Browsing by Author "Muñoz, Daniela"

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    Evaluation of ATM Kinase Inhibitor KU-55933 as Potential Anti-Toxoplasma gondii Agent
    (Frontiers, 2019-02-13) Munera López, Jonathan; Ganuza, Agustina; Bogado, Silvina S.; Muñoz, Daniela; Ruiz, Diego M.; Sullivan, William J., Jr.; Vanagas, Laura; Angel, Sergio O.; Pharmacology and Toxicology, School of Medicine
    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.
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    Histone variant H2B.Z acetylation is necessary for maintenance of Toxoplasma gondii biological fitness
    (Cold Spring Harbor Laboratory, 2023-02-24) Vanagas, Laura; Muñoz, Daniela; Cristaldi, Constanza; Ganuza, Agustina; Nájera, Rosario; Bonardi, Mabel C.; Turowski, Valeria R.; Guzman, Fanny; Deng, Bin; Kim, Kami; Sullivan, William J., Jr.; Angel, Sergio O.; Pharmacology and Toxicology, School of Medicine
    Through regulation of DNA packaging, histone proteins are fundamental to a wide array of biological processes. A variety of post-translational modifications (PTMs), including acetylation, constitute a proposed histone code that is interpreted by “reader” proteins to modulate chromatin structure. Canonical histones can be replaced with variant versions that add an additional layer of regulatory complexity. The protozoan parasite Toxoplasma gondii is unique among eukaryotes in possessing a novel variant of H2B designated H2B.Z. The combination of PTMs and the use of histone variants is important for gene regulation in T. gondii, offering new targets for drug development. In this work, T. gondii parasites were generated in which the 5 N-terminal acetylatable lysines in H2B.Z were mutated to either alanine (c-Myc-A) or arginine (c-Myc-R). c-Myc-A mutant only displayed a mild effect in its ability to kill mice. c-Myc-R mutant presented an impaired ability to grow and an increase in differentiation to latent bradyzoites. This mutant line was also more sensitive to DNA damage, displayed no virulence in mice, and provided protective immunity against future infection. While nucleosome composition was unaltered, key genes were abnormally expressed during in vitro bradyzoite differentiation. Our results show that the N-terminal positive charge patch of H2B.Z is important for these procceses. Pull down assays with acetylated N-terminal H2B.Z peptide and unacetylated one retrieved common and differential interactors. Acetylated peptide pulled down proteins associated with chromosome maintenance/segregation and cell cycle, opening the question of a possible link between H2B.Z acetylation status and mitosis.
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    Histone variant H2B.Z acetylation is necessary for maintenance of Toxoplasma gondii biological fitness
    (Elsevier, 2023) Vanagas, Laura; Muñoz, Daniela; Cristaldi, Constanza; Ganuza, Agustina; Nájera, Rosario; Bonardi, Mabel C.; Turowski, Valeria R.; Guzman, Fanny; Deng, Bin; Kim, Kami; Sullivan, William J., Jr.; Angel, Sergio O.; Pharmacology and Toxicology, School of Medicine
    Through regulation of DNA packaging, histone proteins are fundamental to a wide array of biological processes. A variety of post-translational modifications (PTMs), including acetylation, constitute a proposed histone code that is interpreted by "reader" proteins to modulate chromatin structure. Canonical histones can be replaced with variant versions that add an additional layer of regulatory complexity. The protozoan parasite Toxoplasma gondii is unique among eukaryotes in possessing a novel variant of H2B designated H2B.Z. The combination of PTMs and the use of histone variants are important for gene regulation in T. gondii, offering new targets for drug development. In this work, T. gondii parasites were generated in which the 5 N-terminal acetylatable lysines in H2B.Z were mutated to either alanine (c-Myc-A) or arginine (c-Myc-R). The c-Myc-A mutant displayed no phenotype over than a mild defect in its ability to kill mice. The c-Myc-R mutant presented an impaired ability to grow and an increase in differentiation to latent bradyzoites. The c-Myc-R mutant was also more sensitive to DNA damage, displayed no virulence in mice, and provided protective immunity against future infection. While nucleosome composition was unaltered, key genes were abnormally expressed during in vitro bradyzoite differentiation. Our results show that regulation of the N-terminal positive charge patch of H2B.Z is important for these processes. We also show that acetylated N-terminal H2B.Z interacts with some unique proteins compared to its unacetylated counterpart; the acetylated peptide pulled down proteins associated with chromosome maintenance/segregation and cell cycle, suggesting a link between H2B.Z acetylation status and mitosis.