RODOQUINONA: ¿UN TRANSPORTADOR DE ELECTRONES DE RESPUESTA A HIPOXIA, SULFURO Y CIANURO?

En helmintos, la principal forma de obtención de energía en las condiciones de hipoxia del intestino del hospedero involucra una cadena de transporte de electrones (CTE) mitocondrial alternativa en la que la rodoquinona y no la ubiquinona, es el transportador de electrones lipídico y el fumarato y no el oxígeno es el aceptor final de electrones. Caenorhabditis elegans no experimenta condiciones de hipoxia, a excepción del estadio de resistencia denominado larva Dauer, el cual se ha especulado que podría estar sujeto a condiciones de hipoxia. Sin embargo, este organismo está expuesto a bacterias patógenas productoras de H2S y HCN, inhibidores del complejo IV de la CTE canónica, impidiendo que el oxígeno pueda utilizarse como aceptor final de electrones. Nuestros resultados indican que en C. elegans la rodoquinona no tiene un rol esencial en condiciones de hipoxia y anoxia, pero sí en respuesta a HCN y H2S. Estirpes mutantes que no sintetizan rodoquinona (y sí ubiquinona) no sobreviven a condiciones de HCN y H2S y son más sensibles frente a la bacteria patógena Pseudomonas aeruginosa PA01 (que mata al gusano por generación de HCN), comparado con la estirpe silvestre. Estos resultados sugieren una nueva función de la rodoquinona cumpliendo un rol en la protección del gusano por envenenamiento con HCN y H2S producidos por patógenos presentes en su ambiente natural. Este rol de la rodoquinona también sería relevante en helmintos que habitan el intestino de sus hospederos, en muchos casos, ambientes ricos en H2S generado por la microbiota.Agencia Nacional de Investigación e Innovació

Adjustments, extinction, and remains of selenocysteine incorporation machinery in the nematode lineage

This article is distributed exclusively by the RNA Society for the first 12 months after the full-issue publication date. After 12 months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International).Selenocysteine (Sec) is encoded by an UGA codon with the help of a SECIS element present in selenoprotein mRNAs. SECIS-binding protein (SBP2/SCBP-2) mediates Sec insertion, but the roles of its domains and the impact of its deficiency on Sec insertion are not fully understood. We used Caenorhabditis elegans to examine SBP2 function since it possesses a single selenoprotein, thioredoxin reductase-1 (TRXR-1). All SBP2 described so far have an RNA-binding domain (RBD) and a Secincorporation domain (SID). Surprisingly, C. elegans SBP2 lacks SID and consists only of an RBD. An sbp2 deletion mutant strain ablated Sec incorporation demonstrating SBP2 essentiality for Sec incorporation. Further in silico analyses of nematode genomes revealed conservation of SBP2 lacking SID and maintenance of Sec incorporation linked to TRXR-1. Remarkably, parasitic plant nematodes lost the ability to incorporate Sec, but retained SecP43, a gene associated with Sec incorporation. Interestingly, both selenophosphate synthetase (SPS) genes are absent in plant parasitic nematodes, while only Cys-containing SPS2 is present in Sec-incorporating nematodes. Our results indicate that C. elegans and the nematode lineage provide key insights into Sec incorporation and the evolution of Sec utilization trait, selenoproteomes, selenoproteins, and Sec residues. Finally, our study provides evidence of noncanonical translation initiation in C. elegans, not previously known for this well-established animal model.This work was supported by Universidad de la República, Uruguay (Grant Number 418 to G.S., PhD fellowship to L.O.); Asociación Española de Cooperación Internacional (C/7646/07 to A.M.-V. and G.S.; A/016083/08 to A.M.-V. and G.S.); Asociación Universitaria Iberoamericana de Posgrado and Agencia Nacional de Innovación e Investigación (BE_POS_2009_183 and BE_POS_2010_2160 to L.O.), and was partially funded by FOCEM (MERCOSUR Structural Convergence Fund), [COF 03/11].Peer Reviewe

Alternative splicing of coq-2 controls the levels of rhodoquinone in animals

Parasitic helminths use two benzoquinones as electron carriers in the electron transport chain. In normoxia, they use ubiquinone (UQ), but in anaerobic conditions inside the host, they require rhodoquinone (RQ) and greatly increase RQ levels. We previously showed the switch from UQ to RQ synthesis is driven by a change of substrates by the polyprenyltransferase COQ-2 (Del Borrello et al., 2019; Roberts Buceta et al., 2019); however, the mechanism of substrate selection is not known. Here, we show helminths synthesize two coq-2 splice forms, coq-2a and coq-2e, and the coq-2e-specific exon is only found in species that synthesize RQ. We show that in Caenorhabditis elegans COQ-2e is required for efficient RQ synthesis and survival in cyanide. Importantly, parasites switch from COQ-2a to COQ-2e as they transit into anaerobic environments. We conclude helminths switch from UQ to RQ synthesis principally via changes in the alternative splicing of coq-2.Agencia Nacional de Investigación e InnovaciónCanadian Institutes of Health Researc

Alternative splicing of coq-2 controls the levels of rhodoquinone in animals

Parasitic helminths use two benzoquinones as electron carriers in the electron transport chain. In normoxia, they use ubiquinone (UQ), but in anaerobic conditions inside the host, they require rhodoquinone (RQ) and greatly increase RQ levels. We previously showed the switch from UQ to RQ synthesis is driven by a change of substrates by the polyprenyltransferase COQ-2 (Del Borrello et al., 2019; Roberts Buceta et al., 2019); however, the mechanism of substrate selection is not known. Here, we show helminths synthesize two coq-2 splice forms, coq-2a and coq-2e, and the coq-2e-specific exon is only found in species that synthesize RQ. We show that in Caenorhabditis elegans COQ-2e is required for efficient RQ synthesis and survival in cyanide. Importantly, parasites switch from COQ-2a to COQ-2e as they transit into anaerobic environments. We conclude helminths switch from UQ to RQ synthesis principally via changes in the alternative splicing of coq-2.Agencia Nacional de Investigación e InnovaciónCanadian Institutes of Health Researc

Rodoquinona: una molécula esencial en la respuesta a sulfuro, cianuro y patógenos en Caenorhabditis elegans

Animales que enfrentan condiciones de hipoxia o anoxia necesitan un mecanismo de obtención de energía sin utilizar el oxígeno. Una adaptación bioquímica a la hipoxia en algunos animales involucra una cadena de transporte de electrones mitocondrial (CTEM) alternativa, en la cual la rodoquinona (RQ) y no la ubiquinona (UQ) es el transportador de electrones lipídico y el fumarato, y no el oxígeno, es el aceptor final de electrones. Nuestros resultados sugieren que en C. elegans la RQ no sería esencial en las condiciones de hipoxia examinadas, desencadenando la pregunta de otro posible rol de la RQ en C. elegans. Este gusano en su ambiente natural puede enfrentarse a altas concentraciones de sulfuro de hidrógeno (H2S), así como también a bacterias patógenas que matan al gusano mediante la producción de cianuro de hidrógeno (HCN). Tanto el H2S como el HCN son inhibidores del complejo IV de la CTEM canónica. Así, un posible rol adicional de la RQ podría ser participar en la defensa del gusano frente a estos compuestos tóxicos. De hecho, estirpes mutantes que no sintetizan RQ (y sí UQ) no sobreviven en presencia de altas concentraciones de H2S y de HCN y son mas sensibles a la bacteria patógena Pseudomonas aeruginosa PAO1 (que mata al gusano generando HCN), comparado con las estirpes silvestre y mutante deficiente en UQ. Estos resultados proponen un nuevo rol de la RQ en la protección del gusano del envenenamiento con H2S y HCN producidos por patógenos presentes en su hábitat natural.Agencia Nacional de Investigación e Innovació

Rhodoquinone as a key molecule for sulfide, cyanide and pathogen response in C. elegans

Animals facing hypoxic or anoxic conditions need a mechanism to obtain energy without using oxygen. A biochemical adaptation of some animals to hypoxia is an alternative mitochondrial electron transport chain (CTEM), in which rhodoquinone (RQ) and not ubiquinone (UQ) is the lipidic electron carrier and fumarate, and not oxygen, is the final electron acceptor. Our results suggest that in C. elegans RQ does not have an essential role in the hypoxia conditions examined, raising the question of whether RQ serves another role in C. elegans. In its natural environment, this organism can encounter high concentrations of hydrogen sulfide (H2S), as well as pathogenic bacteria that kill the worm by the production of hydrogen cyanide (HCN). H2S and HCN are inhibitors of complex IV of the canonical CTEM, preventing oxygen from being used as the final electron acceptor. Therefore, a possible additional role for RQ could be its participation in the worm defense against these toxic compounds. In fact, mutant strains that do not synthesize RQ (and do synthesize UQ) do not survive in the presence of both HCN and H2S high concentration and are more sensitive against the pathogenic bacteria Pseudomonas aeruginosa PA01 (which kills the worm by generating HCN), compared to both the wild type strain and UQ-less worms. These results suggest a new role for RQ in protecting the worm from poisoning with HCN and H2S produced by pathogens present in the worm’s habitat.Agencia Nacional de Investigación e Innovació

El H2S como integrador de señales de estrés en C. elegans.

Animales que enfrentan condiciones de hipoxia o anoxia necesitan un mecanismo de obtención de energía sin utilizar el oxígeno. Una adaptación bioquímica a la hipoxia en algunos animales involucra una cadena de transporte de electrones mitocondrial (CTEM) alternativa, en la cual la rodoquinona (RQ) y no la ubiquinona (UQ) es el transportador de electrones lipídico y el fumarato, y no el oxígeno, es el aceptor final de electrones. Nuestros resultados sugieren que en C. elegans la RQ no sería esencial en las condiciones de hipoxia examinadas, desencadenando la pregunta de otro posible rol de la RQ en C. elegans. Este gusano en su ambiente natural puede enfrentarse a altas concentraciones de sulfuro de hidrógeno (H2S), así como también a bacterias patógenas que matan al gusano mediante la producción de cianuro de hidrógeno (HCN). Tanto el H2S como el HCN son inhibidores del complejo IV de la CTEM canónica. Así, un posible rol adicional de la RQ podría ser participar en la defensa del gusano frente a estos compuestos tóxicos. De hecho, estirpes mutantes que no sintetizan RQ (y sí UQ) no sobreviven en presencia de altas concentraciones de H2S y de HCN y son mas sensibles a la bacteria patógena Pseudomonas aeruginosa PAO1 (que mata al gusano generando HCN), comparado con las estirpes silvestre y mutante deficiente en UQ. Estos resultados proponen un nuevo rol de la RQ en la protección del gusano del envenenamiento con H2S y HCN producidos por patógenos presentes en su hábitat natural.Agencia Nacional de Investigación e Innovació

Selenocysteine incorporation in metazoa: the peculiar case of the nematode lineage

Póster presentado en la 19th International Caenorhabditis elegans Meeting, celebrada en Los Ángeles del 26 al 30 de junio de 2013.Selenocysteine (Sec) incorporation into proteins occurs through a non-canonical mechanism: a Sec incorporation sequence (SECIS) present in the selenoprotein mRNA binds to a SECIS-binding protein allowing a UGA codon to be reprogrammed for Sec. Using Caenorhabditis elegans to study Sec incorporation, we found that its SECIS-binding protein (K04G2.11) is significantly shorter than any known SECIS-binding protein and lacks the putative Sec incorporation domain (SID). We amplified the full-length mRNA and confirmed the annotated sequence. A comparative analysis with other SECIS-binding proteins showed extended amino acid homology in the annotated 5'UTR, upstream the first in-frame AUG codon. This suggests that K04G2.11 would have a non-AUG translation initiation. Moreover, the extension of the ORF completes the functional L7Ae domain. Initiation of translation would occur at an AUU codon in an adequate context for translation initiation (GAAAAUU). We generated transgenic strains that overexpress K04G2.11 fused to a polyHis tail. By sequential metal and SECIS affinity chromatography, we isolated from transgenic strains, but not from N2, a 20kDa protein, which corresponds to the expected mass from the proposed AUU start codon. By 75Se metabolic incorporation we demonstrated that this gene is functional and essential for Sec incorporation. The absence of the SID domain in the genome is remarkable and suggests that Sec incorporation mechanism in C. elegans is peculiar. We investigate Sec incorporation in the nematode lineage. The analysis of nematode genomes indicates: i) the SID domain is absent, ii) SECIS-binding proteins consist of the L7Ae domain only, iii) the existence of non-AUG initiation of translation in nematodes SECIS-binding proteins, and iv) the absence of Sec incorporation in plant nematode parasites. Our results indicate that nematoda is an interesting lineage to study the dynamics of the evolution of Sec incorporation. They also point out the occurrence of non-AUG translation initiation in C. elegans, not described before, a finding that needs to be further investigated.Peer Reviewe

Inhibition of Tapeworm Thioredoxin and Glutathione Pathways by an Oxadiazole N-Oxide Leads to Reduced Mesocestoides vogae Infection Burden in Mice

Parasitic flatworms cause serious infectious diseases that affect humans and livestock in vast regions of the world, yet there are few effective drugs to treat them. Thioredoxin glutathione reductase (TGR) is an essential enzyme for redox homeostasis in flatworm parasites and a promising pharmacological target. We purified to homogeneity and characterized the TGR from the tapeworm Mesocestoides vogae (syn. M. corti). This purification revealed absence of conventional TR and GR. The glutathione reductase activity of the purified TGR exhibits a hysteretic behavior typical of flatworm TGRs. Consistently, M. vogae genome analysis revealed the presence of a selenocysteine-containing TGR and absence of conventional TR and GR. M. vogae thioredoxin and glutathione reductase activities were inhibited by 3,4-bis(phenylsulfonyl)-1,2,5-oxadiazole N2-oxide (VL16E), an oxadiazole N-oxide previously identified as an inhibitor of fluke and tapeworm TGRs. Finally, we show that mice experimentally infected with M. vogae tetrathyridia and treated with either praziquantel, the reference drug for flatworm infections, or VL16E exhibited a 28% reduction of intraperitoneal larvae numbers compared to vehicle treated mice. Our results show that oxadiazole N-oxide is a promising chemotype in vivo and highlights the convenience of M. vogae as a model for rapid assessment of tapeworm infections in vivo