Elucidating G protein signaling and ubiquitin conjugation in Entamoeba histolytica Public Deposited

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  • March 22, 2019
  • Bosch, Dustin Eli
    • Affiliation: School of Medicine, Department of Pharmacology
  • The intestinal parasite Entamoeba histolytica is responsible for an estimated 50 million infections and 100,000 deaths per year worldwide. The causative agent of amoebic colitis and systemic amoebiasis is spread primarily through contaminated food and drinking water sources. Although reasonably effective treatments have long been available for invasive amoebiasis, imperfect patient response rates, drug side effects, and concern for emerging drug resistance all warrant exploration of new pharmacological targets in E. histolytica. This work describes structural, biochemical, and cell biological explorations of heterotrimeric G protein and Rho family GTPase signaling and ubiquitination in E. histolytica. The heterotrimeric G protein subunits EhG(alpha)1, EhG(beta)1 and either EhG(gamma)1/2 assembled a typical nucleotide-dependent heterotrimer. Overexpression of wildtype or dominant negative EhG(alpha)1 in E. histolytica altered chemotactic migration, Matrigel transmigration, host cell attachment, and cell killing. Transcriptomic studies linked EhG(alpha)1 expression and altered transcription and secretion of virulence factors. EhG(alpha)1 is distinct from the conventional mammalian G(alpha) subfamilies, as revealed by sequence comparison and a crystalstructure, but shares functionality with mammalian G(alpha)12/13 in engaging an RGS-RhoGEF effector. EhRGS-RhoGEF is apparently autoinhibited, as indicated by structural obstruction of its catalytic domain in the inactive state. However, co-expression with constitutively active EhG(alpha)1 and EhRacC lead to EhRGS-RhoGEF activation in cells. The Rho family GTPase EhRho1 lacks a signature Rho insert helix and sensitivity to C3 exoenzyme. Crystal structures of EhRho1 indicate unique nucleotide-contacting residues that confer fast intrinsic nucleotide exchange activity. However, EhRho1 functions like its homologs in engaging a diaphanous-related formin effector to regulate actin polymerization. EhFormin1 is autoinhibited and is activated by Rho GTPase binding. A crystal structure of the EhRho1/EhFormin1 complex indicates similarity to human RhoC/mDia1, despite an absent secondary binding site at the Rho insert helix. Multiple ubiquitin-proteasome pathway genes were differentially transcribed upon perturbed EhG(alpha)1 expression. EhUbiquitin was activated by the E1 enzyme EhUba1 and conjugated by the E2 enzyme EhUbc5, indicating a conserved ubiquitination cascade in E. histolytica. Crystal structures of EhUbiquitin and EhUbc5 suggested potentially unique polyubiquitin linkages, but a likely conserved mode of ubiquitin chain elongation.
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  • Siderovski, David P.
  • Doctor of Philosophy
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  • 2013

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