Dataset Information

Analysis of the interactome of Schistosoma mansoni histone deacetylase 8.

ABSTRACT: 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.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.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.

SUBMITTER: Caby S

PROVIDER: S-EPMC5722368 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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Analysis of the interactome of Schistosoma mansoni histone deacetylase 8.

Caby Stéphanie S Pagliazzo Lucile L Lancelot Julien J Saliou Jean-Michel JM Bertheaume Nicolas N Pierce Raymond J RJ Roger Emmanuel E

PLoS neglected tropical diseases 20171120 11

<h4>Background</h4>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.<h4>Me ...[more]

PMID: 29155817

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Project description:BackgroundSchistosoma 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 findingsWe 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/significanceOur 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.

Project description:BackgroundSchistosomiasis is a parasitic disease infecting hundreds of millions of people worldwide. Treatment depends on a single drug, praziquantel, which kills the Schistosoma spp. parasite only at the adult stage. HDAC inhibitors (HDACi) such as Trichostatin A (TSA) induce parasite mortality in vitro (schistosomula and adult worms), however the downstream effects of histone hyperacetylation on the parasite are not known.Methodology/principal findingsTSA treatment of adult worms in vitro increased histone acetylation at H3K9ac and H3K14ac, which are transcription activation marks, not affecting the unrelated transcription repression mark H3K27me3. We investigated the effect of TSA HDACi on schistosomula gene expression at three different time points, finding a marked genome-wide change in the transcriptome profile. Gene transcription activity was correlated with changes on the chromatin acetylation mark at gene promoter regions. Moreover, combining expression data with ChIP-Seq public data for schistosomula, we found that differentially expressed genes having the H3K4me3 mark at their promoter region in general showed transcription activation upon HDACi treatment, compared with those without the mark, which showed transcription down-regulation. Affected genes are enriched for DNA replication processes, most of them being up-regulated. Twenty out of 22 genes encoding proteins involved in reducing reactive oxygen species accumulation were down-regulated. Dozens of genes encoding proteins with histone reader motifs were changed, including SmEED from the PRC2 complex. We targeted SmEZH2 methyltransferase PRC2 component with a new EZH2 inhibitor (GSK343) and showed a synergistic effect with TSA, significantly increasing schistosomula mortality.Conclusions/significanceGenome-wide gene expression analyses have identified important pathways and cellular functions that were affected and may explain the schistosomicidal effect of TSA HDACi. The change in expression of dozens of histone reader genes involved in regulation of the epigenetic program in S. mansoni can be used as a starting point to look for possible novel schistosomicidal targets.

Dataset Information

Analysis of the interactome of Schistosoma mansoni histone deacetylase 8.

Publications

Analysis of the interactome of Schistosoma mansoni histone deacetylase 8.

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