ROP18 Is a Rhoptry Kinase Controlling the Intracellular Proliferation of Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular parasite for which the discharge of apical organelles named rhoptries is a key event in host cell invasion. Among rhoptry proteins, ROP2, which is the prototype of a large protein family, is translocated in the parasitophorous vacuole membrane during invasion. The ROP2 family members are related to protein-kinases, but only some of them are predicted to be catalytically active, and none of the latter has been characterized so far. We show here that ROP18, a member of the ROP2 family, is located in the rhoptries and re-localises at the parasitophorous vacuole membrane during invasion. We demonstrate that a recombinant ROP18 catalytic domain (amino acids 243–539) possesses a protein-kinase activity and phosphorylate parasitic substrates, especially a 70-kDa protein of tachyzoites. Furthermore, we show that overexpression of ROP18 in transgenic parasites causes a dramatic increase in intra-vacuolar parasite multiplication rate, which is correlated with kinase activity. Therefore, we demonstrate, to our knowledge for the first time, that rhoptries can discharge active protein-kinases upon host cell invasion, which can exert a long-lasting effect on intracellular parasite development and virulence

Antimicrobial treatment improves mycobacterial survival in nonpermissive growth conditions

Antimicrobials targeting cell wall biosynthesis are generally considered inactive against nonreplicating bacteria. Paradoxically, we found that under nonpermissive growth conditions, exposure of Mycobacterium bovis BCG bacilli to such antimicrobials enhanced their survival. We identified a transcriptional regulator, RaaS (for regulator of antimicrobial-assisted survival), encoded by bcg1279 (rv1219c) as being responsible for the observed phenomenon. Induction of this transcriptional regulator resulted in reduced expression of specific ATP-dependent efflux pumps and promoted long-term survival of mycobacteria, while its deletion accelerated bacterial death under nonpermissive growth conditions in vitro and during macrophage or mouse infection. These findings have implications for the design of antimicrobial drug combination therapies for persistent infectious diseases, such as tuberculosis

A Histone Deacetylase (HDAC) inhibitor with pleiotropic in vitro anti-toxoplasma and anti-plasmodium activities controls acute and chronic toxoplasma infection in mice

© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).Toxoplasmosis is a highly prevalent human disease, and virulent strains of this parasite emerge from wild biotopes. Here, we report on the potential of a histone deacetylase (HDAC) inhibitor we previously synthesized, named JF363, to act in vitro against a large panel of Toxoplasma strains, as well as against the liver and blood stages of Plasmodium parasites, the causative agents of malaria. In vivo administration of the drug significantly increases the survival of mice during the acute phase of infection by T. gondii, thus delaying its spreading. We further provide evidence of the compound's efficiency in controlling the formation of cysts in the brain of T. gondii-infected mice. A convincing docking of the JF363 compound in the active site of the five annotated ME49 T. gondii HDACs was performed by extensive sequence-structure comparison modeling. The resulting complexes show a similar mode of binding in the five paralogous structures and a quite similar prediction of affinities in the micromolar range. Altogether, these results pave the way for further development of this compound to treat acute and chronic toxoplasmosis. It also shows promise for the future development of anti-Plasmodium therapeutic interventions.This research was funded by IDEX Innovation Grant, UGA, 2017 and The GIS ChemBioFranceinfo:eu-repo/semantics/publishedVersio

Oleoyl Coenzyme A regulates interaction of transcriptional regulator RaaS (Rv1219c) with DNA in Mycobacteria

We have recently shown that RaaS (regulator of antimicrobial-assisted survival), encoded by Rv1219c in Mycobacterium tuberculosis and by bcg_1279c in Mycobacterium bovis bacillus Calmette-Guérin, plays an important role in mycobacterial survival in prolonged stationary phase and during murine infection. Here, we demonstrate that long chain acyl-CoA derivatives (oleoyl-CoA and, to lesser extent, palmitoyl-CoA) modulate RaaS binding to DNA and expression of the downstream genes that encode ATP-dependent efflux pumps. Moreover, exogenously added oleic acid influences RaaS-mediated mycobacterial improvement of survival and expression of the RaaS regulon. Our data suggest that long chain acyl-CoA derivatives serve as biological indicators of the bacterial metabolic state. Dysregulation of efflux pumps can be used to eliminate non-growing mycobacteria

Structural and functional characterization of the Mycobacterium tuberculosis uridine monophosphate kinase: insights into the allosteric regulation†

Nucleoside Monophosphate Kinases (NMPKs) family are key enzymes in nucleotide metabolism. Bacterial UMPKs depart from the main superfamily of NMPKs. Having no eukaryotic counterparts they represent attractive therapeutic targets. They are regulated by GTP and UTP, while showing different mechanisms in Gram(+), Gram(–) and archaeal bacteria. In this work, we have characterized the mycobacterial UMPK (UMPKmt) combining enzymatic and structural investigations with site-directed mutagenesis. UMPKmt exhibits cooperativity toward ATP and an allosteric regulation by GTP and UTP. The crystal structure of the complex of UMPKmt with GTP solved at 2.5 Å, was merely identical to the modelled apo-form, in agreement with SAXS experiments. Only a small stretch of residues was affected upon nucleotide binding, pointing out the role of macromolecular dynamics rather than major structural changes in the allosteric regulation of bacterial UMPKs. We further probe allosteric regulation by site-directed mutagenesis. In particular, a key residue involved in the allosteric regulation of this enzyme was identified

Functioning of the dimeric GABA(B) receptor extracellular domain revealed by glycan wedge scanning

The G-protein-coupled receptor (GPCR) activated by the neurotransmitter GABA is made up of two subunits, GABA(B1) and GABA(B2). GABA(B1) binds agonists, whereas GABA(B2) is required for trafficking GABA(B1) to the cell surface, increasing agonist affinity to GABA(B1), and activating associated G proteins. These subunits each comprise two domains, a Venus flytrap domain (VFT) and a heptahelical transmembrane domain (7TM). How agonist binding to the GABA(B1) VFT leads to GABA(B2) 7TM activation remains unknown. Here, we used a glycan wedge scanning approach to investigate how the GABA(B) VFT dimer controls receptor activity. We first identified the dimerization interface using a bioinformatics approach and then showed that introducing an N-glycan at this interface prevents the association of the two subunits and abolishes all activities of GABA(B2), including agonist activation of the G protein. We also identified a second region in the VFT where insertion of an N-glycan does not prevent dimerization, but blocks agonist activation of the receptor. These data provide new insight into the function of this prototypical GPCR and demonstrate that a change in the dimerization interface is required for receptor activation

MM-PBSA and the Importance of the Dielectric Constant for Kinase Drug Design

Predicting the interactions between a set of small molecules and its target plays a critical role in drug discovery and development. Especially in later stages of the drug design process, when a reduced set of molecules is in focus, reliable and accurate binding affinity estimations are important for targeted modifications of given lead molecules.Current limitations in affinity prediction originate from the lack of accurate estimates for solvation energy and entropy. MM-PBSA and the related MM-GBSA aim at providing better estimates.From our studies we infer that the common approach using one dielectric constant for the binding pocket may be misleading (here in the case of a kinase), especially when designed ligands/drugs contain charges. Thus, a range of selected values for the solute dielectric constant is preferred for better and more reliable comparisons.</div

Profilage

Les protéine kinases ont été rapidement identifiées comme favorisant l’apparition de cancers, à travers leur implication dans la régulation du développement et du cycle cellulaire. Il y a une vingtaine d’années, la mise sur le marché des premiers traitements par inhibiteur de protéine kinase, ouvrait la voie vers de nouvelles solutions médicamenteuses plus ciblées contre le cancer. Depuis, nombreuses sont les données structurales et fonctionnelles acquises sur ces cibles thérapeutiques. Les techniques informatiques ont elles aussi évolué, notamment les méthodes d’apprentissage automatique. En tirant parti de la grande quantité d’informations disponibles aujourd’hui, ces méthodes devraient permettre prochainement la prédiction fine de l’interaction d’un inhibiteur donné avec chaque protéine kinase humaine et donc, à terme, la construction d’outils de profilage de leurs inhibiteurs spécifiques. Cette approche intégrative devrait aider la découverte de solutions thérapeutiques anti-cancéreuses plus efficaces et plus sûres

Fragment and Conquer: From Structure to Complexes to Function

In this issue of Structure, Shumilin and colleagues show the power of metabolite screening by means of X-ray crystallography to link an orphan protein domain with an orphan biochemical function. This result paves the way for large-scale functional annotation and sets new objectives for structural genomics