Histone deacetylase 8 interacts with the GTPase SmRho1 in Schistosoma mansoni

PLoS Negl Trop Dis. 2021 Nov 29;15(11):e0009503. doi: 10.1371/journal.pntd.0009503. eCollection 2021 Nov.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylation
  • Animals
  • Female
  • GTP Phosphohydrolases / chemistry*
  • GTP Phosphohydrolases / genetics
  • GTP Phosphohydrolases / metabolism
  • Histone Deacetylases / chemistry*
  • Histone Deacetylases / genetics
  • Histone Deacetylases / metabolism
  • Humans
  • Male
  • Mice
  • Mice, Inbred BALB C
  • Oocytes
  • RNA Interference
  • Schistosoma mansoni / genetics
  • Schistosoma mansoni / metabolism*
  • Tandem Mass Spectrometry
  • Xenopus laevis
  • rhoA GTP-Binding Protein / chemistry*
  • rhoA GTP-Binding Protein / genetics
  • rhoA GTP-Binding Protein / metabolism

Substances

  • RHOA protein, human
  • Histone Deacetylases
  • GTP Phosphohydrolases
  • rhoA GTP-Binding Protein

Grants and funding

This work and the the authors LP, SC, JL, TH, WS, JV and RJP have been supported by funding from the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement no. 602080 (AParaDDisE). Authors LP, SC, JL, S S-D, J-MS, TH, KC, JV and RJP were supported by institutional funds from the CNRS UMR 9017, the Institut Pasteur de Lille and Lille University. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.