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Tag: Roberto Docampo

Further insights of selenium-containing analogues of WC-9 against Trypanosoma cruzi

Donna Huber on February 28, 2019

Graphical abstract

As a continuation of our project aimed at searching for new chemotherapeutic agents against American trypanosomiasis (Chagas disease), new selenocyanate derivatives were designed, synthesized and biologically evaluated against the clinically more relevant dividing form of Trypanosoma cruzi, the etiologic agent of this illness. In addition, in order to establish the role of each part of the selenocyanate moiety, different derivatives, in which the selenium atom or the cyano group were absent, were conceived, synthesized and biologically evaluated. In addition, in order to study the optimal position of the terminal phenoxy group, new regioisomers of WC-9 were synthesized and evaluated against T. cruzi. Finally, the resolution of a racemic mixture of a very potent conformationally rigid analogue of WC-9 was accomplished and further tested as growth inhibitors of T. cruzi proliferation. The results provide further insight into the role of the selenocyanate group in its antiparasitic activity.

 

María N. Chao, María V. Lorenzo-Ocampo, Sergio H. Szajnman, Roberto Docampo, Juan B. Rodriguez. 2019. Bioorganic & Medicinal Chemistry. https://doi.org/10.1016/j.bmc.2019.02.039

5-Diphosphoinositol Pentakisphosphate (5-IP7) Regulates Phosphate Release from Acidocalcisomes and Yeast Vacuoles

Donna Huber on October 12, 2018

Abstract

Acidocalcisomes of Trypanosoma brucei and the acidocalcisome-like vacuoles of Saccharomyces cerevisiae are acidic calcium compartments that store polyphosphate (polyP). Both organelles possess a phosphate sodium symporter (TbPho91, and Pho91p, in T. brucei and yeast, respectively), but the roles of these transporters in growth and orthophosphate (Pi) transport are unclear. We found here that Tbpho91-/- trypanosomes have a lower growth rate under phosphate starvation, and contain larger acidocalcisomes that have increased Pi content. Heterologous expression of TbPHO91 in Xenopus oocytes followed by two-electrode voltage clamp recordings disclosed that myo-inositol polyphosphates stimulate both sodium-dependent depolarization of the oocyte membrane potential and Pi conductance. Deletion of the SPX domain in TbPho91 abolished this stimulation. Inositol pyrophosphates such as 5-diphosphoinositol pentakisphosphate generated outward currents in Na+/Pi -loaded giant vacuoles prepared from wild type or from TbPHO91-expressing pho91Δ strains but not from the pho91Δ strains, or from the pho91Δ strains expressing PHO91 or TbPHO91 with mutated SPX domains. Our results indicate that TbPho91 and Pho91p are responsible for vacuolar Pi and Na+ efflux and that myo-inositol polyphosphates stimulate the Na+/Pi symporter activities through their SPX domains.

Evgeniy Potapenko, Ciro D Cordeiro, Guozhong Huang, Melissa Storey, Christopher Wittwer, Amit K Dutta, Henning J. Jessen, Vincent J. Starai and Roberto Docampo. 2018. Journal of Biological Chemistry; 293:19101-19112.
doi: 10.1074/jbc.RA118.005884

Inorganic Polyphosphate Interacts with Nucleolar and Glycosomal Proteins in Trypanosomatids

Donna Huber on September 19, 2018

Summary

Inorganic polyphosphate (polyP) is a polymer of three to hundreds of phosphate units bound by high‐energy phosphoanhydride bonds and present from bacteria to humans. Most polyP in trypanosomatids is concentrated in acidocalcisomes, acidic calcium stores that possess a number of pumps, exchangers, and channels, and are important for their survival. In this work, using polyP as bait we identified > 25 putative protein targets in cell lysates of both Trypanosoma cruzi and Trypanosoma brucei. Gene ontology analysis of the binding partners found a significant over‐representation of nucleolar and glycosomal proteins. Using the polyphosphate‐binding domain (PPBD) of Escherichia coliexopolyphosphatase (PPX), we localized long‐chain polyP to the nucleoli and glycosomes of trypanosomes. A competitive assay based on the pre‐incubation of PPBD with exogenous polyP and subsequent immunofluorescence assay of procyclic forms (PCF) of T. brucei showed polyP concentration‐dependent and chain length‐dependent decrease in the fluorescence signal. Subcellular fractionation experiments confirmed the presence of polyP in glycosomes of T. brucei PCF. Targeting of yeast PPX to the glycosomes of PCF resulted in polyP hydrolysis, alteration in their glycolytic flux and increase in their susceptibility to oxidative stress.

Raquel S. Negreiros, Noelia Lander, Guozhong Huang, Ciro D. Cordeiro, Stephanie A. Smith, James H. Morrissey, Roberto Docampo. 2018. Molecular Microbiology; 110(6):973-994. https://doi.org/10.1111/mmi.14131

Calcium-sensitive pyruvate dehydrogenase phosphatase is required for energy metabolism, growth, differentiation, and infectivity of Trypanosoma cruzi

Donna Huber on September 19, 2018

Abstract

In vertebrate cells, mitochondrial Ca2+ uptake by the mitochondrial calcium uniporter (MCU) leads to Ca2+-mediated stimulation of an intramitochondrial pyruvate dehydrogenase phosphatase (PDP). This enzyme dephosphorylates serine residues in the E1α subunit of pyruvate dehydrogenase (PDH), thereby activating PDH and resulting in increased ATP production. Although a phosphorylation–dephosphorylation cycle for the E1α subunit of PDH from non-vertebrate organisms has been described, the Ca2+-mediated PDP activation has not been studied. In this work we investigated the Ca2+ sensitivity of two recombinant PDPs from the protozoan human parasites Trypanosoma cruzi (TcPDP) and Trypanosoma brucei (TbPDP) and generated a TcPDP-KO cell line to establish TcPDP’s role in cell bioenergetics and survival. Moreover, the mitochondrial localization of the TcPDP was studied by CRISPR/Cas9-mediated endogenous tagging. Our results indicate that TcPDP and TbPDP both are Ca2+-sensitive phosphatases. Of note, TcPDP-KO epimastigotes exhibited increased levels of phosphorylated TcPDH, slower growth and lower oxygen consumption rates than control cells, an increased AMP:ATP ratio and autophagy under starvation conditions, and reduced differentiation into infective metacyclic forms. Furthermore, TcPDP-KO trypomastigotes were impaired in infecting culture host cells. We conclude that TcPDP is a Ca2+-stimulated mitochondrial phosphatase that dephosphorylates TcPDH and is required for normal growth, differentiation, infectivity and energy metabolism in T. cruzi.  Our results support the view that one of the main roles of the MCU is linked to the regulation of intramitochondrial dehydrogenases.

Noelia Lander, Miguel A. Chiurillo, Mayara S. Bertolini, Melissa Storey, Anibal E. Vercesi and Roberto Docampo. 2018. Journal of Biological Chemistry; 293(45):17402-17417. doi: 10.1074/jbc.RA118.004498