<i>Entamoeba histolytica</i> RacC Selectively Engages p21-Activated Kinase Effectors

Rho family GTPases modulate actin cytoskeleton dynamics by signaling through multiple effectors, including the p21-activated kinases (PAKs). The intestinal parasite <i>Entamoeba histolytica</i> expresses ∼20 Rho family GTPases and seven isoforms of PAK, two of which have been implicated in pathogenesis-related processes such as amoebic motility and invasion and host cell phagocytosis. Here, we describe two previously unstudied PAK isoforms, EhPAK4 and EhPAK5, as highly specific effectors of EhRacC. A structural model based on 2.35 Å X-ray crystallographic data of a complex between EhRacC^Q65L·GTP and the EhPAK4 p21 binding domain (PBD) reveals a fairly well-conserved Rho/effector interface despite deviation of the PBD α-helix. A structural comparison with EhRho1 in complex with EhFormin1 suggests likely determinants of Rho family GTPase signaling specificity in <i>E. histolytica</i>. These findings suggest a high degree of Rho family GTPase diversity and specificity in the single-cell parasite <i>E. histolytica</i>. Because PAKs regulate pathogenesis-related processes in <i>E. histolytica</i>, they may be valid pharmacologic targets for anti-amoebiasis drugs

<i>Entamoeba histolytica</i> Rho1 Regulates Actin Polymerization through a Divergent, Diaphanous-Related Formin

<i>Entamoeba histolytica</i> requires a dynamic actin cytoskeleton for intestinal and systemic pathogenicity. Diaphanous-related formins represent an important family of actin regulators that are activated by Rho GTPases. The <i>E. histolytica</i> genome encodes a large family of Rho GTPases and three diaphanous-related formins, of which EhFormin1 is known to regulate mitosis and cytokinesis in trophozoites. We demonstrate that EhFormin1 modulates actin polymerization through its formin homology 2 domain. Despite a highly divergent diaphanous autoinhibitory domain, EhFormin1 is autoinhibited by an N- and C-terminal intramolecular interaction but activated upon binding of EhRho1 to the N-terminal domain tandem. A crystal structure of the EhRho1·GTPγS–EhFormin1 complex illustrates an EhFormin1 conformation that diverges from mammalian mDia1 and lacks a secondary interaction with a Rho insert helix. The structural model also highlights residues required for specific recognition of the EhRho1 GTPase and suggests that the molecular mechanisms of EhFormin1 autoinhibition and activation differ from those of mammalian homologues

<i>Entamoeba histolytica</i> Rho1 Regulates Actin Polymerization through a Divergent, Diaphanous-Related Formin

<i>Entamoeba histolytica</i> requires a dynamic actin cytoskeleton for intestinal and systemic pathogenicity. Diaphanous-related formins represent an important family of actin regulators that are activated by Rho GTPases. The <i>E. histolytica</i> genome encodes a large family of Rho GTPases and three diaphanous-related formins, of which EhFormin1 is known to regulate mitosis and cytokinesis in trophozoites. We demonstrate that EhFormin1 modulates actin polymerization through its formin homology 2 domain. Despite a highly divergent diaphanous autoinhibitory domain, EhFormin1 is autoinhibited by an N- and C-terminal intramolecular interaction but activated upon binding of EhRho1 to the N-terminal domain tandem. A crystal structure of the EhRho1·GTPγS–EhFormin1 complex illustrates an EhFormin1 conformation that diverges from mammalian mDia1 and lacks a secondary interaction with a Rho insert helix. The structural model also highlights residues required for specific recognition of the EhRho1 GTPase and suggests that the molecular mechanisms of EhFormin1 autoinhibition and activation differ from those of mammalian homologues

<i>Entamoeba histolytica</i> Rho1 Regulates Actin Polymerization through a Divergent, Diaphanous-Related Formin

<i>Entamoeba histolytica</i> requires a dynamic actin cytoskeleton for intestinal and systemic pathogenicity. Diaphanous-related formins represent an important family of actin regulators that are activated by Rho GTPases. The <i>E. histolytica</i> genome encodes a large family of Rho GTPases and three diaphanous-related formins, of which EhFormin1 is known to regulate mitosis and cytokinesis in trophozoites. We demonstrate that EhFormin1 modulates actin polymerization through its formin homology 2 domain. Despite a highly divergent diaphanous autoinhibitory domain, EhFormin1 is autoinhibited by an N- and C-terminal intramolecular interaction but activated upon binding of EhRho1 to the N-terminal domain tandem. A crystal structure of the EhRho1·GTPγS–EhFormin1 complex illustrates an EhFormin1 conformation that diverges from mammalian mDia1 and lacks a secondary interaction with a Rho insert helix. The structural model also highlights residues required for specific recognition of the EhRho1 GTPase and suggests that the molecular mechanisms of EhFormin1 autoinhibition and activation differ from those of mammalian homologues

<i>Entamoeba histolytica</i> Rho1 Regulates Actin Polymerization through a Divergent, Diaphanous-Related Formin

<i>Entamoeba histolytica</i> requires a dynamic actin cytoskeleton for intestinal and systemic pathogenicity. Diaphanous-related formins represent an important family of actin regulators that are activated by Rho GTPases. The <i>E. histolytica</i> genome encodes a large family of Rho GTPases and three diaphanous-related formins, of which EhFormin1 is known to regulate mitosis and cytokinesis in trophozoites. We demonstrate that EhFormin1 modulates actin polymerization through its formin homology 2 domain. Despite a highly divergent diaphanous autoinhibitory domain, EhFormin1 is autoinhibited by an N- and C-terminal intramolecular interaction but activated upon binding of EhRho1 to the N-terminal domain tandem. A crystal structure of the EhRho1·GTPγS–EhFormin1 complex illustrates an EhFormin1 conformation that diverges from mammalian mDia1 and lacks a secondary interaction with a Rho insert helix. The structural model also highlights residues required for specific recognition of the EhRho1 GTPase and suggests that the molecular mechanisms of EhFormin1 autoinhibition and activation differ from those of mammalian homologues

<i>Entamoeba histolytica</i> Rho1 Regulates Actin Polymerization through a Divergent, Diaphanous-Related Formin

<i>Entamoeba histolytica</i> requires a dynamic actin cytoskeleton for intestinal and systemic pathogenicity. Diaphanous-related formins represent an important family of actin regulators that are activated by Rho GTPases. The <i>E. histolytica</i> genome encodes a large family of Rho GTPases and three diaphanous-related formins, of which EhFormin1 is known to regulate mitosis and cytokinesis in trophozoites. We demonstrate that EhFormin1 modulates actin polymerization through its formin homology 2 domain. Despite a highly divergent diaphanous autoinhibitory domain, EhFormin1 is autoinhibited by an N- and C-terminal intramolecular interaction but activated upon binding of EhRho1 to the N-terminal domain tandem. A crystal structure of the EhRho1·GTPγS–EhFormin1 complex illustrates an EhFormin1 conformation that diverges from mammalian mDia1 and lacks a secondary interaction with a Rho insert helix. The structural model also highlights residues required for specific recognition of the EhRho1 GTPase and suggests that the molecular mechanisms of EhFormin1 autoinhibition and activation differ from those of mammalian homologues

<i>Entamoeba histolytica</i> Rho1 Regulates Actin Polymerization through a Divergent, Diaphanous-Related Formin

<i>Entamoeba histolytica</i> requires a dynamic actin cytoskeleton for intestinal and systemic pathogenicity. Diaphanous-related formins represent an important family of actin regulators that are activated by Rho GTPases. The <i>E. histolytica</i> genome encodes a large family of Rho GTPases and three diaphanous-related formins, of which EhFormin1 is known to regulate mitosis and cytokinesis in trophozoites. We demonstrate that EhFormin1 modulates actin polymerization through its formin homology 2 domain. Despite a highly divergent diaphanous autoinhibitory domain, EhFormin1 is autoinhibited by an N- and C-terminal intramolecular interaction but activated upon binding of EhRho1 to the N-terminal domain tandem. A crystal structure of the EhRho1·GTPγS–EhFormin1 complex illustrates an EhFormin1 conformation that diverges from mammalian mDia1 and lacks a secondary interaction with a Rho insert helix. The structural model also highlights residues required for specific recognition of the EhRho1 GTPase and suggests that the molecular mechanisms of EhFormin1 autoinhibition and activation differ from those of mammalian homologues

<i>Entamoeba histolytica</i> Rho1 Regulates Actin Polymerization through a Divergent, Diaphanous-Related Formin

<i>Entamoeba histolytica</i> requires a dynamic actin cytoskeleton for intestinal and systemic pathogenicity. Diaphanous-related formins represent an important family of actin regulators that are activated by Rho GTPases. The <i>E. histolytica</i> genome encodes a large family of Rho GTPases and three diaphanous-related formins, of which EhFormin1 is known to regulate mitosis and cytokinesis in trophozoites. We demonstrate that EhFormin1 modulates actin polymerization through its formin homology 2 domain. Despite a highly divergent diaphanous autoinhibitory domain, EhFormin1 is autoinhibited by an N- and C-terminal intramolecular interaction but activated upon binding of EhRho1 to the N-terminal domain tandem. A crystal structure of the EhRho1·GTPγS–EhFormin1 complex illustrates an EhFormin1 conformation that diverges from mammalian mDia1 and lacks a secondary interaction with a Rho insert helix. The structural model also highlights residues required for specific recognition of the EhRho1 GTPase and suggests that the molecular mechanisms of EhFormin1 autoinhibition and activation differ from those of mammalian homologues

Protective Roles for RGS2 in a Mouse Model of House Dust Mite-Induced Airway Inflammation

<div><p>The GTPase-accelerating protein, regulator of G-protein signalling 2 (RGS2) reduces signalling from G-protein-coupled receptors (GPCRs) that signal via Gαq. In humans, RGS2 expression is up-regulated by inhaled corticosteroids (ICSs) and long-acting β₂-adrenoceptor agonists (LABAs) such that synergy is produced in combination. This may contribute to the superior clinical efficacy of ICS/LABA therapy in asthma relative to ICS alone. In a murine model of house dust mite (HDM)-induced airways inflammation, three weeks of intranasal HDM (25 μg, 3×/week) reduced lung function and induced granulocytic airways inflammation. Compared to wild type animals, <i>Rgs2</i>^-/- mice showed airways hyperresponsiveness (increased airways resistance and reduced compliance). While HDM increased pulmonary inflammation observed on hematoxylin and eosin-stained sections, there was no difference between wild type and <i>Rgs2</i>^-/- animals. HDM-induced mucus hypersecretion was also unaffected by RGS2 deficiency. However, inflammatory cell counts in the bronchoalveolar lavage fluid of <i>Rgs2</i>^-/- animals were significantly increased (57%) compared to wild type animals and this correlated with increased granulocyte (neutrophil and eosinophil) numbers. Likewise, cytokine and chemokine (IL4, IL17, IL5, LIF, IL6, CSF3, CXCLl, CXCL10 and CXCL11) release was increased by HDM exposure. Compared to wild type, <i>Rgs2</i>^-/- animals showed a trend towards increased expression for many cytokines/chemokines, with CCL3, CCL11, CXCL9 and CXCL10 being significantly enhanced. As RGS2 expression was unaffected by HDM exposure, these data indicate that RGS2 exerts tonic bronchoprotection in HDM-induced airways inflammation. Modest anti-inflammatory and anti-remodelling roles for RGS2 are also suggested. If translatable to humans, therapies that maximize RGS2 expression may prove advantageous.</p></div

Effect of RGS2 deficiency on HDM-induced mucus secretion.

<p>Wild type (WT) and <i>Rgs2</i>^-/- (KO) mice were subjected to <i>i</i>.<i>n</i>. instillation of PBS or HDM (25 μg) 3 times/week for 3 weeks. A, Lungs were dissected, inflated and fixed in 10% formalin and embedded in wax. Sections were stained with PAS to allow visualization of mucus. B, PAS-stained sections were scored using a semi-quantitative scoring system where a score of 4 indicated strong staining (more than 75% of the airway epithelium PAS positive), 3 = moderate staining (50–75% of the airway epithelium PAS positive), 2 = mild staining (25–50% of the airway epithelium PAS positive), and a score of 1 = minimal staining (less than 25% of airway epithelium PAS positive). Data (<i>N</i> = 17 for PBS-exposed wild type and 15 for <i>Rgs2</i>^-/-, N = 29 for HDM-exposed wild type and 24 for <i>Rgs2</i>^-/-) are plotted as means ± SE. C, Image J software was used to measure the amount of mucus secreted from the airway epithelium and all measurements were made by an investigator blinded to the study treatments. Data (all <i>N</i> = 8) are plotted as means ± SE. D, RNA was extracted from the right lungs and mucin gene expression analyzed by qPCR. Data (<i>N</i> = 23 for PBS-exposed wild type and 22 for <i>Rgs2</i>^-/-, N = 34 for HDM-exposed wild type and 29 for <i>Rgs2</i>^-/-), normalized to GAPDH, are plotted as means ± SE.</p