A novel protein phosphatase 2A (PP2A) is involved in the transformation of human protozoan parasite Trypanosoma cruzi

Jorge González, Alberto Cornejo, Marcia R M Santos, Esteban M. Cordero, Bessy Gutiérrez, Patricio Porcile, Renato A. Mortara, Hernán Sagua, José Franco Da Silveira, Jorge E. Araya

Resultado de la investigación: Article

31 Citas (Scopus)

Resumen

Here we provide evidence for a critical role of PP2As (protein phosphatase 2As) in the transformation of Trypanosoma cruzi. In axenic medium at pH 5.0, trypomastigotes rapidly transform into amastigotes, a process blocked by okadaic acid, a potent PP2A inhibitor, at concentrations as low as 0.1 μM. 1-Norokadaone, an inactive okadaic acid analogue, did not affect the transformation. Electron microscopy studies indicated that okadaic acid-treated trypomastigotes had not undergone ultrastructural modifications, reinforcing the idea that PP2A inhibits transformation. Using a microcystin-Sepharose affinity column we purified the native T. cruzi PP2A. The enzyme displayed activity against 32P-labelled phosphorylase a that was inhibited in a dose-dependent manner by okadaic acid. The protein was also submitted to MS and, from the peptides obtained, degenerate primers were used to clone a novel T. crud PP2A enzyme by PCR. The isolated gene encodes a protein of 303 amino acids, termed TcPP2A, which displayed a high degree of homology (86%) with the catalytic subunit of Trypanosoma brucei PP2A. Northern-blot analysis revealed the presence of a major 2.1-kb mRNA hybridizing in all T. cruzi developmental stages. Southern-blot analysis suggested that the TcPP2A gene is present in low copy number in the T. cruzi genome. These results are consistent with the mapping of PP2A genes in two chromosomal bands by pulsed-field gel electrophoresis and chromoblot hybridization. Our studies suggest that in T. cruzi PP2A is important for the complete transformation of trypomastigotes into amastigotes during the life cycle of this protozoan parasite.

Idioma originalEnglish
Páginas (desde-hasta)647-656
Número de páginas10
PublicaciónBiochemical Journal
Volumen374
N.º3
DOI
EstadoPublished - 15 sep 2003

Huella dactilar

Protein Phosphatase 2
Trypanosoma cruzi
Parasites
Okadaic Acid
Genes
Phosphorylase a
Trypanosoma brucei brucei
Pulsed Field Gel Electrophoresis
Phosphoprotein Phosphatases
Enzyme activity
Enzymes
Southern Blotting
Life Cycle Stages
Electrophoresis
Northern Blotting
Sepharose
Electron microscopy
Life cycle
Catalytic Domain
Electron Microscopy

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Citar esto

González, J., Cornejo, A., Santos, M. R. M., Cordero, E. M., Gutiérrez, B., Porcile, P., ... Araya, J. E. (2003). A novel protein phosphatase 2A (PP2A) is involved in the transformation of human protozoan parasite Trypanosoma cruzi. Biochemical Journal, 374(3), 647-656. https://doi.org/10.1042/BJ20030215
González, Jorge ; Cornejo, Alberto ; Santos, Marcia R M ; Cordero, Esteban M. ; Gutiérrez, Bessy ; Porcile, Patricio ; Mortara, Renato A. ; Sagua, Hernán ; Da Silveira, José Franco ; Araya, Jorge E. / A novel protein phosphatase 2A (PP2A) is involved in the transformation of human protozoan parasite Trypanosoma cruzi. En: Biochemical Journal. 2003 ; Vol. 374, N.º 3. pp. 647-656.
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abstract = "Here we provide evidence for a critical role of PP2As (protein phosphatase 2As) in the transformation of Trypanosoma cruzi. In axenic medium at pH 5.0, trypomastigotes rapidly transform into amastigotes, a process blocked by okadaic acid, a potent PP2A inhibitor, at concentrations as low as 0.1 μM. 1-Norokadaone, an inactive okadaic acid analogue, did not affect the transformation. Electron microscopy studies indicated that okadaic acid-treated trypomastigotes had not undergone ultrastructural modifications, reinforcing the idea that PP2A inhibits transformation. Using a microcystin-Sepharose affinity column we purified the native T. cruzi PP2A. The enzyme displayed activity against 32P-labelled phosphorylase a that was inhibited in a dose-dependent manner by okadaic acid. The protein was also submitted to MS and, from the peptides obtained, degenerate primers were used to clone a novel T. crud PP2A enzyme by PCR. The isolated gene encodes a protein of 303 amino acids, termed TcPP2A, which displayed a high degree of homology (86{\%}) with the catalytic subunit of Trypanosoma brucei PP2A. Northern-blot analysis revealed the presence of a major 2.1-kb mRNA hybridizing in all T. cruzi developmental stages. Southern-blot analysis suggested that the TcPP2A gene is present in low copy number in the T. cruzi genome. These results are consistent with the mapping of PP2A genes in two chromosomal bands by pulsed-field gel electrophoresis and chromoblot hybridization. Our studies suggest that in T. cruzi PP2A is important for the complete transformation of trypomastigotes into amastigotes during the life cycle of this protozoan parasite.",
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author = "Jorge Gonz{\'a}lez and Alberto Cornejo and Santos, {Marcia R M} and Cordero, {Esteban M.} and Bessy Guti{\'e}rrez and Patricio Porcile and Mortara, {Renato A.} and Hern{\'a}n Sagua and {Da Silveira}, {Jos{\'e} Franco} and Araya, {Jorge E.}",
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González, J, Cornejo, A, Santos, MRM, Cordero, EM, Gutiérrez, B, Porcile, P, Mortara, RA, Sagua, H, Da Silveira, JF & Araya, JE 2003, 'A novel protein phosphatase 2A (PP2A) is involved in the transformation of human protozoan parasite Trypanosoma cruzi', Biochemical Journal, vol. 374, n.º 3, pp. 647-656. https://doi.org/10.1042/BJ20030215

A novel protein phosphatase 2A (PP2A) is involved in the transformation of human protozoan parasite Trypanosoma cruzi. / González, Jorge; Cornejo, Alberto; Santos, Marcia R M; Cordero, Esteban M.; Gutiérrez, Bessy; Porcile, Patricio; Mortara, Renato A.; Sagua, Hernán; Da Silveira, José Franco; Araya, Jorge E.

En: Biochemical Journal, Vol. 374, N.º 3, 15.09.2003, p. 647-656.

Resultado de la investigación: Article

TY - JOUR

T1 - A novel protein phosphatase 2A (PP2A) is involved in the transformation of human protozoan parasite Trypanosoma cruzi

AU - González, Jorge

AU - Cornejo, Alberto

AU - Santos, Marcia R M

AU - Cordero, Esteban M.

AU - Gutiérrez, Bessy

AU - Porcile, Patricio

AU - Mortara, Renato A.

AU - Sagua, Hernán

AU - Da Silveira, José Franco

AU - Araya, Jorge E.

PY - 2003/9/15

Y1 - 2003/9/15

N2 - Here we provide evidence for a critical role of PP2As (protein phosphatase 2As) in the transformation of Trypanosoma cruzi. In axenic medium at pH 5.0, trypomastigotes rapidly transform into amastigotes, a process blocked by okadaic acid, a potent PP2A inhibitor, at concentrations as low as 0.1 μM. 1-Norokadaone, an inactive okadaic acid analogue, did not affect the transformation. Electron microscopy studies indicated that okadaic acid-treated trypomastigotes had not undergone ultrastructural modifications, reinforcing the idea that PP2A inhibits transformation. Using a microcystin-Sepharose affinity column we purified the native T. cruzi PP2A. The enzyme displayed activity against 32P-labelled phosphorylase a that was inhibited in a dose-dependent manner by okadaic acid. The protein was also submitted to MS and, from the peptides obtained, degenerate primers were used to clone a novel T. crud PP2A enzyme by PCR. The isolated gene encodes a protein of 303 amino acids, termed TcPP2A, which displayed a high degree of homology (86%) with the catalytic subunit of Trypanosoma brucei PP2A. Northern-blot analysis revealed the presence of a major 2.1-kb mRNA hybridizing in all T. cruzi developmental stages. Southern-blot analysis suggested that the TcPP2A gene is present in low copy number in the T. cruzi genome. These results are consistent with the mapping of PP2A genes in two chromosomal bands by pulsed-field gel electrophoresis and chromoblot hybridization. Our studies suggest that in T. cruzi PP2A is important for the complete transformation of trypomastigotes into amastigotes during the life cycle of this protozoan parasite.

AB - Here we provide evidence for a critical role of PP2As (protein phosphatase 2As) in the transformation of Trypanosoma cruzi. In axenic medium at pH 5.0, trypomastigotes rapidly transform into amastigotes, a process blocked by okadaic acid, a potent PP2A inhibitor, at concentrations as low as 0.1 μM. 1-Norokadaone, an inactive okadaic acid analogue, did not affect the transformation. Electron microscopy studies indicated that okadaic acid-treated trypomastigotes had not undergone ultrastructural modifications, reinforcing the idea that PP2A inhibits transformation. Using a microcystin-Sepharose affinity column we purified the native T. cruzi PP2A. The enzyme displayed activity against 32P-labelled phosphorylase a that was inhibited in a dose-dependent manner by okadaic acid. The protein was also submitted to MS and, from the peptides obtained, degenerate primers were used to clone a novel T. crud PP2A enzyme by PCR. The isolated gene encodes a protein of 303 amino acids, termed TcPP2A, which displayed a high degree of homology (86%) with the catalytic subunit of Trypanosoma brucei PP2A. Northern-blot analysis revealed the presence of a major 2.1-kb mRNA hybridizing in all T. cruzi developmental stages. Southern-blot analysis suggested that the TcPP2A gene is present in low copy number in the T. cruzi genome. These results are consistent with the mapping of PP2A genes in two chromosomal bands by pulsed-field gel electrophoresis and chromoblot hybridization. Our studies suggest that in T. cruzi PP2A is important for the complete transformation of trypomastigotes into amastigotes during the life cycle of this protozoan parasite.

KW - Enzyme purification

KW - Expression

KW - Gene cloning

KW - Genomic organization

KW - Phosphatase-specific inhibitor

KW - Trypanosome transformation

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U2 - 10.1042/BJ20030215

DO - 10.1042/BJ20030215

M3 - Article

VL - 374

SP - 647

EP - 656

JO - Biochemical Journal

JF - Biochemical Journal

SN - 0264-6021

IS - 3

ER -