thapsigargin and Trypanosomiasis

The Trypanosomatidae family encompasses many unicellular organisms responsible of several tropical diseases that affect humans and animals. Livestock tripanosomosis caused by Trypanosoma brucei brucei (T. brucei), Trypanosoma equiperdum (T. equiperdum) and Trypanosoma evansi (T. evansi), have a significant socio-economic impact and limit animal protein productivity throughout the intertropical zones of the world. Similarly, to all organisms, the maintenance of Ca

Topics: Animals; Boron Compounds; Calcium; Calcium Chelating Agents; Computational Biology; Enzyme Inhibitors; Fluorescent Dyes; Fura-2; Gene Expression; Homeostasis; Hydroquinones; Intracellular Calcium-Sensing Proteins; Manganese; Protozoan Proteins; Thapsigargin; Transient Receptor Potential Channels; Trypanosoma; Trypanosomiasis

In higher eukaryotes, the sarco-endoplasmic reticulum (ER) Ca(2+)-ATPase (SERCA) is characterized for its high sensitivity to low concentrations of thapsigargin (TG), a very specific inhibitor. In contrast, SERCA-like enzymes with different sensitivities to TG have been reported in trypanosomatids. Here, we characterized a SERCA-like enzyme from Trypanosoma evansi and evaluated its interaction with TG. Confocal fluorescence microscopy using BODIPY FL TG and specific anti-SERCA antibodies localized the T. evansi SERCA-like enzyme in the ER and confirmed its direct interaction with TG. Moreover, the use of either 1 μM TG or 25 μM 2',5'-di (tert-butyl)-1,4-benzohydroquinone prevented the reuptake of Ca(2+) and consequently produced a small increase in the parasite cytosolic calcium concentration in a calcium-free medium, which was released from the ER pool. A 3035 bp-sequence coding for a protein with an estimated molecular mass of 110.2 kDa was cloned from T. evansi. The corresponding gene product contained all the invariant residues and conserved motifs found in other P-type ATPases but lacked the calmodulin binding site. Modeling of the three-dimensional structure of the parasite enzyme revealed that the amino acid changes found in the TG-SERCA binding pocket do not compromise the interaction between the enzyme and the inhibitor. Therefore, we concluded that T. evansi possesses a SERCA-like protein that is inhibited by TG.

Topics: Amino Acid Sequence; Animals; Blotting, Western; Calcium-Transporting ATPases; Endoplasmic Reticulum; Enzyme Inhibitors; Horse Diseases; Horses; Ion Pumps; Male; Microscopy, Confocal; Models, Molecular; Molecular Sequence Data; Rats; Rats, Sprague-Dawley; Sequence Alignment; Thapsigargin; Trypanosoma; Trypanosomiasis