Expression and functional characterization of a C-7 cholesterol desaturase from Tetrahymena thermophila in an insect cell line

Autores
Poklépovich Caride, Tomás Javier; Urtasun, Nicolás; Miranda, Maria Victoria; Nusblat, Alejandro David; Nudel, Berta Clara
Año de publicación
2015
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Tetrahymena thermophila transforms exogenous cholesterol into pro-vitamin D3 (7-dehydrocholesterol) with remarkable efficiency in a one-step reaction carried out by a C-7 cholesterol desaturase. The enzyme DES7 is encoded by the gene TTHERM-00310640, identified with RNAi and gene knock-out experiments, but has not yet been heterologously expressed actively in any organism. A model derived from its amino acid sequence classified DES7p as a Rieske-type oxygenase with transmembrane localization. The protein has catalytic activity, sequence and topological similarity to DAF-36/Neverland proteins involved in the synthesis of steroid hormones in insects and nematodes. Due to their structural and functional similarity, we analyzed the expression of a codon optimized DES7 gene from Tetrahymena in the insect Sf9 cell line, identified and measured the steroid metabolites formed, and extended the actual knowledge on its localization. We found that the accumulation of 7-dehydrocholesterol could be increased 16-40-fold in Spodoptera frugiperda, depending on physiological conditions, by overexpression of T. thermophila DES7. The protein was detected in the microsomal fraction, in accordance with previous reports. Although the electron transfer chain for Des7p/DAF-36/Neverland Rieske-type oxygenases is presently unknown, we identified possible donors in the ciliate and insect genomes by bioinformatic analysis. In spite of the large evolutionary distance between S. frugiperda and T. thermophila, the results indicate that there is significant functional conservation of the electron donors, since the ciliate's sterol desaturase can function in the context of the insect electron transport system. The results achieved demonstrate that DES7 is the first gene from a ciliate, coding for a microsomal enzyme, expressed in active form in an insect cell line.
Fil: Poklépovich Caride, Tomás Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; Argentina
Fil: Urtasun, Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; Argentina
Fil: Miranda, Maria Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; Argentina
Fil: Nusblat, Alejandro David. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; Argentina
Fil: Nudel, Berta Clara. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; Argentina
Materia
7- Colesterol Desaturasa
Tetrahymena
Celulas de Insecto
Expresion Genetica
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/38089
Acceder
id CONICETDig_60bc5c784bee819647ed08db37b52bbe
oai_identifier_str oai:ri.conicet.gov.ar:11336/38089
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Expression and functional characterization of a C-7 cholesterol desaturase from Tetrahymena thermophila in an insect cell linePoklépovich Caride, Tomás JavierUrtasun, NicolásMiranda, Maria VictoriaNusblat, Alejandro DavidNudel, Berta Clara7- Colesterol DesaturasaTetrahymenaCelulas de InsectoExpresion Geneticahttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Tetrahymena thermophila transforms exogenous cholesterol into pro-vitamin D3 (7-dehydrocholesterol) with remarkable efficiency in a one-step reaction carried out by a C-7 cholesterol desaturase. The enzyme DES7 is encoded by the gene TTHERM-00310640, identified with RNAi and gene knock-out experiments, but has not yet been heterologously expressed actively in any organism. A model derived from its amino acid sequence classified DES7p as a Rieske-type oxygenase with transmembrane localization. The protein has catalytic activity, sequence and topological similarity to DAF-36/Neverland proteins involved in the synthesis of steroid hormones in insects and nematodes. Due to their structural and functional similarity, we analyzed the expression of a codon optimized DES7 gene from Tetrahymena in the insect Sf9 cell line, identified and measured the steroid metabolites formed, and extended the actual knowledge on its localization. We found that the accumulation of 7-dehydrocholesterol could be increased 16-40-fold in Spodoptera frugiperda, depending on physiological conditions, by overexpression of T. thermophila DES7. The protein was detected in the microsomal fraction, in accordance with previous reports. Although the electron transfer chain for Des7p/DAF-36/Neverland Rieske-type oxygenases is presently unknown, we identified possible donors in the ciliate and insect genomes by bioinformatic analysis. In spite of the large evolutionary distance between S. frugiperda and T. thermophila, the results indicate that there is significant functional conservation of the electron donors, since the ciliate's sterol desaturase can function in the context of the insect electron transport system. The results achieved demonstrate that DES7 is the first gene from a ciliate, coding for a microsomal enzyme, expressed in active form in an insect cell line.Fil: Poklépovich Caride, Tomás Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Urtasun, Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Miranda, Maria Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Nusblat, Alejandro David. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Nudel, Berta Clara. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaElsevier Science Inc2015-02info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/38089Poklépovich Caride, Tomás Javier; Urtasun, Nicolás; Miranda, Maria Victoria; Nusblat, Alejandro David; Nudel, Berta Clara; Expression and functional characterization of a C-7 cholesterol desaturase from Tetrahymena thermophila in an insect cell line; Elsevier Science Inc; Steroids; 96; 2-2015; 132-1390039-128XCONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://www.sciencedirect.com/science/article/pii/S0039128X15000574info:eu-repo/semantics/altIdentifier/doi/10.1016/j.steroids.2015.02.001info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T10:35:27Zoai:ri.conicet.gov.ar:11336/38089instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-09-29 10:35:27.298CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Expression and functional characterization of a C-7 cholesterol desaturase from Tetrahymena thermophila in an insect cell line
title Expression and functional characterization of a C-7 cholesterol desaturase from Tetrahymena thermophila in an insect cell line
spellingShingle Expression and functional characterization of a C-7 cholesterol desaturase from Tetrahymena thermophila in an insect cell line
Poklépovich Caride, Tomás Javier
7- Colesterol Desaturasa
Tetrahymena
Celulas de Insecto
Expresion Genetica
title_short Expression and functional characterization of a C-7 cholesterol desaturase from Tetrahymena thermophila in an insect cell line
title_full Expression and functional characterization of a C-7 cholesterol desaturase from Tetrahymena thermophila in an insect cell line
title_fullStr Expression and functional characterization of a C-7 cholesterol desaturase from Tetrahymena thermophila in an insect cell line
title_full_unstemmed Expression and functional characterization of a C-7 cholesterol desaturase from Tetrahymena thermophila in an insect cell line
title_sort Expression and functional characterization of a C-7 cholesterol desaturase from Tetrahymena thermophila in an insect cell line
dc.creator.none.fl_str_mv Poklépovich Caride, Tomás Javier
Urtasun, Nicolás
Miranda, Maria Victoria
Nusblat, Alejandro David
Nudel, Berta Clara
author Poklépovich Caride, Tomás Javier
author_facet Poklépovich Caride, Tomás Javier
Urtasun, Nicolás
Miranda, Maria Victoria
Nusblat, Alejandro David
Nudel, Berta Clara
author_role author
author2 Urtasun, Nicolás
Miranda, Maria Victoria
Nusblat, Alejandro David
Nudel, Berta Clara
author2_role author
author
author
author
dc.subject.none.fl_str_mv 7- Colesterol Desaturasa
Tetrahymena
Celulas de Insecto
Expresion Genetica
topic 7- Colesterol Desaturasa
Tetrahymena
Celulas de Insecto
Expresion Genetica
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv Tetrahymena thermophila transforms exogenous cholesterol into pro-vitamin D3 (7-dehydrocholesterol) with remarkable efficiency in a one-step reaction carried out by a C-7 cholesterol desaturase. The enzyme DES7 is encoded by the gene TTHERM-00310640, identified with RNAi and gene knock-out experiments, but has not yet been heterologously expressed actively in any organism. A model derived from its amino acid sequence classified DES7p as a Rieske-type oxygenase with transmembrane localization. The protein has catalytic activity, sequence and topological similarity to DAF-36/Neverland proteins involved in the synthesis of steroid hormones in insects and nematodes. Due to their structural and functional similarity, we analyzed the expression of a codon optimized DES7 gene from Tetrahymena in the insect Sf9 cell line, identified and measured the steroid metabolites formed, and extended the actual knowledge on its localization. We found that the accumulation of 7-dehydrocholesterol could be increased 16-40-fold in Spodoptera frugiperda, depending on physiological conditions, by overexpression of T. thermophila DES7. The protein was detected in the microsomal fraction, in accordance with previous reports. Although the electron transfer chain for Des7p/DAF-36/Neverland Rieske-type oxygenases is presently unknown, we identified possible donors in the ciliate and insect genomes by bioinformatic analysis. In spite of the large evolutionary distance between S. frugiperda and T. thermophila, the results indicate that there is significant functional conservation of the electron donors, since the ciliate's sterol desaturase can function in the context of the insect electron transport system. The results achieved demonstrate that DES7 is the first gene from a ciliate, coding for a microsomal enzyme, expressed in active form in an insect cell line.
Fil: Poklépovich Caride, Tomás Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; Argentina
Fil: Urtasun, Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; Argentina
Fil: Miranda, Maria Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; Argentina
Fil: Nusblat, Alejandro David. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; Argentina
Fil: Nudel, Berta Clara. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; Argentina
description Tetrahymena thermophila transforms exogenous cholesterol into pro-vitamin D3 (7-dehydrocholesterol) with remarkable efficiency in a one-step reaction carried out by a C-7 cholesterol desaturase. The enzyme DES7 is encoded by the gene TTHERM-00310640, identified with RNAi and gene knock-out experiments, but has not yet been heterologously expressed actively in any organism. A model derived from its amino acid sequence classified DES7p as a Rieske-type oxygenase with transmembrane localization. The protein has catalytic activity, sequence and topological similarity to DAF-36/Neverland proteins involved in the synthesis of steroid hormones in insects and nematodes. Due to their structural and functional similarity, we analyzed the expression of a codon optimized DES7 gene from Tetrahymena in the insect Sf9 cell line, identified and measured the steroid metabolites formed, and extended the actual knowledge on its localization. We found that the accumulation of 7-dehydrocholesterol could be increased 16-40-fold in Spodoptera frugiperda, depending on physiological conditions, by overexpression of T. thermophila DES7. The protein was detected in the microsomal fraction, in accordance with previous reports. Although the electron transfer chain for Des7p/DAF-36/Neverland Rieske-type oxygenases is presently unknown, we identified possible donors in the ciliate and insect genomes by bioinformatic analysis. In spite of the large evolutionary distance between S. frugiperda and T. thermophila, the results indicate that there is significant functional conservation of the electron donors, since the ciliate's sterol desaturase can function in the context of the insect electron transport system. The results achieved demonstrate that DES7 is the first gene from a ciliate, coding for a microsomal enzyme, expressed in active form in an insect cell line.
publishDate 2015
dc.date.none.fl_str_mv 2015-02
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/38089
Poklépovich Caride, Tomás Javier; Urtasun, Nicolás; Miranda, Maria Victoria; Nusblat, Alejandro David; Nudel, Berta Clara; Expression and functional characterization of a C-7 cholesterol desaturase from Tetrahymena thermophila in an insect cell line; Elsevier Science Inc; Steroids; 96; 2-2015; 132-139
0039-128X
CONICET Digital
CONICET
url http://hdl.handle.net/11336/38089
identifier_str_mv Poklépovich Caride, Tomás Javier; Urtasun, Nicolás; Miranda, Maria Victoria; Nusblat, Alejandro David; Nudel, Berta Clara; Expression and functional characterization of a C-7 cholesterol desaturase from Tetrahymena thermophila in an insect cell line; Elsevier Science Inc; Steroids; 96; 2-2015; 132-139
0039-128X
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/https://www.sciencedirect.com/science/article/pii/S0039128X15000574
info:eu-repo/semantics/altIdentifier/doi/10.1016/j.steroids.2015.02.001
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
application/pdf
application/pdf
application/pdf
application/pdf
application/pdf
dc.publisher.none.fl_str_mv Elsevier Science Inc
publisher.none.fl_str_mv Elsevier Science Inc
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
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