title: Presence of a thapsigargin-sensitive calcium pump in Trypanosoma evansi: Immunological, physiological, molecular and structural evidences.
abstract: 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.
authors: Instituto de Biología Experimental (IBE), Universidad Central de Venezuela (UCV), Caracas, Venezuela.
authors: M C
authors: MC
authors: Pérez-Gordones
authors: Unidad de Química Medicinal, Facultad de Farmacia, Universidad Central de Venezuela (UCV), Caracas, 1041A, Venezuela.
authors: M L
authors: ML
authors: Serrano
authors: Centro de Biofísica y Bioquímica (CBB), Instituto Venezolano de Investigaciones Científicas (IVIC), Caracas, Venezuela.
authors: H
authors: H
authors: Rojas
authors: Centro de Biociencias y Medicina Molecular, Instituto de Estudios Avanzados (IDEA), Caracas, Venezuela.
authors: J C
authors: JC
authors: Martínez
authors: Centro de Biociencias y Medicina Molecular, Instituto de Estudios Avanzados (IDEA), Caracas, Venezuela; Centro Internacional de Zoonosis (CIZ), Universidad Central del Ecuador, Ecuador.
authors: G
authors: G
authors: Uzcanga
authors: Centro de Estudios Biomédicos y Veterinarios, Instituto de Estudios Científicos y Tecnológicos (IDECYT), Universidad Nacional Experimental Simon Bolivar, Venezuela. Electronic address: mendozamarta17@gmail.com.
authors: M
authors: M
authors: Mendoza
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000187:drug effects* / Q000235:genetics / Q000276:immunology; D004721:Endoplasmic Reticulum / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006734:Horse Diseases / Q000469:parasitology; D006736:Horses; D016623:Ion Pumps / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D018613:Microscopy, Confocal; D008958:Models, Molecular; D008969:Molecular Sequence Data; D051381:Rats; D017207:Rats, Sprague-Dawley; D016415:Sequence Alignment; D019284:Thapsigargin / Q000494:pharmacology*; D014345:Trypanosoma / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D014352:Trypanosomiasis / Q000469:parasitology / Q000662:veterinary
chemical_list: D004791:Enzyme Inhibitors; D016623:Ion Pumps; D019284:Thapsigargin; D000252:Calcium-Transporting ATPases
keywords: SERCA; Thapsigargin; Trypanosoma evansi