Calpain-like: A Ca(2+) dependent cystein protease in Entamoeba histolytica cell death.
Entamoeba histolytica programmed cell death (PCD) induced by G418 is characterized by the release of important amounts of intracellular calcium from reservoirs. Nevertheless, no typical caspases have been detected in the parasite, the PCD phenotype is inhibited by the cysteine protease inhibitor E-64. These results strongly suggest that Ca(2+)-dependent proteases could be involved in PCD. In this study, we evaluate the expression and activity of a specific dependent Ca(2+) protease, the calpain-like protease, by real-time quantitative PCR (RTq-PCR), Western blot assays and a enzymatic method during the induction of PCD by G418. Alternatively, using cell viability and TUNEL assays, we also demonstrated that the Z-Leu-Leu-Leu-al calpain inhibitor reduced the rate of cell death. The results demonstrated 4.9-fold overexpression of calpain-like gene 1.5 h after G418 PCD induction, while calpain-like protein increased almost two-fold with respect to basal calpain-like expression after 3 h of induction, and calpain activity was found to be approximately three-fold higher 6 h after treatment compared with untreated trophozoites. Taken together, these results suggest that this Ca(2+)-dependent protease could be involved in the executory phase of PCD.
Laboratorio de Biomedicina Molecular I, Programa Institucional de Biomedicina Molecular, Escuela Nacional de Medicina y Homeopatía, IPN, Guillermo Massieu Helguera No.239, Fracc. La Escalera, Col. Ticomán, D.F, C.P.07320, Mexico; Laboratorio Multidisciplinario de Investigación, Escuela Militar de Graduados de Sanidad, UDEFA, Lomas de San Isidro, DF, CP 11620, Mexico.
Virginia Sánchez
VS
Monroy
Laboratorio de Biomedicina Molecular I, Programa Institucional de Biomedicina Molecular, Escuela Nacional de Medicina y Homeopatía, IPN, Guillermo Massieu Helguera No.239, Fracc. La Escalera, Col. Ticomán, D.F, C.P.07320, Mexico.
Olivia Medel
OM
Flores
Laboratorio de Biomedicina Molecular I, Programa Institucional de Biomedicina Molecular, Escuela Nacional de Medicina y Homeopatía, IPN, Guillermo Massieu Helguera No.239, Fracc. La Escalera, Col. Ticomán, D.F, C.P.07320, Mexico.
Consuelo Gómez
CG
García
Facultad de Estudios Superiores Cuautitlán Izcalli, UNAM, Cuautitlán Izcalli, Estado de México CP.54740, Mexico.
Yesenia Chávez
YC
Maya
Laboratorio de Biomedicina Molecular I, Programa Institucional de Biomedicina Molecular, Escuela Nacional de Medicina y Homeopatía, IPN, Guillermo Massieu Helguera No.239, Fracc. La Escalera, Col. Ticomán, D.F, C.P.07320, Mexico.
Tania Domínguez
TD
Fernández
Laboratorio de Biomedicina Molecular I, Programa Institucional de Biomedicina Molecular, Escuela Nacional de Medicina y Homeopatía, IPN, Guillermo Massieu Helguera No.239, Fracc. La Escalera, Col. Ticomán, D.F, C.P.07320, Mexico; Centro de Investigación en Ciencia Aplicada. Instituto Politécnico Nacional, Tepetitla de Lardizabal, Tlaxcala, Doctorado en Biotecnología, Red de Investigación en Biotecnología IPN, Mexico. Electronic address: ishiwaramx@yahoo.com.mx.
D Guillermo
DG
Pérez Ishiwara
D000563:Amebicides / Q000494:pharmacology; D000595:Amino Acid Sequence; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D053938:DNA Fragmentation; D003720:Densitometry; D004748:Entamoeba histolytica / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D005839:Gentamicins / Q000494:pharmacology; D020287:In Situ Nick-End Labeling; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D007976:Leupeptins / Q000494:pharmacology; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D060888:Real-Time Polymerase Chain Reaction
D000563:Amebicides; D015853:Cysteine Proteinase Inhibitors; D005839:Gentamicins; D007976:Leupeptins; C010680:antibiotic G 418; D002154:Calpain; D003546:Cysteine Endopeptidases; D007930:Leucine; C024974:E 64; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; D002118:Calcium
Calpain-like; Entamoeba histolytica; G418; PCD