Sobre-expresión de ciclofilinas en el parásito unicelular Trypanosoma cruzi: Desarrollo de herramientas para el análisis de la función de proteínas.

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Browsing Sobre-expresión de ciclofilinas en el parásito unicelular Trypanosoma cruzi: Desarrollo de herramientas para el análisis de la función de proteínas. by Author "Bustos, Patricia L."
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    A homolog of cyclophilin D is expressed in Trypanosoma cruzi and is involved in the oxidative stress–damage response
    (Cell Death Differentiation Association (ADMC), 2017-2-6) Bustos, Patricia L. ; Volta, Viviana J. ; Perrone, Alina E ; Milduberger, Natalia A. ; Bua, Jaqueline
    Mitochondria have an important role in energy production, homeostasis and cell death. The opening of the mitochondrial permeability transition pore (mPTP) is considered one of the key events in apoptosis and necrosis, modulated by cyclophilin D (CyPD), a crucial component of this protein complex. In Trypanosoma cruzi, the protozoan parasite that causes Chagas disease, we have previously described that mitochondrial permeability transition occurs after oxidative stress induction in a cyclosporin A-dependent manner, a well-known cyclophilin inhibitor. In the present work, a mitochondrial parasite cyclophilin, named TcCyP22, which is homolog to the mammalian CyPD was identified. TcCyP22-overexpressing parasites showed an enhanced loss of mitochondrial membrane potential and loss of cell viability when exposed to a hydrogen peroxide stimulus compared with control parasites. Our results describe for the first time in a protozoan parasite that a mitochondrial cyclophilin is a component of the permeability transition pore and is involved in regulated cell death induced by oxidative stress
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    Improved immuno-detection of a low-abundance cyclophilin allows the confirmation of its expression in a protozoan parasite
    (Hilaris, 2015-10-6) Bustos, Patricia L. ; Perrone, Alina E ; Milduberger, Natalia A. ; Bua, Jaqueline
    Protein samples can be challenging to analyze due to the presence of high-abundance proteins masking low abundance proteins of interest, such as biomarkers and novel physiological mediators. Cyclophilins are chaperones involved in the cis/trans isomerization of peptidyl-prolyl bonds in peptides or proteins and have been found in every organism sequenced to date. Although considerable progress has been made in the characterization of some cyclophilins expressed in diverse parasites invading humans, the main aspects of low-abundance members of this family remain unknown. In the present work, we present that the combined strategy of using more specific antibodies and increasing the presence of subcellular proteins in the sample, allowed us to confirm the expression of a 21.1 kDa cyclophilin for the first time in Trypanosoma cruzi.
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    Oxidative stress damage in the protozoan parasite Trypanosoma cruzi is inhibited by cyclosporin A
    (Parasitology, Cambridge University Press, 2015-2-6) Bustos, Patricia L. ; Perrone, Alina E ; Milduberger, Natalia A. ; Postan, Miriam ; Bua, Jaqueline
    Cyclosporin A (CsA) specifically inhibits the mitochondrial permeability transition pore (mPTP). Opening of the mPTP, which is triggered by high levels of matrix [Ca2+] and/or oxidative stress, leads to mitochondrial dysfunction and thus to cell death by either apoptosis or necrosis. In the present study, we analysed the response of Trypanosoma cruzi epimastigote parasites to oxidative stress with 5 mM H2O2, by studying several features related to programmed cell death and the effects of pre-incubation with 1 µM of CsA. We evaluated TcPARP cleavage, DNA integrity, cytochrome c translocation, Annexin V/propidium iodide staining, reactive oxygen species production. CsA prevented parasite oxidative stress damage as it significantly inhibited DNA degradation, cytochrome c translocation to cytosol and TcPARP cleavage. The calcein-AM/CoCl2 assay, used as a selective indicator of mPTP opening in mammals, was also performed in T. cruzi parasites. H2O2 treatment decreased calcein fluorescence, but this decline was partially inhibited by pre-incubation with CsA. Our results encourage further studies to investigate if there is a mPTP-like pore and a mitochondrial cyclophilin involved in this protozoan parasite.