Different proteomic strategies to identify genuine Small Ubiquitin-like MOdifier targets and their modification sites in Trypanosoma brucei procyclic forms

Autores
Iribarren, Paula Ana; Berazategui, Maria Agustina; Bayona, Julio César; Almeida, I. C.; Cazzulo, Juan Jose; Alvarez, Vanina Eder
Año de publicación
2015
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
SUMOylation is an important post-translational modification conserved in eukaryotic organisms. In Trypanosoma brucei, SUMO (Small Ubiquitin-like MOdifier) is essential in procyclic and bloodstream forms. Furthermore, SUMO has been linked to the antigenic variation process, as a highly SUMOylated focus was recently identified within chromatin-associated proteins of the active variant surface glycoprotein expression site. We aimed to establish a reliable strategy to identify SUMO conjugates in T.brucei. We expressed various tagged variants of SUMO from the endogenous locus. His-HA-TbSUMO was useful to validate the tag functionality but SUMO conjugates were not enriched enough over contaminants after affinity purification. A Lys-deficient SUMO version, created to reduce contaminants by Lys-C digestion, was able to overcome this issue but did not allow mapping many SUMOylation sites. This cell line was in turn useful to demonstrate that polySUMO chains are not essential for parasite viability. Finally, a His-HA-TbSUMOT106K version allowed the purification of SUMO conjugates and, after digestion with Lys-C, the enrichment for diGly-Lys peptides using specific antibodies. This site-specific proteomic strategy led us to identify 45 SUMOylated proteins and 53 acceptor sites unambiguously. SUMOylated proteins belong mainly to nuclear processes, such as DNA replication and repair, transcription, rRNA biogenesis and chromatin remodelling, among others.
Fil: Iribarren, Paula Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina
Fil: Berazategui, Maria Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina
Fil: Bayona, Julio César. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina
Fil: Almeida, I. C.. University of Texas at El Paso; Estados Unidos
Fil: Cazzulo, Juan Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina
Fil: Alvarez, Vanina Eder. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina
Materia
TRYPANOSOMA BRUCEI
SUMO
PROTEOMIC
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:11336/181239
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/181239
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Different proteomic strategies to identify genuine Small Ubiquitin-like MOdifier targets and their modification sites in Trypanosoma brucei procyclic formsIribarren, Paula AnaBerazategui, Maria AgustinaBayona, Julio CésarAlmeida, I. C.Cazzulo, Juan JoseAlvarez, Vanina EderTRYPANOSOMA BRUCEISUMOPROTEOMIChttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1SUMOylation is an important post-translational modification conserved in eukaryotic organisms. In Trypanosoma brucei, SUMO (Small Ubiquitin-like MOdifier) is essential in procyclic and bloodstream forms. Furthermore, SUMO has been linked to the antigenic variation process, as a highly SUMOylated focus was recently identified within chromatin-associated proteins of the active variant surface glycoprotein expression site. We aimed to establish a reliable strategy to identify SUMO conjugates in T.brucei. We expressed various tagged variants of SUMO from the endogenous locus. His-HA-TbSUMO was useful to validate the tag functionality but SUMO conjugates were not enriched enough over contaminants after affinity purification. A Lys-deficient SUMO version, created to reduce contaminants by Lys-C digestion, was able to overcome this issue but did not allow mapping many SUMOylation sites. This cell line was in turn useful to demonstrate that polySUMO chains are not essential for parasite viability. Finally, a His-HA-TbSUMOT106K version allowed the purification of SUMO conjugates and, after digestion with Lys-C, the enrichment for diGly-Lys peptides using specific antibodies. This site-specific proteomic strategy led us to identify 45 SUMOylated proteins and 53 acceptor sites unambiguously. SUMOylated proteins belong mainly to nuclear processes, such as DNA replication and repair, transcription, rRNA biogenesis and chromatin remodelling, among others.Fil: Iribarren, Paula Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Berazategui, Maria Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Bayona, Julio César. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Almeida, I. C.. University of Texas at El Paso; Estados UnidosFil: Cazzulo, Juan Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Alvarez, Vanina Eder. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaWiley Blackwell Publishing, Inc2015-06info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/181239Iribarren, Paula Ana; Berazategui, Maria Agustina; Bayona, Julio César; Almeida, I. C.; Cazzulo, Juan Jose; et al.; Different proteomic strategies to identify genuine Small Ubiquitin-like MOdifier targets and their modification sites in Trypanosoma brucei procyclic forms; Wiley Blackwell Publishing, Inc; Cellular Microbiology (print); 17; 10; 6-2015; 1413-14221462-5814CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/doi/10.1111/cmi.12467info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-10-29T11:43:10Zoai:ri.conicet.gov.ar:11336/181239instacron: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:34982025-10-29 11:43:10.388CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Different proteomic strategies to identify genuine Small Ubiquitin-like MOdifier targets and their modification sites in Trypanosoma brucei procyclic forms
title Different proteomic strategies to identify genuine Small Ubiquitin-like MOdifier targets and their modification sites in Trypanosoma brucei procyclic forms
spellingShingle Different proteomic strategies to identify genuine Small Ubiquitin-like MOdifier targets and their modification sites in Trypanosoma brucei procyclic forms
Iribarren, Paula Ana
TRYPANOSOMA BRUCEI
SUMO
PROTEOMIC
title_short Different proteomic strategies to identify genuine Small Ubiquitin-like MOdifier targets and their modification sites in Trypanosoma brucei procyclic forms
title_full Different proteomic strategies to identify genuine Small Ubiquitin-like MOdifier targets and their modification sites in Trypanosoma brucei procyclic forms
title_fullStr Different proteomic strategies to identify genuine Small Ubiquitin-like MOdifier targets and their modification sites in Trypanosoma brucei procyclic forms
title_full_unstemmed Different proteomic strategies to identify genuine Small Ubiquitin-like MOdifier targets and their modification sites in Trypanosoma brucei procyclic forms
title_sort Different proteomic strategies to identify genuine Small Ubiquitin-like MOdifier targets and their modification sites in Trypanosoma brucei procyclic forms
dc.creator.none.fl_str_mv Iribarren, Paula Ana
Berazategui, Maria Agustina
Bayona, Julio César
Almeida, I. C.
Cazzulo, Juan Jose
Alvarez, Vanina Eder
author Iribarren, Paula Ana
author_facet Iribarren, Paula Ana
Berazategui, Maria Agustina
Bayona, Julio César
Almeida, I. C.
Cazzulo, Juan Jose
Alvarez, Vanina Eder
author_role author
author2 Berazategui, Maria Agustina
Bayona, Julio César
Almeida, I. C.
Cazzulo, Juan Jose
Alvarez, Vanina Eder
author2_role author
author
author
author
author
dc.subject.none.fl_str_mv TRYPANOSOMA BRUCEI
SUMO
PROTEOMIC
topic TRYPANOSOMA BRUCEI
SUMO
PROTEOMIC
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv SUMOylation is an important post-translational modification conserved in eukaryotic organisms. In Trypanosoma brucei, SUMO (Small Ubiquitin-like MOdifier) is essential in procyclic and bloodstream forms. Furthermore, SUMO has been linked to the antigenic variation process, as a highly SUMOylated focus was recently identified within chromatin-associated proteins of the active variant surface glycoprotein expression site. We aimed to establish a reliable strategy to identify SUMO conjugates in T.brucei. We expressed various tagged variants of SUMO from the endogenous locus. His-HA-TbSUMO was useful to validate the tag functionality but SUMO conjugates were not enriched enough over contaminants after affinity purification. A Lys-deficient SUMO version, created to reduce contaminants by Lys-C digestion, was able to overcome this issue but did not allow mapping many SUMOylation sites. This cell line was in turn useful to demonstrate that polySUMO chains are not essential for parasite viability. Finally, a His-HA-TbSUMOT106K version allowed the purification of SUMO conjugates and, after digestion with Lys-C, the enrichment for diGly-Lys peptides using specific antibodies. This site-specific proteomic strategy led us to identify 45 SUMOylated proteins and 53 acceptor sites unambiguously. SUMOylated proteins belong mainly to nuclear processes, such as DNA replication and repair, transcription, rRNA biogenesis and chromatin remodelling, among others.
Fil: Iribarren, Paula Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina
Fil: Berazategui, Maria Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina
Fil: Bayona, Julio César. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina
Fil: Almeida, I. C.. University of Texas at El Paso; Estados Unidos
Fil: Cazzulo, Juan Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina
Fil: Alvarez, Vanina Eder. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina
description SUMOylation is an important post-translational modification conserved in eukaryotic organisms. In Trypanosoma brucei, SUMO (Small Ubiquitin-like MOdifier) is essential in procyclic and bloodstream forms. Furthermore, SUMO has been linked to the antigenic variation process, as a highly SUMOylated focus was recently identified within chromatin-associated proteins of the active variant surface glycoprotein expression site. We aimed to establish a reliable strategy to identify SUMO conjugates in T.brucei. We expressed various tagged variants of SUMO from the endogenous locus. His-HA-TbSUMO was useful to validate the tag functionality but SUMO conjugates were not enriched enough over contaminants after affinity purification. A Lys-deficient SUMO version, created to reduce contaminants by Lys-C digestion, was able to overcome this issue but did not allow mapping many SUMOylation sites. This cell line was in turn useful to demonstrate that polySUMO chains are not essential for parasite viability. Finally, a His-HA-TbSUMOT106K version allowed the purification of SUMO conjugates and, after digestion with Lys-C, the enrichment for diGly-Lys peptides using specific antibodies. This site-specific proteomic strategy led us to identify 45 SUMOylated proteins and 53 acceptor sites unambiguously. SUMOylated proteins belong mainly to nuclear processes, such as DNA replication and repair, transcription, rRNA biogenesis and chromatin remodelling, among others.
publishDate 2015
dc.date.none.fl_str_mv 2015-06
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/11336/181239
Iribarren, Paula Ana; Berazategui, Maria Agustina; Bayona, Julio César; Almeida, I. C.; Cazzulo, Juan Jose; et al.; Different proteomic strategies to identify genuine Small Ubiquitin-like MOdifier targets and their modification sites in Trypanosoma brucei procyclic forms; Wiley Blackwell Publishing, Inc; Cellular Microbiology (print); 17; 10; 6-2015; 1413-1422
1462-5814
CONICET Digital
CONICET
url http://hdl.handle.net/11336/181239
identifier_str_mv Iribarren, Paula Ana; Berazategui, Maria Agustina; Bayona, Julio César; Almeida, I. C.; Cazzulo, Juan Jose; et al.; Different proteomic strategies to identify genuine Small Ubiquitin-like MOdifier targets and their modification sites in Trypanosoma brucei procyclic forms; Wiley Blackwell Publishing, Inc; Cellular Microbiology (print); 17; 10; 6-2015; 1413-1422
1462-5814
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/doi/10.1111/cmi.12467
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
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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
dc.publisher.none.fl_str_mv Wiley Blackwell Publishing, Inc
publisher.none.fl_str_mv Wiley Blackwell Publishing, 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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score 13.10058

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