Exploring protein myristoylation in Toxoplasma gondii

Toxoplasma gondii is an important human and veterinary pathogen and the causative agent of toxoplasmosis, a potentially severe disease especially in immunocompromised or congenitally infected humans. Current therapeutic compounds are not well-tolerated, present increasing resistance, limited efficacy and require long periods of treatment. On this context, searching for new therapeutic targets is crucial to drug discovery. In this sense, recent works suggest that N-myristoyltransferase (NMT), the enzyme responsible for protein myristoylation that is essential in some parasites, could be the target of new anti-parasitic compounds. However, up to date there is no information on NMT and the extent of this modification in T. gondii. In this work, we decided to explore T. gondii genome in search of elements related with the N-myristoylation process. By a bioinformatics approach it was possible to identify a putative T. gondii NMT (TgNMT). This enzyme that is homologous to other parasitic NMTs, presents activity in vitro, is expressed in both intra- and extracellular parasites and interacts with predicted TgNMT substrates. Additionally, NMT activity seems to be important for the lytic cycle of Toxoplasma gondii. In parallel, an in silico myristoylome predicts 157 proteins to be affected by this modification. Myristoylated proteins would be affecting several metabolic functions with some of them being critical for the life cycle of this parasite. Together, these data indicate that TgNMT could be an interesting target of intervention for the treatment of toxoplasmosis.Fil: Alonso, Andrés Mariano. Universidad Nacional de San Martin. Instituto Tecnológico de Chascomús - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Tecnológico de Chascomús; Argentina. Universidad Nacional de La Plata. Centro Regional de Estudios Genómicos; ArgentinaFil: Turowski, Valeria Rosana. Universidad Nacional de La Plata. Centro Regional de Estudios Genómicos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; ArgentinaFil: Ruiz, Diego Mario. Universidad Nacional de La Plata. Centro Regional de Estudios Genómicos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; ArgentinaFil: Orelo, Barbara D.. The Scripps Research Institute; Estados UnidosFil: Moresco, James J.. The Scripps Research Institute; Estados UnidosFil: Yates, John R.. The Scripps Research Institute; Estados UnidosFil: Corvi, Maria Martha. Universidad Nacional de San Martin. Instituto Tecnológico de Chascomús - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Tecnológico de Chascomús; Argentin

Predicted subcellular localization of AtFH (At4g03240).

<p>^a TargetP 1.1 Server (<a href="http://www.cbs.dtu.dk/services/TargetP" target="_blank">http://www.cbs.dtu.dk/services/TargetP</a>)</p><p>^b MitoProt II–v1.101 (<a href="http://ihg.gsf.de/ihg/mitoprot.html" target="_blank">http://ihg.gsf.de/ihg/mitoprot.html</a>)</p><p>^c ChloroP 1.1 Server (<a href="http://www.cbs.dtu.dk/services/ChloroP/" target="_blank">http://www.cbs.dtu.dk/services/ChloroP/</a>)</p><p>^d GTP—Green Targeting Predictor & ATP2—Ambiguous Targeting Predictor 2 (<a href="http://www.plantco.de/gtp" target="_blank">http://www.plantco.de/gtp</a>).</p><p>Predicted subcellular localization of AtFH (At4g03240).</p

Analysis of the presence of AtFH in isolated chloroplasts and mitochondria from <i>A</i>. <i>thaliana</i> by western blot in wt and <i>as-AtFH-1</i> plants.

<p>AtFH was detected using anti-AtFH antibodies. Anti-ADPGlc PPase and anti-NAD9 antibodies were used as controls to asses the purity of chloroplasts and mitochondria, respectively.</p

Photosynthetic parameters in <i>as-AtFH</i> and wt leaves.

<p>Fv/Fm, Maximum quantum yield of PSII; ϕ PSII, Quantum yield of PSII; qP, Photochemical quenching; NPQ, Non-photochemical quenching. Values are the mean ± standard deviation of at least three leaves from 10 individual plants.</p><p>Asterisks (*) indicate statistically different results (P < 0.05).</p><p>Photosynthetic parameters in <i>as-AtFH</i> and wt leaves.</p

Subcellular localization of AtFH.

<p>The plasmid pZP212 containing the coding sequence of AtFH-GFP was introduced into Arabidopsis thaliana Col-0 plants by the floral dip method. Protoplasts were isolated from the resulting transgenic plants and analyzed by confocal microscopy. (A) Mitotracker staining showing mitochondria (excitation 543 nm/emission 576 nm); (B) GFP fluorescence (excitation 488 nm/emission 510 nm); (C) Chlorophyll autofluorescence (excitation 488 nm/emission 650 nm); (D) Overlay of A and B showing coincidence of GFP localization and Mitotracker (yellow); (E) Overlay of B and C showing the coincidence of GFP localization and chlorophyll autofluorescence (yellow); (F) Phase contrast image of the protoplast analyzed.</p

Quantification of chlorophyll and iron contents.

<p>(A) Quantification of chlorophyll a, b and total chlorophyll in wt (black bars), <i>as-AtFH-1</i> (grey bars), and <i>as-AtFH-2</i> (white bars) plants. (B) Analysis of total iron content in wt and <i>as-AtFH</i> plants. Asterisks indicate a statistically different result from the control value (P < 0.05). Values are the mean ± standard deviation of at least four independent replicates.</p

Frataxin Is Localized to Both the Chloroplast and Mitochondrion and Is Involved in Chloroplast Fe-S Protein Function in Arabidopsis.

Frataxin plays a key role in eukaryotic cellular iron metabolism, particularly in mitochondrial heme and iron-sulfur (Fe-S) cluster biosynthesis. However, its precise role has yet to be elucidated. In this work, we studied the subcellular localization of Arabidopsis frataxin, AtFH, using confocal microscopy, and found a novel dual localization for this protein. We demonstrate that plant frataxin is targeted to both the mitochondria and the chloroplast, where it may play a role in Fe-S cluster metabolism as suggested by functional studies on nitrite reductase (NIR) and ferredoxin (Fd), two Fe-S containing chloroplast proteins, in AtFH deficient plants. Our results indicate that frataxin deficiency alters the normal functioning of chloroplasts by affecting the levels of Fe, chlorophyll, and the photosynthetic electron transport chain in this organelle