RESUMEN
BACKGROUND: Schistosoma mansoni histone deacetylase 8 (SmHDAC8) has elicited considerable interest as a target for drug discovery. Invalidation of its transcripts by RNAi leads to impaired survival of the worms in infected mice and its inhibition causes cell apoptosis and death. To determine why it is a promising therapeutic target the study of the currently unknown cellular signaling pathways involving this enzyme is essential. Protein partners of SmHDAC8 were previously identified by yeast two-hybrid (Y2H) cDNA library screening and by mass spectrometry (MS) analysis. Among these partners we characterized SmRho1, the schistosome orthologue of human RhoA GTPase, which is involved in the regulation of the cytoskeleton. In this work, we validated the interaction between SmHDAC8 and SmRho1 and explored the role of the lysine deacetylase in cytoskeletal regulation. METHODOLOGY/PRINCIPAL FINDINGS: We characterized two isoforms of SmRho1, SmRho1.1 and SmRho1.2. Co- immunoprecipitation (Co-IP)/Mass Spectrometry (MS) analysis identified SmRho1 partner proteins and we used two heterologous expression systems (Y2H assay and Xenopus laevis oocytes) to study interactions between SmHDAC8 and SmRho1 isoforms. To confirm SmHDAC8 and SmRho1 interaction in adult worms and schistosomula, we performed Co-IP experiments and additionally demonstrated SmRho1 acetylation using a Nano LC-MS/MS approach. A major impact of SmHDAC8 in cytoskeleton organization was documented by treating adult worms and schistosomula with a selective SmHDAC8 inhibitor or using RNAi followed by confocal microscopy. CONCLUSIONS/SIGNIFICANCE: Our results suggest that SmHDAC8 is involved in cytoskeleton organization via its interaction with the SmRho1.1 isoform. The SmRho1.2 isoform failed to interact with SmHDAC8, but did specifically interact with SmDia suggesting the existence of two distinct signaling pathways regulating S. mansoni cytoskeleton organization via the two SmRho1 isoforms. A specific interaction between SmHDAC8 and the C-terminal moiety of SmRho1.1 was demonstrated, and we showed that SmRho1 is acetylated on K136. SmHDAC8 inhibition or knockdown using RNAi caused extensive disruption of schistosomula actin cytoskeleton.
Asunto(s)
GTP Fosfohidrolasas/química , Histona Desacetilasas/química , Schistosoma mansoni/metabolismo , Proteína de Unión al GTP rhoA/química , Acetilación , Animales , Femenino , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Oocitos , Interferencia de ARN , Schistosoma mansoni/genética , Espectrometría de Masas en Tándem , Xenopus laevis , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
In this work, we show that Not4 and Not5 from the Ccr4-Not complex modulate translation elongation dynamics and change ribosome A-site dwelling occupancy in a codon-dependent fashion. These codon-specific changes in not5Δ cells are very robust and independent of codon position within the mRNA, the overall mRNA codon composition, or changes of mRNA expression levels. They inversely correlate with codon-specific changes in cells depleted for eIF5A and positively correlate with those in cells depleted for ribosome-recycling factor Rli1. Not5 resides in punctate loci, co-purifies with ribosomes and Rli1, but not with eIF5A, and limits mRNA solubility. Overexpression of wild-type or non-complementing Rli1 and loss of Rps7A ubiquitination enable Not4 E3 ligase-dependent translation of polyarginine stretches. We propose that Not4 and Not5 modulate translation elongation dynamics to produce a soluble proteome by Rps7A ubiquitination, dynamic condensates that limit mRNA solubility and exclude eIF5A, and a moonlighting function of Rli1.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Factor 5 Eucariótico de Iniciación/metabolismo , Extensión de la Cadena Peptídica de Translación , Factores de Iniciación de Péptidos/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Represoras/metabolismo , Subunidades Ribosómicas Pequeñas/metabolismo , Ribosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Factor 5 Eucariótico de Iniciación/genética , Regulación Fúngica de la Expresión Génica , Factores de Iniciación de Péptidos/genética , ARN de Hongos/genética , ARN de Hongos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas Represoras/genética , Subunidades Ribosómicas Pequeñas/genética , Ribosomas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transducción de Señal , Factores de Transcripción/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación , Factor 5A Eucariótico de Iniciación de TraducciónRESUMEN
BACKGROUND: Histone deacetylase 8 from Schistosoma mansoni (SmHDAC8) is essential to parasite growth and development within the mammalian host and is under investigation as a target for the development of selective inhibitors as novel schistosomicidal drugs. Although some protein substrates and protein partners of human HDAC8 have been characterized, notably indicating a role in the function of the cohesin complex, nothing is known of the partners and biological function of SmHDAC8. METHODOLOGY/PRINCIPAL FINDINGS: We therefore employed two strategies to characterize the SmHDAC8 interactome. We first used SmHDAC8 as a bait protein in yeast two-hybrid (Y2H) screening of an S. mansoni cDNA library. This allowed the identification of 49 different sequences encoding proteins. We next performed co-immunoprecipitation (Co-IP) experiments on parasite extracts with an anti-SmHDAC8 antibody. Mass spectrometry (MS) analysis allowed the identification of 160 different proteins. CONCLUSIONS/SIGNIFICANCE: SmHDAC8 partners are involved in about 40 different processes, included expected functions such as the cohesin complex, cytoskeleton organization, transcriptional and translational regulation, metabolism, DNA repair, the cell cycle, protein dephosphorylation, proteolysis, protein transport, but also some proteasome and ribosome components were detected. Our results show that SmHDAC8 is a versatile deacetylase, potentially involved in both cytosolic and nuclear processes.
