Histone deacetylase 8 interacts with the GTPase SmRho1 in Schistosoma mansoni

Abstract

BACKGROUNDSchistosoma mansoni histone deacetylase 8 (SmHDAC8) is a privileged target for drug discovery. Invalidation of its transcription 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 have been 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-IP/Mass Spectrometry 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 SmRho interaction in adult worms and schistosomula, we performed co-immunoprecipitation (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. A specific interaction between SmHDAC8 and the C-terminal moiety of this isoform was demonstrated, and we showed that SmRho1 is acetylated on lysine K136. SmHDAC8 inhibition or knockdown using RNAi caused massive disruption of schistosomula actin cytoskeleton. A specific interaction between SmRho1.2 and SmDia suggested the existence of two signaling pathways that could regulate cytoskeleton organization via the two SmRho1 isoforms. Author summarySchistosoma mansoni is the major parasitic platyhelminth species causing intestinal schistosomiasis, for which around 200 million people are in need of treatment. Currently one drug, praziquantel, is the treatment of choice and its use in mass treatment programs, rendered imperative the development of new therapeutic agents. As new potential targets, we have focused on lysine deacetylases, and in particular Schistosoma mansoni histone deacetylase 8 (SmHDAC8). Previous studies showed that invalidation of the transcription of SmHDAC8 by RNAi led to the impaired survival of the worms after the infection of mice. The analysis of the 3D structure of SmHDAC8 by X-ray crystallography showed that the catalytic domain structure diverges significantly from that of human HDAC8 and this was exploited to develop novel anti-schistosomal drugs. Biological roles of SmHDAC8 are unknown. For this reason, we previously characterized its protein partners and identified the schistosome orthologue of the human RhoA GTPase, suggesting the involvement of SmHDAC8 in the modulation of cytoskeleton organization. Here, we investigated the interaction between SmHDAC8 and SmRho1 and identified two SmRho1 isoforms (SmRho1.1 and SmRho1.2). Our study showed that SmHDAC8 is indeed involved in schistosome cytoskeleton organization.