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3. Biophysical analysis of the membrane-proximal Venus Flytrap domain of ESAG4 receptor-like adenylate cyclase from Trypanosoma brucei.

Author

Alves, Desirée O., Geens, Rob, da Silva Arruda, Hiam R., Jennen, Lisa, Corthaut, Sam, Wuyts, Ellen, de Andrade, Guilherme Caldas, Prosdocimi, Francisco, Cordeiro, Yraima, Pires, José Ricardo, Vieira, Larissa Rezende, de Oliveira, Guilherme A.P., Sterckx, Yann G.-J., and Salmon, Didier

Subjects
*ADENYLATE cyclase, *SMALL-angle scattering, *GUANYLATE cyclase, *LIGANDS (Chemistry), *CATALYTIC domains
Abstract

The protozoan parasite Trypanosoma brucei possesses a large family of transmembrane receptor-like adenylate cyclases (RACs), primarily located to the flagellar surface and involved in sensing of the extracellular environment. RACs exhibit a conserved topology characterized by a large N-terminal extracellular moiety harbouring two Venus Flytrap (VFT) bilobate structures separated from an intracellular catalytic domain by a single transmembrane helix. RAC activation, which typically occurs under mild acid stress, requires the dimerization of the intracellular catalytic domain. The occurrence of VFT domains in the RAC's extracellular moiety suggests their potential responsiveness to extracellular ligands in the absence of stress, although no such ligands have been identified so far. Herein we report the biophysical characterization of the membrane-proximal VFT2 domain of a bloodstream form-specific RAC called ESAG4, whose ectodomain 3D structure is completely unknown. The paper describes an AlphaFold2-based optimisation of the expression construct, enabling facile and high-yield recombinant production and purification of the target protein. Through an interdisciplinary approach combining various biophysical methods, we demonstrate that the optimised VFT2 domain obtained by recombination is properly folded and behaves as a monomer in solution. The latter suggests a ligand-binding capacity independent of dimerization, unlike typical mammalian VFT receptors, as guanylate cyclase. In silico VFT2 genomic analyses shows divergence among cyclase isoforms, hinting at ligand specificity. Taken together this improved procedure enabling facile and high-yield recombinant production and purification of the target protein could benefit researchers studying trypanosomal RAC VFT domains but also any trypanosome domain with poorly defined boundaries. Additionally, our findings support the stable monomeric VFT2 domain as a useful tool for future structural investigations and ligand screening. [Display omitted] • AlphaFold2 predictions can be used for optimize trypanosomal domain expression. • The membrane-proximal VFT2 domain of ESAG4 behaves as a monomer in solution. • Structural divergence among cyclase isoforms suggests ligand specificity. • Stable monomer suggests dimerization-independent ligand-binding capacity. [ABSTRACT FROM AUTHOR]

Published
2024
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9. Levamisole and ryanodine receptors (I): A contraction study in Ascaris suum

Author

Robertson, Alan P., Clark, Cheryl L., and Martin, Richard J.

Subjects
*RYANODINE receptors, *LEVAMISOLE, *ASCARIS, *DRUG resistance in microorganisms, *PHYSIOLOGICAL effects of caffeine, *NEUROPEPTIDES, *DANTROLENE, *ELECTROPHYSIOLOGY
Abstract

Abstract: Cholinergic anthelmintics (like levamisole) are important drugs but resistance with reduced responses by the parasite to these compounds is a concern. There is a need to study and understand mechanisms that affect the amplitude of the responses of parasites to these drugs. In this paper, we study interactions of levamisole and ryanodine receptors on contractions of Ascaris suum body muscle flaps. In our second paper, we extend these observations to examine electrophysiological interactions of levamisole, ryanodine receptors (RyRs) and AF2. We report that the maximum force of contraction, g max , was dependent on the extracellular concentration of calcium but the levamisole EC 50 (0.8μM) was not. The relationship between maximum force of contraction and extracellular calcium was described by the Michaelis–Menten equation with a K m of 1.8mM. Ryanodine inhibited g max without effect on EC 50 ; ryanodine inhibited only 44% of the maximum contraction (K i of 40nM), revealing a ryanodine-insensitive component in the levamisole excitation–contraction pathway. Dantrolene had the same effect as ryanodine but was less potent. The neuropeptide AF2 (1μM) decreased the levamisole EC 50 to 0.2μM without effect on g max ; 0.1μM ryanodine and 100μM dantrolene, inhibited the g max of the AF2-potentiated levamisole response. High concentrations of caffeine, 30mM, produced weak contraction of the body-flap preparation. Caffeine behaved like ryanodine in that it inhibited the maximum force of contraction, g max , without effects on the levamisole EC 50 . Thus, RyRs play a modulatory role in the levamisole excitation–contraction pathway by affecting the maximum force of contraction without an effect on levamisole EC 50 . The levamisole excitation–contraction coupling is graded and has at least two pathways: one sensitive to ryanodine and one not. [Copyright &y& Elsevier]

Published
2010
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15. Purification of native Sigma class glutathione transferase from Fasciola hepatica.

Author

Duncan, Joshua, Cutress, David, Morphew, Russell M., and Brophy, Peter M.

Subjects
*FASCIOLA hepatica, *GLUTATHIONE transferase, *FASCIOLIASIS, *VACCINE effectiveness, *PREVENTION, *THERAPEUTICS
Abstract

Fascioliasis is a parasitic disease of grazing livestock and a threat to global food security by significantly reducing the production value of sheep, goats and cattle. Moreover, the zoonotic parasite is also a re-emerging food borne threat to human populations. Driven by climate change, the prevalence of fascioliasis is set to increase. Efforts to control the main causative agent, Fasciola hepatica , are hampered by short lived chemotherapy approaches that are becoming increasingly obsolete due to therapeutic failure and resistance. A protective vaccine is urgently needed. A recombinant form of Sigma class glutathione transferase (Hematopoietic Prostaglandin D synthase) from F. hepatica ( Fh GSTS1) with confirmed prostaglandin synthase activity shows immune-modulation activity via suppression of Th17 responses in host dendritic cells. In vaccine trials recombinant Fh GSTS1 reduces liver pathology but not worm burden. Native Fh GSTS1 is yet to be tested for immune-modulation activities or for vaccine potential, primarily due to the technical difficulty in purifying Fh GST-S1 away from the other more abundant GST members in F. hepatica cytosol. This paper reports a pipeline for the purification of native Fh GSTS1 using a two-step process consisting of glutathione-agarose affinity and cationic exchange chromatography. The methodology allows for the isolation of purified and active Fh GSTS1 or Sigma GSTs from other sources for analytical biochemical and immunological studies. [ABSTRACT FROM AUTHOR]

Published
2018
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25. Transcriptomic analyses of the avirulent protozoan parasite Trypanosoma rangeli

Author

Grisard, Edmundo C., Stoco, Patrícia H., Wagner, Glauber, Sincero, Thaís C.M., Rotava, Gianinna, Rodrigues, Juliana B., Snoeijer, Cristiane Q., Koerich, Leonardo B., Sperandio, Maísa M., Bayer-Santos, Ethel, Fragoso, Stenio P., Goldenberg, Samuel, Triana, Omar, Vallejo, Gustavo A., Tyler, Kevin M., Dávila, Alberto M.R., and Steindel, Mário

Published
2010
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