vitellogenin concentration in the hemolymph from 13-d-old bees and protein concentration of fat bodies from 9-d-old bees could be good indicators of nutritional status of honey bees. Fil: Basualdo, Marina. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Barragán, Santiago. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias; Argentina Fil: Vanagas, Laura. Instituto Nacional de Tecnología Agropecuaria; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: García, C.. Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias; Argentina Fil: Solana, Hugo Daniel. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias; Argentina Fil: Rodriguez, Edgardo Mario. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Bedascarrasbure, Enrique Luciano. Instituto Nacional de Tecnología Agropecuaria; Argentina

Published
2013

14. Epichromatin is conserved in Toxoplasma gondii and labels the exterior parasite chromatin throughout the cell cycle

Author

Donald E. Olins, Maria Carolina Dalmasso, Enrique Leo Portiansky, Laura Vanagas, Sergio O. Angel, and Jean François Dubremetz

Subjects
DNA Replication, Nuclear Envelope, Immunoelectron microscopy, Protozoan Proteins, Antibodies, Protozoan, Toxoplasma gondii, Replication, Cell cycle, Article, Epitope, Nuclear envelope, Toxoplasma Gondii, Ciencias Biológicas, Histones, purl.org/becyt/ford/1 [https], Epitopes, Mice, parasitic diseases, medicine, Animals, Humans, purl.org/becyt/ford/1.6 [https], Mitosis, Epichromatin, Cell Nucleus, biology, Ciencias Veterinarias, Cell Cycle, Antibodies, Monoclonal, Bioquímica y Biología Molecular, DNA, Protozoan, Fibroblasts, biology.organism_classification, Molecular biology, Chromatin, Recombinant Proteins, Cell nucleus, Infectious Diseases, medicine.anatomical_structure, Histone, biology.protein, Animal Science and Zoology, Parasitology, Toxoplasma, CIENCIAS NATURALES Y EXACTAS, Toxoplasmosis
Abstract

Toxoplasma gondii is an apicomplexan intracellular protozoan parasite responsible for toxoplasmosis, a disease with considerable medical and economic impact worldwide. Toxoplasma gondii cells never lose the nuclear envelope and their chromosomes do not condense. Here, we tested the murine monoclonal antibody PL2-6, which labels epichromatin (a conformational chromatin epitope based on histones H2A and H2B complexed with DNA), in T. gondii cultured in human fibroblasts. This epitope is present at the exterior chromatin surface of interphase nuclei and on the periphery of mitotic chromosomes in higher eukaryotes. PL2-6 reacted with T. gondii H2A and H2B histones in Western blot (WB) assays. In addition, the antibody reacted with the nuclear fraction of tachyzoites, as a single band coincident with H2B histone. In the T. gondii tachyzoite stage, PL2-6 also had peripheral nuclear localization, as observed by epifluorescence/confocal microscopy and immunoelectron microscopy. Confocal analysis showed that epichromatin is slightly polarized to one face of the parasite exterior chromatin surface. In replicating tachyzoites, PL2-6 also labels the exterior chromatin surface, covering the face of both segregating nuclei, facing the plasma membrane of the mother cell. The possible role of epichromatin in T. gondii is discussed., Facultad de Ciencias Veterinarias

Published
2013

15. Differential Effects of G- and F-Actin on the Plasma Membrane Calcium Pump Activity

Author

María Candelaria de la Fuente, Irene C. Mangialavori, Rolando C. Rossi, Emanuel E. Strehler, Laura Vanagas, Marianela G Dalghi, Mariela S. Ferreira-Gomes, and Juan Pablo F.C. Rossi

Subjects
Erythrocytes, Protein Conformation, Otras Ciencias Biológicas, Biophysics, Arp2/3 complex, macromolecular substances, Calcium-Transporting ATPases, CYTOSKELETON, Biology, Biochemistry, Article, Polymerization, ACTIN, Ciencias Biológicas, PMCA, Humans, Calcium Signaling, Phosphorylation, Cytoskeleton, Integral membrane protein, Actin, Erythrocyte Membrane, Actin remodeling, Membrane Proteins, Cell Biology, General Medicine, Actin cytoskeleton, Actins, Cell biology, Enzyme Activation, Actin Cytoskeleton, Membrane protein, biology.protein, REGULATION, Plasma membrane Ca2+ ATPase, Calcium, CIENCIAS NATURALES Y EXACTAS
Abstract

We have previously shown that plasma membrane calcium ATPase (PMCA) pump activity is affected by the membrane protein concentration (Vanagas et al., Biochim Biophys Acta 1768:1641-1644, 2007). The results of this study provided evidence for the involvement of the actin cytoskeleton. In this study, we explored the relationship between the polymerization state of actin and its effects on purified PMCA activity. Our results show that PMCA associates with the actin cytoskeleton and this interaction causes a modulation of the catalytic activity involving the phosphorylated intermediate of the pump. The state of actin polymerization determines whether it acts as an activator or an inhibitor of the pump: G-actin and/or short oligomers activate the pump, while F-actin inhibits it. The effects of actin on PMCA are the consequence of direct interaction as demonstrated by immunoblotting and cosedimentation experiments. Taken together, these findings suggest that interactions with actin play a dynamic role in the regulation of PMCA-mediated Ca2+ extrusion through the membrane. Our results provide further evidence of the activation-inhibition phenomenon as a property of many cytoskeleton-associated membrane proteins where the cytoskeleton is no longer restricted to a mechanical function but is dynamically involved in modulating the activity of integral proteins with which it interacts. © 2012 Springer Science+Business Media New York. Fil: Vanagas, Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina Fil: de la Fuente, Maria Candelaria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina Fil: Dalghi, Marianela Gisela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina Fil: Ferreira Gomes, Mariela Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina Fil: Rossi, Rolando Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina Fil: Strehler, Emanuel E.. Mayo Clinic College of Medicine; Estados Unidos Fil: Mangialavori, Irene Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina Fil: Rossi, Juan Pablo Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina

Published
2012
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16. Plasma membrane calcium pump activity is affected by the membrane protein concentration. Evidence for the involvement of the actin cytoskeleton

Author

Rolando C. Rossi, Emanuel E. Strehler, Ariel J. Caride, Juan Pablo F.C. Rossi, Laura Vanagas, and Adelaida G. Filoteo

Subjects
Cytochalasin D, Erythrocytes, Calcium pump, Biophysics, Biology, Spodoptera, Biochemistry, Article, Cell Line, Cell membrane, 03 medical and health sciences, chemistry.chemical_compound, Plasma Membrane Calcium-Transporting ATPases, PMCA, Calmodulin, medicine, Animals, Humans, Cytochalasin, Integral membrane protein, Cytoskeleton, 030304 developmental biology, 0303 health sciences, 030302 biochemistry & molecular biology, Cell Membrane, Membrane, Biological Transport, Cell Biology, Actin cytoskeleton, Cell biology, medicine.anatomical_structure, chemistry, Membrane protein, Plasma membrane Ca2+ ATPase, Calcium, Electrophoresis, Polyacrylamide Gel
Abstract

Plasma membrane calcium pumps (PMCAs) are integral membrane proteins that actively expel Ca 2+ from the cell. Specific Ca 2+ -ATPase activity of erythrocyte membranes increased steeply up to 1.5–5 times when the membrane protein concentration decreased from 50 μg/ml to 1 μg/ml. The activation by dilution was also observed for ATP-dependent Ca 2+ uptake into vesicles from Sf9 cells over-expressing the PMCA 4b isoform, confirming that it is a property of the PMCA. Dilution of the protein did not modify the activation by ATP, Ca 2+ or Ca 2+ -calmodulin. Treatment with non-ionic detergents did not abolish the dilution effect, suggesting that it was not due to resealing of the membrane vesicles. Pre-incubation of erythrocyte membranes with Cytochalasin D under conditions that promote actin polymerization abolished the dilution effect. Highly-purified, micellar PMCA showed no dilution effect and was not affected by Cytochalasin D. Taken together, these results suggest that the concentration-dependent behavior of the PMCA activity was due to interactions with cytoskeletal proteins. The dilution effect was also observed with different PMCA isoforms, indicating that this is a general phenomenon for all PMCAs.

Published
2007

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